National Institute of Science and Technology in. Edital Nº 15/2008 MCT/CNPq/FNDCT/CAPES/FAPEMIG/FAPERJ/FAPESP

Transcription

1 National Institute of Science and Technology Edital Nº 15/2008 MCT/CNPq/FNDCT/CAPES/FAPEMIG/FAPERJ/FAPESP National Institute of Science and Technology in Stem Cell and Cell Therapy INCTC Coordinator: Roberto Passetto Falcão Vice-Coordinator: Dimas Tadeu Covas Coordinating Institution: Faculdade de Medicina e Hemocentro de Ribeirão Preto, Universidade de São Paulo

2 Associated Research Laboratories and Groups Laboratório de Hematologia da Faculdade de Medicina de Ribeirão Preto da USP. Laboratório de Morfofisiologia Molecular e do Desenvolvimento da Faculdade de Zootecnia e Engenharia de Alimentos de Pirassunga, USP Centro de Transplantes de Medula Óssea do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP Departamento de Cirurgia, setor de Anatomia dos Animais Domésticos e Silvestres da Faculdade de Medicina Veterinária da USP Instituto de Ciências Biomédicas da UFRJ Laboratórios de Terapia Celular do Hemocentro de Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Biologia Celular do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Biologia Molecular do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Transferência Gênica do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Criobiologia I e II do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Biotecnologia do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de HLA do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Anemias Hereditárias do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Genética Molecular do Hemocentro de Ribeirão Preto HCFMRP-USP Laboratório de Citometria de Fluxo do Hemocentro de Ribeirão Preto HCFMRP-USP Centro Químico de Proteínas do Hemocentro de Ribeirão Preto HCFMRP-USP Centro de Primatologia do Instituto Evandro Chagas de Belém, Pará. Departamento de Genética e Biologia Evolutiva do Instituto de Biociências USP Laboratório de Genética da Faculdade de Medicina de Ribeirão Preto USP Departamento de Biologia Celular, Molecular e Bioagentes Patogênicos USP Departamento de Clínica Médica, Divisão de Imunologia Clínica - FMRP-USP Principal Researchers Dimas Tadeu Covas Fundação Hemocentro de Ribeirão Preto Eduardo Magalhães Rego Faculdade de Medicina de Ribeirão Preto/USP Flavio Vieira Meirelles - Faculdade de Zootecnia e Engenharia de Alimentos Júlio Cesar Voltarelli - Faculdade de Medicina de Ribeirão Preto/USP João Palermo Neto Faculdade de Medicina Veterinária e Zootecnia/USP Klena Sarges Marruaz da Silva Centro de Primatas do Institudo Evandro Chagas Belém-PA Lewis Joel Greene - Faculdade de Medicina de Ribeirão Preto/USP Lygia da Veiga Pereira - Instituto de Biociências/USP Maria Angélica Miglino - Faculdade de Medicina Veterinária e Zootecnia/USP Roberto Passetto Falcão - Faculdade de Medicina de Ribeirão Preto/USP Stevens Kastrup Rehen - Instituto de Ciências Biomédicas da UFRJ Wilson Araújo da Silva Jr - Faculdade de Medicina de Ribeirão Preto/USP

3 Table of Contents National Institute of Science and Technology in Stem Cell and Cell Therapy... 4 A) Program Description of the Institute... 4 The subject: Stem cells and Cell Therapy... 5 Justification B) Objetives and Goals C) Detailing the Research Program Lines of research and subprojects Studies with Pluripotential stem cells Induced Pluripotent Stem Cells Somatic Stem Cells Estabishment of Animal Models and Pre-clinical trials INDICATORS D) Program for High Qualified Human Resources Formation E) Detailed Description of the Actions for the Transfer of Knowledge to Society F) Actions detailing for transfer of knowledge for the Business Sector or for the Public Policy formation G) Detailed Description of the Proposing Group H) Activities Specification that must be done by the Team members I) Mechanisms that will be used to promote the Interaction of the Research Groups J) Forms of Interaction with Foreign Groups K) Definition of the Specific Tasks of Each Entity L) Comparative Analysis between the Current and Expected Statuses M) Justified Budget N) Potential for the Generation of Patents O) List of the Projects Financed over the last 5 years P) Formal Agreement by the Institutions Involved Q) Institutional Contribution R) Time table S) Indication of Management Committée T) Organization and Functional Structure of the Institute References

4 National Institute of Science and Technology in Stem Cell and Cell Therapy A) Program Description of the Institute The present proposal of creation a National Institute of Science and Technology (NISCT) is to continue and to amplify the activities of Center for Cellbased Therapy (CTC-CEPID-FAPESP), one Center of Excellence in Research, Innovation and Diffusion created by FAPESP in 2001 with the aim objective to be a new paradigm to organize research at Sao Paulo state. The CTC-CEPID, as we expected to be NISCT in the future, develops activities in three sectors which constitute the project scope: scientific project on the frontiers of knowledge, diffusion of knowledge for society and technological innovation with close collaboration with productive sector. The best way to demonstrate our capacity to perform the proposed objectives consists on demonstrating what we are doing at the present time. The CTC-FAPESP is formed by eight principal investigators (PI) which had good scientific research until 2000 but with very low interactions among of them. The impact of creation of CTC is evaluated at figure 1. The academic production related to Cell Therapy from the eight PIs increased gradually each year which resulted in 70 publications in The article citations of these PIs also increase significantly and became higher than 600 citations in The score h of the group is, at the present time, 42 which demonstrate the quality of scientific production. The collaborations among of PIs, which is measured by the presence of co-authors at the publications, also increased. All together, this demonstrates that CTC has established a unique plan, promoting the convergence and integration of the research areas among their PIs. Moreover, stimulate the researchers to actively participate to the diffusion of scientific knowledge. As example, is the edition of the book: Stem Cell the new frontier of Medicine which became a reference in this area in the country and received the Jabuti Premium in The creation of a National Institute for Stem Cell and Cell Therapy (INCTC) will allow for the consolidation and amplification of the experience obtained with 4

5 CTC, and will allow the mobilization and aggregation with the best research groups about stem cells and cell therapy in the country which will act as a network to allow the best performance of each specific competence and guarantee an accentuated advance capable to place Brazil in forefront of international scenario. Figura 1. Evolution of number of papers and cited papers obtained from the eight PI from CTC. The subject: Stem cells and Cell Therapy Cell therapy, by definition, consists on the use of cells with therapeutic applications. These cells can be used in several ways: by intravenous injections to have systemic actions or to protected organs or tissues such as bone marrow or central nervous system (CNS); used locally or injected directly into the damage tissue or organ to promote some regenerative effect or to protect. The cells used on cell therapy strategies are different in relation to their state of maturation or differentiation. They can be used as mature cells from peripheral blood such as eritrocytes, leukocytes and platelets as occurs, for example, in blood tranfusions, as well as, lymphocytes or dendritic cells which are in vitro sensibilized to be used in the cell vaccine protocols; also, it can be used stem cells or progenitor cells with the objective to repair or even total reconstitution of damage organ or tissue. As example of the latter, we have the consolidate example from more of 40 years of experience, the hematopoietic stem cells transplantation derived by bone marrow or peripheral blood or umbilical cord blood and which are able to reconstitute the total hematopoietic system. 5

6 Other examples of cell therapies well succeed with stem cells, either embryonary or somatic stem cells in animal models and in the man, have emerged in the last years and the search for new cell therapies more effective have occupied, in a relevant form, the scientific literature. The objective National Institute for Stem Cell and Cell Therapy (INCTC) is to develop an extensive program with basic and clinic researchs to understand, isolate, culture and with therapeutic application in animal models, as well as, in humans stem cells from different sources: embryonary stem cells, somatic stem animals and induced pluripotent stem cells. To better understand the mechanism involved in the maintenance or re-initiation of pluripotency it will be studied neoplasic stem cells derived from several tissues, in special stem cells from bone marrow which give rises to leukemia and lymphoma. In general, the scope of the scientific project is illustrated by figure 2.. The stem cells which will be studied can be classified into two groups: pluripotent stem cells and somatic stem cells. In the first group includes the embryonary stem cells and the induced pluripotent stem cells. In the second group will include hematopoietic stem cells (HSC), mesenchymal stem cells (MSC), endothelial progenitor cells and neoplasic stem cells (NSC). Additionally, it will be studied stem cells poorly characterized, such as, epithelial cells from placenta and stem cells from different regions of yolk sac. These stem cells will be isolated, characterized morphologically and functionally and cultured to become the source for several studies with focus on understanding the functional properties and to identify genetic and epigenetic molecular mechanisms involved in the induction and control of cell differentiation. Several strategies and tools dominated by the participants will be used in these studies, including: genomic, proteomic, cytogenetic, genetic, epigenetic, immunology, embryology, cell biology and culture and the sistemic biology. These cells also, will be cultured in large scale to be used in pre-clinical and clinical protocols with animals and humans. Specifically, for the studies with animals the Institute has the proposal of creation a Center for Pre-Clinical Studies and a Bank of Stem Cells to allow studies in animal of several sizes (rat, histricomorfs, rabitts, ovine, canine, equine, bovine and monkeys). This Center will be formed with the participation of Faculty of Veterinary Medicine and Zootecnia of São Paulo USP, 6

7 Faculty of Zootecnia and Food Engineer in Pirassununga USP, National Center of Primatas from Institute Evandro Chagas from Belém-PA and Laboratory for animal model studies from CTC. The infra-structure and scientific and technologic competence is present in all of these Institutions and will allow the formation of a network to perform pre-clinical and clinical studies without precedents in the history of the country. Specially, the studies with primatas (monkeys from old and new world) will add an important differential in the pre-clinical studies with stem cells developed in Brazil. In humans, three clinical studies at phase I and II will be conducted using mesenchymal stem cells: for diabets type I and to treat acute graft versus host disease (agvhd), developed by patients submitted to bone marrow transplantation. To execute this ambitious work plan a group of specialists and Institutions with comproved scientific experience in several areas will put together: molecular and cell biology, genetic, embryology, immunology, hematology, systemic biology, chemistry of protein, veterinary and bioinformatics. The participation of these scientists and their respective groups performed in an integrative and complementary way. The proposal objectives and expected results are the result of a multidisciplinary approach which focus on the achievement scientific objectives, but at the same way integrative relative to more relevant and extensive questions. We expected that this scientific multifaceted and multidisciplinary approach in this specific subject will allow the fast grow of cell therapy program, consolidating and amplifying the participation of Brazil in the international community and placing in the first line of evidence concerning to this subject. The research activities are described with detail at item C. 7

8 Somatic Stem cells Pluripotent Stem cells MSC EPC HSC Cancer Embryonic Induced (ips) Placenta Epithelial cells Yolk Sac stem cells Functional Properties and Mechanisms Genomics Proteomics Cytogenetics Genetics Cell Biology Epigenetics B. Systemic Immunology Embryology Culture Engineering Diferenciation Controle and Induction Production of Cell to Clinical Use Embryonic Lineages Stablishment Neoplasic Lineages Stablishment Neoplasic Mechanism and tranformation Clinical and Pre-Clinical Studies Animal Models Mice Hystricomorphus Rabbits Sheep Suine Canine Equine Bovine Monkeys Humans Diabetes DECHA (1) DECHA (2) Figure 2. Scope of Project In parallel with scientific program we intend to develop intense activity about scientific knowledge transfer for the society involving the public in general, but also, and in special the professors from high school and their students. The NIST intend to amplify the transfer activities which are made by the Cell-based Therapy Center (CTC-CEPID-FAPESP), and will extend these activities to the other 8

9 associated laboratories (São Paulo, Pirassununga, Rio de Janeiro e Belém) as well as will perform new activities with greater abrangency. The knowledge dissemination activities were always relevant in the CTC program. In 2000, as part of the program it was constructed the House of Science at the Hemocentro de Ribeirão Preto do HCFMRP-USP (Coordinating Institution of the CTC). The House of Science is an area of 50 m 2 which allocates 8 professionals with exclusive dedication to the knowledge diffusion and Science teaching are allocated. This team is coordinated by Profa. Dra. Marisa Barbieri, a retired professor from the University of São Paulo who has large experience in teaching and training science or biology teachers. The construction of this place was supported by several Institutions: Fundação Hemocentro de Ribeirão Preto, FAPESP, Fundação Vitae and the University of São Paulo (scholarships linked to the COSEAS program). Among the principal activities developed we can mentioned: Courses of specialization termed The cells, the genome and you, professor administrated together with USA and the Education Secretary of São Paulo State. This course was offer to more than 200 professor of High School from public and private schools The university extension presential or distance courses of with duration of 30 hours; the projects of scientific initiation from FAPESP; the courses of scientific initiation for students of public schools termed Talented students and Adopt a Scientist, being the first one a project with weekly meetings among senior investigators from CTC, students and professoars and the second one, a project which consists to develop simple research projects, liderated by graduated students from São Paulo University with have the responsability to teach to a small group of students and to generate educational material for science diffusion as the Journal of Science. This Journal is at the 18 a. eddition and is produced by students and distributed for several schools of the region. The production of education games, theatrical play, such as, A cell s agony, confection brochures, such as, Talking about HIV and AIDS which was financed by Ministry of Health and distributed to Schools of all country, as well as, the publication of manuscripts about the CTC educational project at Brazilian and international journals are other diffusion material educational from the House of Science. 9

10 In 2004, the House of Science had the amplification of their activities with the construction, supported by the University of São Paulo, which gave a House from the University Campus of Ribeirão Preto, of MULEC -Museu e Laboratório de Ensino de Ciências (from English - Museum and Laboratory for Science Teaching) which contains a permanent exhibition with educational material produced during the activities developed at House of Science. This new place receive visitations of students and public in general. Other activities performed are: the participation during the National Week of Science and Technology (2004, 2005, 2006, 2007), in Scientific Conferences (SBPC), in programs of electronic media (radio and TV) and in the impressa midia. All these activities and divulgation of science which are promoved and supported by CTC are described with details at the site of House of Science ( and it will be part of the educational program from INCTC. The figure 3 illustrate a summary of the activities of knowlegded transfer from CTC. Other educational activities and formation of human resources will include summer courses offered annually and courses of professional formation. The CTC offers annually, at January month, a summer course with destination to undergraduate students from biology area from all the country. Until this moment, it was offered 8 editions with the participations of more than 280 students. The thematic of these courses have been: genomic, proteomic and stem cells. The course comprehends two weeks and include several theoretical and fundamented practical classes within this thematic. Moreover, CTC offers specialization courses for physician and no physicians in the Field of hemotherapy, nurse, social assistance and gestor in health service. The CTC is linked to São Paulo University by the Faculty of Medicine of Ribeirão Preto and other units situated at Ribeirao Preto Campus and their professors and researchers which are activally engaged at undergraduated courses of medicine, pharmacy, chemistry, biomedical informatic. Also, participate regularly at pos-graduation courses from these departments. 10

11 The cells, the genome and you Teacher Talent-Hunting Precongress Site Journal of Sciences Junior scientific initiation Curso On-Line Course MuLEC National Week of Science and Technology Adopt a Scientist Wheel of Science Events and Activities Wall I and II Tuesday of Science Fridays of Wall land Science II Folhetins PIPOC PIPAS Site to Teacher Science for Everyone Space Blood Panel Congress of Pediatrics Safety Blood Partners on the Science Dissemination Agony of a Cell - Brasília Stem Cell Exposition Sara and the Blood preciousness Pre scientific initiation - USP Science with the youth Building the future Retro Room Institutes of Support and Funding: FAPESP; CNPq; Fundação Vitae; Pró-Reitoria de Cultura e Extensão USP Figure 3. History of the Transfer Activities. In this present proposal, the objective is to amplify the CTC experience and to extend these described activities to the laboratories and units which are associated in the INCTC proposal. Also, we plan to scale up these activities by offering specialization courses via internet. The target public will be students and science or biology teachers from High Schools. During the first year the INCTC has planned to offer a specialization course of 360 hours to train 900 science or biology teachers. As an integrant part of the activities, each professor will need to register a classroom from a public school with at least 40 regular students. This initiative will result in the participation of 36,000 students during a 10 month period. The activities with the school teachers will be predominantly performed at distance using the TIDIA plataform. Approximately 20% of the activities will be performed on 11

12 site at distance-education training places situated at different units from universities of the State of São Paulo. The course will be authorized by the USP and developed within the Program Virtual University of the State of São Paulo (UNIVESP). The official documentation of the course is underway and a preliminary approval from the Secretary of Higher Education of the State of São Paulo and from the Pro-Rector of Culture and Educational Extension of the University of São Paulo was already obtained. After the second year of the project, the course could be extended to the entire country. Moreover, we intend to organize an Inter-Units post-graduation course (FMRP-USP, FMV-USP, FZ-USP, ICB-USP e UFRJ) on Stem cell and Cell Therapy and a professionalizing master course for training specialized technicians in this field. In addition to these activities we are proposing an ambitious plan of science diffusion and specialized personnel training by offering post-graduation strictu e latu senso courses. The divulgation of scientific relevant productions generated within the INCTC will be performed by scientific publications and additional supplementary means. The main goal is to reach several segments of the society in order to promote the interest for science and demonstrate the importance of continuous scientific education. These new initiatives proposed by the INCTC are described with more details in the Item E. Finally, the innovation activities, together with the research and education transfer programs, constitute the triple helix that characterizes this project proposal. The triple helix represents the ideal organization model of Universities and Research Centers at the XXI century (Etzkowitz, 1996). The INCTC intends to amplify the experience of the CTC-CEPID in implementing business incubators within the Coordinating Institution. The first business incubator denominated INBIOS (Business Incubator in Health Biotechnology) was successfully implemented in The objective of INBIOS is to give support to business people to create or amplify biotechnology business companies or to support special P&D areas of middle and large companies interested in developing products or services at the incubator company. The preferential working areas of INBIOS are: biotechnology, biomedicine, medical and odontological materials and equipments, tools, informatics technology, environment, chemistry and nuclear technicians. In 2006, the INBIOS, was integrated to the SUPERA business incubator of Technology of 12

13 Ribeirão Preto, which was then denominated SUPERA-HEMOCENTRO ( At this moment, SUPERA gives support to 09 resident companies, 03 preresident companies, 01 associated companies and 20 projects in the modality of Hotel of Projects. The SUPERA-HEMOCENTRO is installed in the space of 128 m 2 and offers space to companies, such as: Rad Tech Sistemas Médicos Ltda, Capelli Fabris Desenvolvimento e Pesquisa Ltda, Lychnoflora Pesquisa e Desenvolvimento de Produtos Naturais Ltda, CG Brasil Consultoria e Informática Ltda e Innolution Sistemas de Informática Ltda. The INCTC project will incorporate this previous innovation experience of the CTC-CEPID into the productive sector. Additionally, it will be developed new initiatives focusing in technology development and its transfer to the productive sector, as detailed described in the Section F. Justification In the year 2005, the United Kingdom promoted a panel of specialists diagnose and plan activities related to stem cells and cell therapy for 10 the next years ( ). In the final document, UK Stem Cell Initiative Report and Recommendations short to long term actions were proposed to place the UK in the leading position worldwide in cell therapy, which was considered strategic due to its innovative potential in the healing of illnesses. In chapter 5 of the document, a fluxogram describing the critical steps that would rule the development of products and processes with stem cells and cell therapy was presented. In Figure 4 we reproduce that fluxogram. The development of new cellular therapies starts with an effort in basic research in order to isolate, characterize and understand the biological mechanisms involved in the maintenance of the undifferentiated status of the stem cells. The second step in this fluxogram consists of the establishment of well-characterized and homogeneous Stem Cell lines, which are to be deposited in a stem cell bank specifically created for that purpose. The bank would be responsible for storing, exporting and distributing the cells lines to research laboratories. In UK that suggestion led to the creation of the UK Stem Cell Bank. The third step consists in the large scale production of well-characterized stem 13

14 cells to be used in pre-clinical and clinical studies. In the UK, several initiatives sponsored by both the government and by the private initiative have begun. At this level, the generation of patents of products and processes is highly likely which explains the great interest of the private sector in this area all over the world. In the fourth step of the fluxogram, there are the clinical studies, which require specialized physical and professional structures in that area. In the final stage, there are the registration and launching of the product for general use. In Brazil we can say for sure that most of the studies and initiatives are found at phase 1 of the fluxogram below. The current proposal takes that track. We propose a group of activities that are distributed throughout the described phases described in Figure 4: basic research, the creation of a stem cell bank, the development of large scale cultivation systems under GMP conditions, and preclinical and clinical tests. This coordinated effort will put Brazil at the leading edge of research in cell therapy. THERAPIES PRODUCED FROM STEM CELL LINES Research & Development Stem Cell Banking Cell Therapy Production Stem Cell Clinical Research Marketing Approval Basic Stem Cell Research Cell Biology Imunology Animal Modeling Stem Cell Bank Research grade cell banking and characterisation Clinical grade cell banking and characterisation Stem Cell Therapy Production Unit Production process development Plot scale capacity to supply stem cell clinical trials Stem Cell Clinical Trials Specialist Bed and Facilities Safety and Efficacy Epigenetics Bioengineering Coordination 1,3,5,6,7,8,9,10, 11,12,13,14,15, 16,17,18,19,20, 4,6,7,26, e 27* 2 e 23* 28, 29 e 30* e 25 * Licensed Product Manufacture Stem Cell Therapy Commercial or Plubic Sponsor Therapy Surveilance Figure 4. Fluxogram of the phases under development of cell therapies. (Adapted from UK Stem Cell Initiative, 2005).* number of the subprojects. 14

15 In the last line of Figure 4 we show the relationship of the phases with subprojects proposed here. The major differential of this Project is that it expands the scope of the stem cells studies and cell therapies that are currently underway in Brazil. Although there are several phase I and II clinical studies underway in the country, we note that these are generally based on the use of minimally manipulated bone marrow mononuclear cells and follow experimental protocols established by other studies, which became widespread worldwide. These protocols were embraced by many opportunistic research groups which had no previous experience in studies with stem cells simply because these cells are easily obtained. That phase was rapidly exhausted, and it was demonstrated that the revolutionary clinical advances expected from the use of the mononuclear fraction of bone marrow were not realized, this reduce the priority of this kind of study in cell therapy. Cell therapy, as we have previously defined, has great therapeutic potential, being really able to revolutionize the treatment of countless chronic-degenerative diseases. However, in order to reach such potential, both scientific and regulatory activities are necessary. Unfortunately, this does not exist in a consolidated way in Brazil at the present moment. The Project of the National Institute for Stem Cells and Cell Therapy proposes a model of coordinated performance, similar those adopted by the countries who have become leaders in this area, especially the United Kingdom. As shown in Figure 4, our proposal is constituted by a group of subprojects that aim to accomplish both scientific and technical, so that effective cell therapy be available for wider sectors of the population in a time span that ranges from 8 to 15 years. In this context, a first group of subprojects is directed to the study the study of the basic properties of stem cells (cellular biology, immunology, epigenetic, genomics, cytogenomics, proteomics, bioengineering, etc.). Included in this group, there is also the development of animal models, and here we emphasize the utilization, for the first time in Brazil, of non-human primates for the development of experimental models to test cellular therapies. We intend to form a Center of Pre-Clinical Studies to meet the demand that will be generated by the project. Simultaneously, a second group of subprojects will be developed who objective is the establishment of homogeneous pluripotent and somatic stem cell 15

16 lines. Our goal is to obtain well-characterized stem cell lines that can be expanded culture systems without losing their primitive properties. They will be made available to several research laboratories and for pre-clinical and clinical studies. To reach this goal, we intend to establish two stem cell banks: one for animal stem cells, which will be coordinated by teams of Prof. Maria Angélica Miglino at Faculty of Veterinary Medicine of the University of Sao Paulo (FMV-USP-SP) and by Dr. Klena Sarges Marruaz da Silva at the Belem s Primate National Center (Belem-PA). The second bank for human stem cells which already exists at our Center for Cellbased Therapy (CTC) - Hemocentro shall be enlarged in its processing capacity to accomplish the needs required by the Institute. The major premise of this phase of the Project is that the therapeutic effects obtained with the animal models and the clinical studies need to be directly correlated with specific cell populations in order to be reproducible and trustworthy and also be capable of expansion in culture, so that the progress in this area can be scientifically consistent. A third group of subprojects is directed to the development of large scale stem cell production system to extend the availability of cellular therapies to a greater number of patients. The limited expansion capacity of stem cells is today the greatest obstacle to implement effective cell therapies. For instance, in the clinical study of phase I we have been using mesenchymal stromal cells for the treatment of the acute graft-versus-host disease (GVHD) post-transplant of bone marrow, the treatment of one patient weighing 70 Kg demands the culture of cells in 30 one liter culture flasks for 20 to 30 days to achieve a final number of about 7 x 10 7 cells. Without the development of more efficient cell culture system, it is impossible to imagine that this kind of treatment may be extended to those who need it. An alternative approach to circumvent this problem is to produce cells in bioreactors. In the present Project, we will explore the use of bioreactors not only for the expansion of embryonic stem cells but also for mesenchymal stem cells. The fourth group of subprojects involves clinical studies using mesenchymal stem cells to treat patients who have type 1 diabetes and for the treatment of acute graft-versus-host disease (GVHD). Our group is the only in Brazil using mesenchymal cells cultivated under GMP conditions for therapy. In the proposal group, this Project of INCTC promotes great advances to the country in studies involving stem cells and cell therapy. 16

17 B) Objetives and Goals To aggregate renomated Brazilian researchers involved in the study of stem cells, currently working in different parts of the country, in a network called Instituto Nacional de Ciencia, Tecnologia e Inovação (INCTI). The set up of this INCTI will allow the interchange of scientific information, reagents, methods, human resources and logistic, as well as equipment already available at the different centers, in order to foster academic and technological advancement, along with training of specialized personnel in the field of Biotechnology, Health and Agrary sciences with national and international relevance. One of our aims is to address essential questions such the characterization of stem cells from different sources and species, determination of genetic, epigenetic and cellular mechanisms regulating the maintenance of the pool and the differentiation of stem cells. In addition, we propose to create technical protocols, a tissue and cell bank, bioinformatics tools, cell lineages, genetically modified animal which will be used in the subprojects listed below. The coordinated effort of the different researchers and centers will be a unique opportunity to approach the scientific problems using several strategies. As aims in applied science we propose to use stem cells in the treatment of patients with diabetes mellitus, multiple sclerosis and graft versus host disease. Our proposal also aims to generate human resources in the field of biotechnology, health and agrarian sciences. Moreover, we will create the learning opportunities for teachers of biology and sciences working in middle and high public schools, as well as arouse the interest of children and adolescents attending these schools in the study of Sciences. The general population and technology companies (some of them working in the SUPERA) will have access to the main achievements of the Project. To allow the monitoring of the development of the project, we list below the specific objectives of each subproject (item C). This list, chronograms and supplementary material may be found in specific sections and in the website 17

18 C) Detailing the Research Program The research program of the INCTC is composed by 30 subprojects which are presented here with the objective and goals of each one. In the second part of this item (C2) these subprojects are briefly described as part of a rational organization indicating, preliminarily, the integrator character of the proposal. The rationalization for this program was shown in figure 4. In the H item of this project (Activities Specification that Must be Done by the Time Members) we present a table which shows the integration of the involved research groups (represented by the coordinator s names) in these subprojects. C1) Subprojects, Objectives and Goals Subproject 1: Evaluation of the epigenetic stability on embryonic stem cell lines established under different conditions Coordinator: Lygia da Veiga Pereira 1. The establishment of hesc lines from the inner cell mass of blastocyst; 2. Evaluation of different conditions for cell line establishment (in the presence or not of murine fibroblasts; with fetal bovine serum or serum replacement; in a defined medium) in relation to its epigenetic state; 3. Characterization of the plasticity of hesc lineages; 4. Analysis of the state of activity of X chromosomes in trophoblasts cells removed from embryos; 5. Evaluation of the effect of chromosomal instability on capacity of hesc. 18

19 Subproject 2: Scale-up of human embryonic stem cell production in bioreactors for application in regenerative medicine Coordinator: Stevens Kastrup Rehen Specific goals are: 1. To evaluate comparatively the growth and pluripotency of human embryonic stem cells cultured in stirred systems as embryoid bodies or using microcarriers as a support. 2. To characterize chromosomal stability and differentiation properties of human embryonic stem cells maintained in stirred systems for long periods of time. 3. To scale-up the expansion of human embryonic stem cells by the use of bioreactors with the objective to use them for transplantation in experimental animals and, eventually, in humans. 4. To develop an economically viable protocol for the expansion of human embryonic stem cells for the production of large amounts of cells. Subproject 3: Molecular basis of hematopoietic differentiation from embryonic stem cells Coordinator: Dimas Tadeu Covas 1. Standardization of an in vitro hematopoietic differentiation protocol for human embryonic stem cell (hesc) lines; 5. Immunomagnetic selection of CD34+ hematopoietic stem cells (HSC) derived from hesc and umbilical cord blood (UCB) and subsequent RNA isolation; 6. Standardization of in vitro T-lymphocyte differentiation protocol from CD34+ HSC derived from hesc or UCB; 7. Evaluation of differentiation protocols by flow cytometry; 19

20 8. To perform microarrays to obtain the transcriptional profiles (mrna and microrna); 9. Analysis of the results using bioinformatics and specific databases for identification of putative regulatory mechanisms; 10. Validation of results by real-time PCR (mrna and microrna results). Subproject 4: Culture of adult and embryonic stem cells of animals and human beings for use in cellular therapies Coordinator: Maria de Fátima Lima de Assis 1. Bibliographical survey for the choice of the protocol to be adopted for cell culture; 2. Selection, collection and identification of the cells; 3. To culture adult and embryonic stem cells with minimum chemical interference to conserve the genome integrity, keeping the cultures cryopreserved in the first stage to prevent the occurrence of polyploidy in vitro; 4. Culture, expansion and cryopreservation of embryonic and adult stem cells; 5. Karyotypic identification of adult and embryonic stem cells; 6. In vitro differentiation of the stem cells, into specific tissues, for purposes of cell therapy; 7. Stimulate adult stem cells to develop function specific of embryonic stem cells (cellular dedifferentiation). 20

21 Subproject 5: Gene Modification of stem cells Coordinator: Dimas Tadeu Covas 1. To construct lentiviral vectors carrying the stemness genes followed by IRES and GFP; 2. To transduce mesenchymal and endothelial cells with the viral vectors to induce the enhancement of the transcription factors Oct3/4, Sox2 and Nanog. 3. To evaluate the changes in the gene expression profile to elucidate the mechanism of action of stemness genes and to describe new therapeutic targets. 4. To select the mesenchymal cell clones transformed by the lentiviral vectors, assess the viability and observe the capacity to differentiate into different cell lines. Subproject 6: Establishment of induced pluripotent stem cell lines (ips) in large animal models Coordinator: Lygia da Veiga Pereira 1. Development of pluripotency induction vectors in non-human primates and canine cells; 2. Establishment of ips lines from animal model fibroblasts; 3. Neural differentiation of ips and pre-clinical assays in animal models of spinal cord injury. 21

22 Subproject 7: Establishment of induced pluripotent stem cell lines (ips) from fibroblasts of patients with Mendelian and multifactorial genetic diseases (with genetic component) Coordinator: Lygia da Veiga Pereira 1. Standardization of methods for the generation of human ips from skin fibroblasts, peripheral blood and bone marrow cells; 2. Establishment of ips lines from patients with genetic diseases of interest to the groups, in order to study their molecular mechanisms; 3. To establish ips from human fibroblast cell lines GM1662, 46 XX, heterozigous for a HPRT gene mutation, in order to use this cell line to study epigenetic modifications associated with human X chromosome inactivation; 4. To develop adenoviral bi-cistronic vectors aiming to (i) increase ips production efficiency and (ii) generate non-genetically modified through the elimination of vectors after the induction. Subproject 8: Autologous pluripotent cells generated from differentiated somatic cells Coordinator: Flavio Vieira Meirelles 1. Dedifferentiation of differentiated cells using the following methods: 2. Genetic insertion of known genetic factors (ips) 3. Nuclear transfer using fibroblasts as nuclear donor cells; 4. Nuclear transfer using ips as nuclear donor cells; 5. Derivation of pluripotent cells with the aforementioned methods, as well as from embryonic stem cells in vitro generated; 6. Morphological, gene expression and epigenetic comparison of undifferentiated cells obtained using the methods above. 22

23 7. To determine the method that most resembles the undifferentiated cells of the control group (transcriptome, epigenomic and proteomic profiles) and apply it in the interespecific model; 8. To perform dedifferentiation functional assays with the cells generated by the interespecific model: bovine cytoplast and monkey cell as nuclear donor cell. Subproject 9: Animal models to study mitochondrial inheritance intra- and interspecies Coordinator: Flavio Vieira Meirelles 1. To generate bovine blastocysts (Bos taurus) with mtdna of somatic origin intra- (B. indicus) or interspecies (Homo sapiens) in heteroplasmia with mtdna of embryonic origin (oocyte inherited) 2. To develop a method to increase the percentage of inherited somatic mtdna in the blastocysts (B. indicus or H. sapiens). Subproject 10: Basic knowledge of MSC biology by means of comparison with pericytes Coordinator: Dimas Tadeu Covas 1. Compare freshly isolated pericytes, pericytes cultured under MSC conditions, and MSCs in terms of their behavior when subjected to differentiation conditions described for MSCs, and different culture conditions in vitro; 2. Compare freshly isolated pericytes, pericytes cultured under MSC conditions, and MSCs in terms of their ability to contribute to diverse tissues when injected into blastocysts; 3. Study the surface marker profile of freshly isolated pericytes, and compare it directly to that of pericytes cultured under MSC conditions, and cultured MSCs; 23

24 4. Study the profile of cytokines with immunomodulatory potential expressed by freshly isolated pericytes, and compare it directly to that of pericytes cultured under MSC conditions, and cultured MSCs; 5. Observe the behavior of pericytes infused systemically in an animal model of tissue injury, using cultured MSCs as a control, and investigate the role of pericytes during the tissue repair/regeneration process. Subproject 11: Investigation of angiogenic process and molecular interactions between mesenchymal stem cells or pericytes with endothelial cells using developed in vitro and in vivo systems Coordinator: Dimas Tadeu Covas 1. Phenotypic characterization of mesenchymal stem cells from bone marrow (BM-MSC) and umbilical cord vein (UCV-MSC) and central nervous system microvascular pericytes (CNS-P) using 6 CD markers: CD271, CD140B, 3G5, NG2, Stro-1 and CD146. Among of them, to select the surface antigen which is more expressed in MSC and CNS-P; 2. Clonogenic assays and morphological characterization of FACS-sorted BM- MSC, UCV-MSC and CNS-P using a specific marker previously selected; 3. Morphological and phenotypical characterization of FACS-sorted cell populations; 4. Isolation, morphologic and phenotypic characterization of mesenchymal stem cells from canine yolk sac (cys-msc); 5. Investigation of multipotential potential of FACS sorted and expanded stem cells using standard conditions for osteogenic, chondrogenic and adipogenic differentiation; 6. Genetic modification of human and canine MSC with firefly luciferase (luc) and and DsRed2 fluorescent protein using transposon-mediated gene transfer; 24

25 7. Morphological and phenotypical characterization of FACS-sorted cell population and DsRed2 fluorescent protein positive cells; 8. Isolation and culture of human umbilical vein endothelial cell (HUVEC) and canine yolk sac endothelial cells (cys-ec) FACS-sorted according to CD31 high level expression; 9. Generation of mature endothelial cells from human bone marrow - and cord blood derived endothelial progenitor cells (CD133+KDR+); 10. Morphological and phenotypical characterization of FACS-sorted CD31+ cell population; 11. The influence of endothelial soluble factors, as well as, the requirement of direct cell-cell contact between endothelial cells and BM-MSC, UCV-MSC or NS-P using co-culture in vitro systems; 12. Morphological, phenotypical and gene expression analysis of both cell populations before and after co-culture systems, as well as, the formation of vascular like structure on matrigel for the evaluation of in vitro angiogenesis; 13. Assessment of the role of BM-MSC,UCV-MSC, CNS-P and cys-msc in angiogenesis after co-infusion with HUVEC or cys-ec using the Matrigel plug assay followed by subcutaneous injection in NODSCID mice. Image of new blood vessels in live animals with IVIS Xenogen system will be performed; 14. Characterization of new blood vessel formation by confocal microscopy; 15. Analyze the contribution of mesenchymal stem cells or pericyte together with endothelial cells to the recovery of the hind limb ischemic injury in NODSCID mice by confocal microscopy.. 25

26 Subproject 12: Comparison of proteins Expression of human mesenchymal stem cells (MSC) obtained from bone marrow and umbilical vein cord Coordinator: Lewis Joel Greene 1. To obtain human MSC from bone marrow and umbilical cord vein and expansion of these cells in vitro. 2. Immunophenotypic characterization and MSC differentiation into adipocytes, condrocytes and osteocytes. 3. Extraction and quantification of soluble cytoplasmatic proteins of MSC. 4. Bidimensional electrophoresis and comparison of the proteins in the MSC extracts with fluorescent dyes (DIGE). 5. Gel 2D quantitative analysis using the DeCyder software. 6. Protein identification of the spots of interest obtained from preparative gel after trypsin digestion by mass spectrometry (MALDI-TOF-TOF). 7. Proteins identifications using data banks such as the Gene Ontology. 8. Comparison the Proteome results to the published data obtained by SAGE Subproject 13: Differences in gene expression and immunophenotypic profile of amniotic epithelial cells obtained with and without animal substances Coordinator: Wilson Araújo da Silva-Jr 1. - To obtain two amniotic epithelial cell populations using different methods of isolation and culture, with and without serum; 2. - To evaluate typical markers of embryonic stem cells in these populations by flow cytometry; 3. - To assess the expression of surface markers related to immunogenicity and immunomodulatory properties of amniotic epithelial cells (flow cytometry); 26

27 4. - Comparison of gene expression profiles of the different cell populations obtained; 5. - To compare the differentiation potential into 3 germinal layers: endoderm (hepatic); mesoderm (cardiomyogenic) and ectoderm (neurogenic). Subproject 14: Isolation and functional characterization of pluripotent stem cell - side population Coordinator: Dimas Tadeu Covas 1. Isolation and characterization of human side population of stem cells from bone marrow, umbilical cord vein and blood capillaries from central nervous system (day 0); 2. Isolation and characterization of human side population of stem cells obtained from cultured mesenchymal stem cells (MSC-SP) isolated from bone marrow, umbilical cord vein, as well as, cultured pericytes isolated from central nervous system blood capillaries using the classical plastic adherence protocol; 3. Isolation and characterization of ovine side population stem cells in bone marrow and in blood capillaries from central nervous system (day 0); 4. Isolation and characterization of canine side population stem cells from yolk sac (day 0); 5. Isolation and characterization of side population stem cells in bone marrow and umbilical cord vein from transgenic fetal bovine, which express GFP (day 0); 6. Analysis of side population frequency from human, dog, sheep and bovine tissues at day 0 by flow cytometry and in vitro clonogenic assay; 7. Analysis of side population frequency from human, dog, sheep and bovine cultured mesenchymal stem cells tissues obtained by the classical plastic adherence protocol; 27

28 8. Culture and expansion of side-population of stem cells isolated at day 0, as well as, SP cells isolated from MSC cultures using four different culture media: a) culture medium for MSC expansion; b) culture medium for expansion of hematopoietic stem cell (HSC-M); c) culture medium for expansion of endothelial progenitor cells and d) culture medium for expansion of human embryonic stem cells; 9. Analysis of karyotype and cytogenetic characteristics of human SP; 10. Analysis by microarray of the gene expression profile of human SP culture and expanded under MSC conditions; 11. Culture and expansion of side-population stem cells isolated sheep, canine and bovine tissues using medium from MSC expansion; 12. Morphologic and immunophenotypic characterization of side population stem cells undergoing expansion using different conditions; 13. Differentiation potential of mesenchymal stem cells derived from human and animal SP; 14. Analysis of the gene expression profile of human SP cultured under with culture media (EPC, HSC and hes), as well as, bovine, canine and ovine SP cells cultured with several media by real time PCR; 15. Investigation of therapeutic potential of human and animal SP using three murine models: 1) long-term engraftment of hematopoietic stem cells after bone marrow transplantation with human and animal SP expanded under HSC medium conditions; b) engraftment of human and animal SP cultured and expanded under EPC and MSC medium after endothelial hepatic injury by monocrotaline administration and c) in vivo angiogenesis potential using the Matrigel plug assay followed by subcutaneous injection in NODSCID mouse; 16. Investigation of therapeutic potential of culture SP cells using two largeanimal models: 1) ability of canine bone marrow SP cells cultured under HSC conditions to stimulate hematopoietic reconstitution in a canine bone marrow aplasia model and 2) ability of ovine brain capillaries SP cells cultured under MSC and EPC conditions to engraft and persist in brain ischemic injury. 28

29 Subproject 15: Functional evaluation of micrornas in mesenchymal stem cells differentiation Coordenador: Dimas Tadeu Covas 17. Isolation and expansion of bone marrow derived mesenchymal stem cells from; 18. Differentiation under appropriate in vitro conditions into osteoblasts; 19. Quantification of microrna expression during distinct stages of in vitro osteoblasts induction; 20. Identification of potential gene targets for mirnas by in silico analysis; 21. Functional evaluation of mirnas that were differentially expressed. Subproject 16: Gene expression profile of mesenchymal stem cells of patients with Osteogenesis Imperfecta during in vitro osteogenic differentiation. Coordinator: Wilson Araújo da Silva Junior 1. Analyze mesenchymal stem cells gene expression profile by microarray assay at least in four points of osteogenic differentiation. These analyses will be applied in two samples of patients with Osteogenesis Imperfecta and two normal samples. 2. Select genes with differential expression to be evaluated by Real Time PCR. 3. To perform functional analyses of genes selected as potential regulators of normal and abnormal osteogenesis evaluating cell proliferation, migration, apoptosis and cell death and to verify if these processes are modified in patients with Osteogenesis Imperfecta. Subproject 17: Genetic, proteomic and functional analyses of mesenchymal stem cell (MSC) from patients with autoimmune diseases 29

30 Coordinator: Julio Cesar Voltarelli 1. To isolate, expand and characterize (number of colony forming unitsfibroblast, immunophenotype, cell expansion, doubling time and differentiation potential) MSC isolated from bone marrow of T1D and MS patients; 2. To isolate, expand and characterize (number of colony forming unitsfibroblast, immunophenotype, cell expansion, doubling time and differentiation potential) MSC isolated from bone marrow of T1D and MS patients; 3. To evaluated the in vitro immunossupressive capacity of MSC from T1D and MS patients 4. To evaluated the effect of the high dose immunossupression on the MSC from T1D and MS patients; 5. To analyze the gene expression of MSC from T1D and MS patients by cdna microarrays; 6. To analyze the proteomic expression of MSC from T1D and MS patients by two-dimensional gel electrophoresis; 7. To validate the gene expression results obtained by cdna microarrays by real time RT-PCR; 8. To study relevant genes/proteins, whose expression were found altered in the gene and proteomic expression studies, by functional studies using RNA interference. 30

31 Subproject 18: Determination of some proteins related to apoptotic process and cell cycle control of hematopoietic and leukemic progenitors Coordinator: Lewis Joel Greene 1. Isolation, separation, characterization and As2O3 treatment of normal precursor cells, leukemic stem cell and leukemic blasts. 2. 2DE Gel standardization 3. Spots analyses by densitometry 4. Immune detection of proteins involved in the apoptotic process using mono and polyclonal antibodies. 5. Protein identification by MALDI-TOF and ESI-MS/MS 6. Data analyze using bioinformatic approaches Subproject 19: Evaluation of the function of dyskerin in early hematopoietic differentiation Coordinator: Eduardo Magalhães Rego 1. To examine the role of dyskerin in hematopoietic stem cells (HSC) of Dkc1 mutant mice will be tested against their wild-type counterparts in their capacity for repopulating lethally irradiated recipients C57/BL6; 2. To examine the role of dyskerin in early lymphoid differentiation by competitive repopulation assays using rag-/- mice, which do not have mature T and B cells; 3. To quantify the expression of the Dkc1 gene in isolated progenitors by Real Time PCR isolates by FACS Sorting in Dkc1m and wild type mice; 4. To analyze apoptosis and cell growth of Dkcm lymphoid progenitors and in correlated cells in wild type animals. 31

32 Subproject 20: Acute Leukemia Model in Chlorocebus aethiops (Cercopithecus aethiops) using haematopoetic stem cells transduced with retroviral vector containing the hibrid gene CALM-AF10 Coordinator: Eduardo Magalhães Rego 1. To establish the acute leukemia model in Chlorocebus aethiops using haematopoetic stem cells transduced with retroviral vector containing the hybrid gene CALM-AF10 2. To establish the collection protocol and bone marrow hematopoietic stem cell culture 3. To study the haematology, cytogenetic, and molecular characteristics 4. Determination of bone marrow immunessuppression and transplantation protocols 5. To establish the gene expression model by retroviral vector in bone marrow haematopoetic stem cells from Chlorocebus aethiops 6. To study the haematological cytogenetic and molecular alterations due to the CALM-AF-10 retroviral gene expression in the bone marrow of Chlorocebus aethiops patients after the autologous transplantation. 7. To establish the leukemia treatment protocol in Chlorocebus aethiops. Subproject 21: Evaluation of the therapeutic effect of mesenchymal stem cells on regeneration of extensive thermal burn wounds in animal model Coordinator: Júlio César Voltarelli 1. To compare therapeutic and regenerative potential of xenogenic and allogenic MSC; 2. To compare therapeutic and regenerative potential of MSC administered locally or systemically; 32

33 3. To compare therapeutic and regenerative potential of xenogenic MSC isolated from bone marrow of wild- type C57BL/6 mice (Gal-1+/+) or Gal-1 deficient C57BL/6 mice (Gal-1-/-); 4. To evaluate the thermal burn wound severity and the skin regeneration process by histological analyses of the wounded skin biopsies; 5. To evaluate the in situ expression of angiogenic and growth factors, metalloproteinases, cytokines, chemokines in the wounded skin biopsies; 6. To analyze the differential protein expression of treated or non-treated wounds by proteomic analyses. Subproject 22: Use of mesenchymal stem cells genetically modified with human recombinant coagulation factor IX in Hemophilic B mice Coordinator: Dimas Tadeu Covas 1. Isolation, expansion and characterization of murine hepatic mesenchymal stem cells (mmsc); 2. Culture, expansion and characterization of human, hepatic mesenchymal cell line termed LX2; 3. Gene modification of murine CTM (mmsc/fix) and human MSC (hlx-2/fix) with human recombinant coagulation factor IX using retrovirus system; 4. Cell cloning of both recombinant cell lines to obtain a population with high level of recombinant factor IX; 5. Characterization of both recombinant cell lines mmsc/fix and hlx-2/fix about the expression level of rfix by RT-PCR, as well as, the protein level presents at the supernatant of cell cultures by ELISA; 6. Evaluation of biological activity of recombinant FIX present at the supernatant of these cell lines by APTT assay; 33

34 7. Infusion of recombinant cells lines mmsc/fix and hlx-2/fix in NOD/SCID mice and Hemophilic B mice; 8. Evaluation of engraftment level in the liver different times after the infusion of each recombinant cell line; 9. Evaluation of FIX kinetics in the plasma mice different times after infusion of each recombinant cell line; 10. Evaluation of the period of maintenance of FIX protein expression. Subproject 23: Bioprocess development for mesenchymal stem cell expansion on microcarriers Coordinator: Dimas Tadeu Covas 1. Isolation, culture and expansion of bone marrow and umbilical cord mesenchymal stem cells in static culture as control procedure; 2. MSC culture in spinner bioreactor on microcarriers 3. Comparison MSC cultured on microcarriers in spinner bioreactor and static culture using five parameters: a) morphology; b) immunophenotypic profile; c) cytogenetic profile; d) differentiation potential on adipocyte, osteocyte and chrondocyte and e) analysis of gene expression; 4. Comparison the MSC culture performance in spinner bioreactor and static culture related to: a) Population Doubling; b) Doubling time; c) cellular viability and d) metabolic activity (glucose and glutamine consumption, lactate and ammonium production among others); Subproject 24: Functional Proteomic: analyses of the nucleofosmine in the gliomagenesis Coordinator: Lewis Joel Greene 1. To extend the obtaining of protein maps by bidimensional eletrophoresis of the new patient samples. 34

35 2. Protein identification by mass spectrometry by MALDI-TOF-TOF 3. Introduction of the shotgun peptide sequencing proteomic strategy to obtain additional the data from those of the bidimensional gels. 4. Proliferation, migration and apoptosis assays under EGF stimulation in T98G and U87MG cell lines. 5. To obtain the bidimensional maps of protein extracts of T98G and U87MG cell lines under EGF stimulation. 6. Identification of the differentially expressed proteins between the EGF stimulated and non-stimulated cells. 7. Silencing or modulation of the nucleofosmine gene expression by interference RNA (irna) in T98G and U87MG cells line with or without EGF stimulation and functional studies (proliferation, migration and apoptosis assays) 8. To obtain of the bidimensional maps T98G and U87MG cell lines proteic extracts under EGF stimulation and silenced to NPM. 9. Study of the differentially expressed proteins (item 5 ) in EGF stimulated cells and silenced to NPM. Subproject 25: Cytogenomic tools applied to the investigation of chromossomic instability in chronic lymphocytic leukemia (CLL) Coordinator: Roberto Passetto Falcão 1. Obtain metaphasic cells to use with the SKY technique 2. Hybridization of the metaphasic cells carry out to spectral analysis (Applied Spectral Imaging) 3. Extraction of blood DNA from patients and healthy donors and the evaluation of the change in the copy numbers of the genetic region by CGH array method using the Agilent platform. 35

36 4. Identification and characterization of the breaking points related to the presence of the chromossomic alterations; detection and identification of the probably enrolled genes. 5. Identification of the submicroscopics deletions in patients with normal kariotype. Subprojects 26: Center of pre-clinical studies and animal stem cell bank Coordinator: Maria Angélica Miglino 1. Identify, isolate and test culture protocols for olfactory neural epithelial cells 2. Develop methods for cellular differentiation and establishment of the ideal markers for olfactory neural epithelial cells; 3. Isolation and characterization of mesenchymal stem cells of the amniotic fluid and, yolk sac of canine fetuses to be applied in the treatment of genetic "defects", in intrauterine environment. 4. Standardization of a protocol for genotyping using amniotic fluid and the yolk sac of embryos/fetuses of dogs to detect the presence of genetic alterations in prenatal period 5. Study the potential for differentiation in vitro of mesenchymal stem cells from amniotic fluid and yolk sac of canine fetus, as well as, its potential for the intrauterine injection and, study of biodistribution and differentiation into multiple cell types 6. Application of the immature dental pulp cells in animal models of patients suffering from total limbic deficiency and, demonstrating resistance to the conventional surgical procedure D reconstruction of critical defects in tibia of sheep and, characterization of cell differentiation by immunohistochemistry. 8. Quantify osteoblasts and osteocytes in the selected 3-D complex after application in vivo, using histological morphometry. 36

37 9. Establishment of primary culture of canine and feline osteosarcoma; 10. Characterization of tumor cells culture and analysis of proliferative potential and cells markers; 11. Osteosarcoma in vitro regenerative therapy; 12. Establishment of human extravillus trophoblast cell; 13. Produce virus recombinants of retrovirus vectors derived from the Moloney Murine Leukemia Virus (MoMLV) carrying the genes reporters LacZ and egfp. 14. Transduce stem cells acquired from placenta using retrovirus vector carrying the genes reporters LacZ and egfp to analyze the expression of the genes in vitro. 15. To evaluate the tracing of the transducing cells in mdx and nude mice, the potentiality of differentiation and marking in the different tissues and, its pathogenicity; 16. Trace the implanted stem cells in mice after euthanasia and histopathologic analysis using the product of genes LacZ and egfp. 17. Analyze the potentiality of immune modulated response against the human extravillous trophoblast cells. 18. In collaboration to Prof. Dr. Dimas T. Covas (Dra. Kamilla Swiech) group, knowledge of specific techniques for culture and expansion using microcarriers will be acquired; 19. In collaboration to the researcher Aparecida Maria Fontes (member of Prof. Dr. Dimas Tadeu Covas group) side population cells from of canine s yolk sac and liver will be established to test its the vasculogenesis potentiality. Specifically to characterize the phenotype profile and genes expression using specific markers. The responsibilities of the pre-clinical group will be to establish the cerebral ischemic model and to establish in vitro the molecular interactions of the relations with endothelium mesenchymal. 37

38 20. In collaboration with the researcher Dra.Lygia da Veiga Pereira, the group is going to test chronic spinal lesions (canine model) the application of the ips (induced pluripotent stem cells) cells; 21. In collaboration with Dr. Lindolfo da Silva Meirelles, Dra. Irina Kerkis will perform the injection of pericytes and mice Rosa26 mesenchymal stem cell into blastocyst and, murines to evaluate the capacity of interaction in different types of cells in embryos tissue. 22. In collaboration with Dra. Adriana Santos Moreno (member of Prof. Dimas T. Covas group), we intend to inject GFP-neural cells from embryonic cells or pluripotent stem cells into canine model with chronic medullar disease, and/or in murine models with induced lesion, to verify the adhesion of these cells in the injured tissue and, the development of the necessary synaptic connections to the re-establishment of the functions; 23. In collaboration with the PhD student Carla Kaneto (member of Prof. Dr. Wilson Araujo s group), we intend to establish the mechanisms involved in the imperfect osteogenesis that occurs during the differentiation process of the osteoblasts. The results obtained from animal testing would identify the genes involved in this process and, could be applied in human therapy. Subproject 27: To make Neotropical primates (new world) and Chlorocebus aetiops (old world) to stem cell research as animal model for cellular therapies Coordinator: Klena Sarges Marruaz da Silva 1. Indentify good large animal models to stem cell research. 2. To establish clinical and pre-clinical model protocols of Neotropical primates and Chlorocebus aethiops to test therapies using stem cells. 38

39 Subproject 28: Treatment of Type 1 diabetes (DM-1) with mesenchymal stem cell (MSCs) Coordinator: Júlio César Voltarelli 1. To evaluate the safety (toxic effects, infectious and neoplasic complication, mortality) of the intravenous infusion of the allogeneic MCS from relateddonor in recent-onset MD-1 patients; 2. To evaluate the therapeutic effect of MSC on the pancreatic autoimmune injury of the MD-1 patients (determination of the levels of glycemia fasting, C-peptide and glycosylated hemoglobin before and after MSC infusions; exogenous insulin needs before and after MSC infusions); 3. To evaluate the immune response (naive, memory and regulatory T cell subsets, serum cytokine quantification, T cell repertoire, frequency of antipancreatic β cells autoreactive T cells) in the patients in many periods after MSC infusion; 4. To evaluate the immunological mechanisms (immunomodulation of the pathogenic responses by the MSC; role of T regulatory cells) involved in the therapeutic response of NOD mice (genetically determined diabetes model) and C57BL/6 mice treated with streptozotocin (chemically-induced diabetes model) to human MSC infusion; 5. To evaluate the regenerative mechanisms (role in the pancreatic tissue regeneration; human MCS fusion with murine pancreatic β cells) involved in the therapeutic response of NOD mice (genetically determined model) and C57BL/6 mice treated with streptozotocin (chemically-induced diabetes model) to human MSC infusion. 39

40 Subproject 29: Treatment of multiple sclerosis with hematopoietic stem cells: evaluation of clinical response and immune mechanisms of action Coordinator: Júlio César Voltarelli 1. To evaluate the safety (toxic effects, infectious complications, mortality) of the HDI/AHSCT for treatment of relapsing-remitting MS patients; 2. To determine if the HDI/AHSCT could stop the disease progression in relapsing-remitting multiple sclerosis patients, refractory to the immunomodulatory action of Interferon; 3. To evaluate the efficacy of the HDI/AHSCT therapy in comparison to the conventional treatment (Interferon, Copaxone or Mitoxantrone) for treatment of relapsing-remitting MS patients; 4. To analyze the large scale gene expression by cdna microarrays, of peripheral blood CD4+ and CD8+ T cells from relapsing-remitting MS patients, isolated at pre- and post-ahsct; 5. To validate the gene expression results obtained by cdna microarrays by real time RT-PCR; 6. To study relevant genes/proteins, whose expression were found altered in the gene and proteomic expression studies, by functional studies using RNA interference. Subproject 30: Mesenchymal stem cell (CTM) for treatment and prevention of graft versus host disease (GVHD) in transplanted patients with hematopoietic stem cells Coordinator: Dimas Tadeu Covas 1- Isolation and MSC expansion in large scale using culture standard medium 15% FBS; 2- Comparison of the MSC culture and expansion with human serum or platelets lysate versus FBS; 40

41 3- Establish criteria for selection of donor MSC suitable for transplantation; 4- To determine the frequency of MSC using clonogenic assay to quantify the number of colony forming units (CFU-F); 5- Characterization of immunophenotypic and cytogenetic profile, differentiation capacity and immunomodulator potential; 6- Infusion of two MSC doses (1-2 x 10 6 CTM/Kg) in patients prone to develop GVHD; 7- Infusion of 1 to 6 MSC doses (1-2 x 10 6 CTM/Kg) in acute GVHD patients non responsive to steroids treatment; 8- Evaluate the early and late side effects of the MSCs infusion; 9- Analyze the engraftment time of the bone marrow; 10- To evaluate the incidence of the acute and chronic GVHD in patients which received prophylactic MSCs 11- To evaluate the relapse of the hematologic disease after the transplantation 12- To analyze the immunologic recovery and leukocyte activation by flow cytometry to T, B, NK lymphocytes and memory cells 13- Serum immunoglobulins quantification; 14- Evaluation of the acute and chronic GVHD development; 15- T cell and mesenchymal stem cell chimerism evaluation. 41

42 Lines of research and subprojects Studies with Pluripotential stem cells Pluripotential stem cells are capable of originating cells and tissues from the three embryonic cell layers: ectoderm, mesoderm and endoderm. Because of this characteristic, these cells are the most attractive from the therapeutic point of view, once they would work in a wide range of diseases. Cells having this potential can be obtained from the inner mass of blastocyst, fetal gonadal fold from 6 to 9 weeks old and from teratocarcinomas in adults. Recently, pluripotential stem cells have been obtained from mature fibroblasts by the insertion of genes considered to be inducers of pluripotentiality (Oct4, c-myc, Klf4 e Sox2), what gave birth to the so called induced pluripotent stem cells (IPS). In the present proposal of INCTC one of the focuses of study will be the pluripotential stem cells derived from the inner cell mass of humans and animals and the IPS. For that, we are placing together four groups of Brazilian researchers who have previous proven experience in this area. The first group is headed by Dra. Lygia da Veiga Pereira, professor of Instituto de Biociências da Universidade de São Paulo that has experience in cultivating embryonic stem cells and other pluripotential stem cells, as the ones derived from dental pulp (Sukoyan, Kerkis et al., 2002; Kerkis, Kerkis et al., 2006). The second group is led by Stevens K. Rehen da Universidade Federal do Rio de Janeiro who has great experience in the cultivation and neural differentiation of ESC (Mcconnell, Kaushal et al., 2004; Kingsbury, Friedman et al., 2005; Rehen, Yung et al., 2005; Rehen, Kingsbury et al., 2006). To this initial nucleus, there is the incorporation of the group from Faculdade de Zootecnia de Pirassununga da Universidade de São Paulo, which has great experience in the derivation of embryonic stem cells, in embryology, and in manipulation techniques of oocytes, including the transfer of somatic nucleus and the manipulation of mitochondrial content of the zygote (Meirelles e Smith, 1998; Meirelles, Bordignon et al., 2001; Meirelles, Caetano et al., 2004; Ripamonte, Merighe et al., 2005; Ferreira, Meirelles et al., 2007; Miglino, Pereira et al., 2007; Biase, Fonseca Merighe et al., 2008; Paneto, Ferraz et al., 2008). So far, in addition to the experience of the groups cited above, there is also the experience of the group from Ribeirão Preto regarding cultivation, characterization and expansion of stem cells under conditions of good manufacturing practices (GMP) e in intrexpansion of stem cells in cellular lineages (Covas, Siufi et al., 2003; Silva, Covas et al., 2003; Panepucci, Siufi et al., 2004; 42

43 Covas, Piccinato et al., 2005; Pereira, Faca et al., 2005; Carrara, Orellana et al., 2007; Picanco, Heinz et al., 2007; Covas, Panepucci et al., 2008; Picanco-Castro, Fontes et al., 2008; Picanco-Castro, Russo-Carbolante et al., 2008). This group of laboratories and researchers have complementary experiences and competences, what will allow the consolidation of a strong national group dedicated to the study of embryonic and induced stem cells (IPS) and, certainly, will place Brazil among the countries in the world that are in the vanguard of this field. After what has been discussed, the group coordinated by Dra. Lygia da Veiga Pereira of USP (USP-SP) through subproject 1, which is entitled Evaluation of the epigenetic stability on embryonic stem cell lines established under different conditions, will have as general goal, to evaluate the epigenetic state of new lines of human ESC, to establish conditions and characterize their plasticity in vitro and in vivo. In addition, the trophoblast cells removed from the blastocyst during the isolation will be used to study the beginning of the inactivation process of X chromosome. For that matter, the following technical approaches will be used: Micromanipulation of human blastocysts for isolation of ICM and trophoblasts; cultivation of ICM; immunofluorescence and FACS studies of pluripotent cell markers (OCT4, NANOG, SSEA-1,2,3,4); differentiation embryo bodies and characterization by immunofluorescence; formation of teratomas in rats SCID; analysis of the activity state of chromosome x in CTEh 46,XX pre and post differentiation; FISH for detection of RNA of XIST; ChIP for modified histons; allelespecific expression of X-linked genes; global methylation analysis of hesc by methylation microscope. Pre-clinical studies and the perspective of cell therapy in human require quantities of cultivated stem cells in vitro. Therefore, defining good conditions for cultivation, both in a small scale or large scale, is essential to the feasibility of this and other procedures to be developed. Few advances have been reported relative to the propagation of embryonic stem cells in large scale (Thomson, 2007). The optimization of cultivation conditions is usually performed in static dishes, that is, in low scale. However, such procedure needs serial passages, which makes difficult the adequate control of cellular physiology, driving the cells to frequently suffer; and also 43

44 manipulator/researcher interference which increases the risks of contamination and makes expansion a hard process. For those reasons, it is essential to develop new methods of expansion for human embryonic stem cells that will allow an adequate control of environmental conditions, in a way that large scale cultivation preserves the phenotypic characteristics, keeping the human embryonic cells as pluripotent and homogeneous population. Cultivation in agitated systems seems to be in particular most promising once they provide a homogeneous environment, in which conditions such as ph, oxygen content and temperature can be constantly monitored and controlled, allowing better control of the cellular physiology. Furthermore, an agitated system eliminates the heterogeneity of the dish environment, a static surface of cultivation. Due to its well known dependence in cell-to-cell adhesion and formation of aggregates for proliferation, there are two alternatives for the cultivation of embryonic stem cells in agitated systems: the cultivation in suspension as aggregates and embryonic bodies, or the cultivation when adhering to microcarriers. Microcarriers are porous particles, usually spherical, showing high surface area and low density, which keeps itself in suspension under soft agitating conditions, acting like a cell adhesion support. Analysis of viability of human embryonic stem cells cultivated in agitated system, comparing different matrices for microcarriers and the influence of different substrates in large scale expansion, becomes extremely interesting. To this end, the group coordinated by Dr. Stevens Kastrup Rehen at Universidade Federal do Rio de Janeiro proposes subproject 2, entitled Scale-up of human embryonic stem cell production in bioreactors for application in Regenerative Medicine, which has the large-scale production of human embryonic stem cells for use in preclinical and clinical trials as its general objective. To achieve this goal, human embryonic stem cells obtained as a gift from the Scripps Research Institute USA, and from Harvard University (Cowan et al. 2004; Klimanskaya et al., 2004) will be initially cultured over a monolayer of mouse embryonic fibroblasts (MEFs; feeder layer), and submitted to different expansion methods that will be compared to each other. Stirred culture systems will be used to expand these stem cells as embryoid bodies in suspension as well as in the presence of commercially available microcarriers (Cytodex 1, Cytodex 3, and Cytopore GE Healthcare). Conical flasks (erlenmeyers) and spinner flasks (Bellco) will be used in stirred culture assays. For assays using automated stirred 44

45 bioreactors, a Bioflo 110 bioreactor system (New Brunswick Scientific) will be used, and its native vessel, which has a standard geometry (0.4 to 1 L working volume), will be compared with a vessel especially designed for stem cell culture (DASGIP, 0.1 L working volume). Operational variances of the process, e.g., stirring, aeration rate and operation mode (batch, feed-batch and perfusion) will be evaluated. Cell growth kinetics will be evaluated and molecular and immunocytochemical assays will be performed with the purpose of evaluating and quantifying the cells pluripotency and degree of differentiation. The total cell concentration of the cultures will be determined by counting cell nuclei in a Neubauer chamber under the microscopy (crystal violet method). Glucose and lactate concentrations in the supernatant will be determined using an YSI model 2700 automatic analyzer. Chromosomal stability and other pluripotency parameters of ESCs cultured in bioreactors will be compared with those of ESCs cultured under standard conditions (MEFs; feeder layers) using newly developed protocols and methodologies for the analysis of morphology and karyotyping (Martin, Muotri et al., 2005). Chromosomes stained with 4,6-diamino-2-phenylindole (DAPI) will be counted according to the methodology developed by the team (Rehen, Mcconnell et al., 2001), in a minimum of 80 chromosomal spreads. Molecular karyotyping and fluorescent in situ hybridization (FISH) probes will be used if required (Rehen, Yung et al., 2005). Alterations in proliferation, differentiation and survival of ESCs maintained in culture dishes or bioreactors will be examined by immunohistochemical assays for the bromodeoxyuridine (BrdU), phospho H3 (mitosis), Tuj-1, caspase-3, Ki67 and DNA fragmentation, as previously described (Rehen, Varella et al., 1996; Rehen, Neves et al., 1999; Rehen, Mcconnel et al., 2001; Rehen, Kingsbury et al., 2002, Kingsbury, Rehen et al., 2003). Despite the establishment, propagation and characterization of the cell lines are a fundamental step in the eventual establishment of treatment protocols, to the safety use of hesc it s necessary that efficient and practical approaches be developed to promote the desired differentiation. The success in this task could avoid the appearance of unwanted cells or tissues, with uncontrolled growth or other unexpected behave. Thus, the study of different processes of differentiation is crucial for the possible implementation of ESC in clinical practice. Different adult tissues have the capacity for renewal and repair, such as blood, epidermis and liver tissue. The ability to repair or to renew some adult 45

46 tissues is related to the presence of adult stem cells that have a limited potential for differentiation. Among these cells, the hematopoietic stem cells (HSC) are highlighted because of the volume of theoretical and applied research in medicine. The HSC are used for over 30 years as a form of hematopoietic reconstitution treatment through the bone marrow (BM) transplantation. Similarly, newborns blood that remains in the umbilical cord (UCB) and placenta after birth can be used as a source of CTH. The success of the transplant depends, among other factors, on the existence and compatibility between donor and recipient, since incompatibility in this case may result in the disease graft versus host disease (GVHD). However, the available cell number in the cord blood represents a limit that results in further delay in restoring the hematopoietic system, increasing the infection risk in the patient. Front to these restrictions, the production of HSC from ESC represents an attractive alternative, which would eliminate this kind of restrictions. However, HSC derived from ESC have differences concerning to the engraftment potential (Shojaei e Menendez, 2008), which reflect the deficiencies in homing, repopulation in long periods, and also in the differentiation potential, specially the lymphocytic line (Martin, Woll et al., 2008). The comparison between the gene expression profile of HSC derived from the BM or UCB have been successively done by our group to characterize the molecular differences related to the biological differences among these cells (Panepucci, Calado et al., 2007). Similarly, the comparison among the adults HSC and ESC expression profile could help in understanding the molecular base related to these existing differences, and in the differentiation process optimization (Martin e Kaufman, 2005) The group coordinated by Dr. Dimas Tadeu Covas of USP in Ribeirão Preto (USP) through subproject 3 entitled Molecular basis of hematopoietic differentiation from embryonic stem cells, will have as general goal, the study of transcriptional profile of the ESC and of the HSC derived from those, comparing them to the profile previously obtained from adult HSC. In addition to that, the differentiation of the HSC (derived from ESC or adults) in T lymphocyte will be also evaluated. This way, molecular differences identified can indicate which processes of signaling are altered and their linkage to the deficiencies presented by HSC derived from ESC. With this objective, commercial lines of ESC (or derived in other projects) will be differentiated in HSC, using co-culture system with OP9 stromal cells. The HSC will then be selected by immunomagnetic techniques using anti- 46

47 CD34 antibodies. Populations of HSC CD34+ of UCB will be obtained by the same way. These cells (ESC and HSC) will have the transcriptional profile acquired by using microarrays of oligonucleotides covering the whole human genome. The same RNA of these cells will be used to obtain of the profile of the expression of micrornas. Additionally, HSC derived from ESC or UCB will be submitted to a differentiation process in T lymphocyte, through the co-culture with OP9-DL1 cells. These cells, the agonist ligand of the NOTCH (Delta-like 1) pathway induced to differentiation Co-cultured cells will also be evaluated in terms of mrna e microrna using microarrays. Bioinformatics methods and specific databases will be used to integrate the information obtained so that it will identify potential regulatory mechanisms involved in existing functional differences. Specific antibodies will be used to evaluate the differentiation processes through flow cytometry. Real Time PCR will be used afterwards to validate these differences found in the mrna as well as in microrna. The future of the cell therapy with stem cells will depend on the diffusion and expansion of the area as a whole. This expansion depends on the establishment of new centers of research, experienced on propagating the knowledge and the techniques used in the study of the stem cells, forming specialized personal in this area. With this in mind, the center intends to support the consolidation of the group headquartered in the Instituto Nacional de Primatas Evandro Chagas in Belém (Pará), as an operating group in the research with stem cells. The Seção of Meio Ambiente of the Instituto Evandro Chagas makes use of a bank of cultivated primary cells from the biopsy of humans and of other mammals, belonging to Brazilian Amazon region. The bank is composed of cultures of some tissue and organs such as: skin, kidney, epiphyses, brain, cornea, spleen and parotid gland. The bank contains human cells of prepuce, cornea, sclera, quotes to umbilical and placenta, beyond gotten stem cells from deciduous tooth (Miura et al., 2003). This previous experience in the cell culture of distinct animal tissues, successfully allows the training and implantation of the techniques for ESC culture. In such a way, the Laboratory of Cell Culture of the Seção de Meio Ambiente/IEC/SVS/MS of the Instituto Evandro Chagas kept for Maria de Fátima Lima de Assis, through subproject 4, entitled Culture of animals and humans adult and embryonic stem cells for use in cell therapies, it will have as general objective to establish lineage of ESCs of animals and to characterize them. 47

48 Induced Pluripotent Stem Cell When eventually mastered, the processes involved in the obtaining, cultivating and differencing ESC into cells of clinical interest, another practical limitation should be taken into account. In spite of the fact that HSC Banks may eventually be created allowing the establishment of large collections, the ESC would have their use restricted to hystocompatible receptor. One of the alternatives to the creation of an ESC bank involves the reprogramming of the nucleus of somatic cells in the case of a patient with no donor, by the cytoplasm of an oocyte to obtain autologous hesc. This approach is also known as therapeutic cloning, since it does not want to generate a cloned individual, but only ESC for therapeutic use. The reprogramming of the somatic nucleus induced by oocyte cytoplasm resulted from the presence of different molecules, including transcription factors and other proteins. Recently, several groups reported the induction of pluripotency in human primary fibroblasts through their transduction with viral vectors expressing the OCT4, C-MYC, KLF4 and SOX2 genes. The so called induced pluripotential stem cells (ips) have the characteristic morphology of ESCs. Express pluripotential cell markers and are capable of differentiation in vitro and in tissues derived from the three embryonic layers. This way, the generation of ips from somatic cell induction by specific factors could represent an alternative to obtain of hystocompatible stem cells. Preliminary results of Dr. Dimas Tadeu Covas group from Hemocentro de Ribeirão Preto (USP) show the efficiency in the lentiviral production, in the stem cell transduction (mesenchymal and endothelial progenitor) and also in the endothelial progenitor expression profile with Nanog. Thus, this group, with the subproject 5, named Gene Modification of stem cell, has as general objective to genetically modify mesenchymal stem cell and endothelial progenitor cells with lentiviral vectors carrying the stemness genes, in order to transform multipotent cells into pluripotent ones with greater in vitro expansion capacity. The project has the following approaches: a) To transduce MSC with 1054-CIGWS lentiviral vector carrying one of the transcription factors (Nanog, Oct3/4, Sox 2, β-catenina, Kfl4, c-myc, Esrrb, Tcl 1and Tbx 3) together with the GFP gene sort out the positive cells by flow cytometry; b) transductions will be made one at a time and 48

49 afterwards four vectors at the same time will be used to induce de ips; c) Evaluation of the changes in the gene expression profile of the transduced cells; d) Evaluation of the morphological changes of the transformed cells; e) assess the specific embryonic stem cells markers (alkaline phosphatase and/or SSEA-1 antigens) e) evaluate if the modified cells have acquired pluripotenciality. A great limitation of pre-clinical studies with ESCs is the lack of large animal models on which these test can be performed since a series of technical/ biological difficulties prevent the establishment of ESCs from embryos of those models. Keeping this in mind, the group coordinated by Dr. Lygia da Veiga Pereira of USP (USP-SP) through subproject 6, entitled Establishment of induced pluripotent stem cell lines (ips) in large animal models, intends to develop a methodology to establish lines of ips from large animal models specifically from non-human primates and canine. The cells generated will be used for pre-clinical studies of lesion of spinal cord in the respective animal models. For that matter, lentiviral vectors of AddGene enterprise (EUA) will be transferred to 293 cells by cotransfection with packing vectors. Cultures of dogs and monkeys will be transduced with lentivirus expressing the reporter gene GFP for evaluation of transduction efficiency with different packing systems; fibroblast transduction animals with induction vectors according to conditions established above, and isolation of ips in culture medium specific for ESC (in case we are not able to establish the ips by using vectors with human genes of induction, the homologous of each species will be isolated by RT-PCR from embryos, pre-implantation and new species-specific vectors will be constructed and used for induction); characterization of pluripotentiality of ips by immunofluorescence, in vitro differentiation in embryonic bodies and in vivo by the measurement of the formation of teratomas; neural differentiation of animal ips by culture in media with retinoic acid; transplant of differentiated cells in spine cord lesion models. Despite the fact that is inadequate for clinical use, the ips are an important tool of basic research, mainly for cells obtained from individuals with genetic diseases Thus, the group headed by Dr. Lygia da Veiga Pereira, through subproject 7, entitled Establishment of induced pluripotent stem cell lines (ips) from fibroblasts of patients with Mendelian and multifactorial genetic diseases (with genetic component), intends to implement the methodology of generation of human ips so that it is possible to establish ips from different tissues of patients 49

50 with genetic diseases of interest to the group, particularly patients who have osseous dysplasia, type 1 diabetes, multiple sclerosis and acute promyielocytic leukemia. The ips established will be used as experimental model for the study of basic mechanisms behind the respective diseases. Furthermore, new vectors based on adenovirus will be constructed, which will not integrate to the genome, so nonmodified ips will be generated, which is better suited for clinical use. For that purpose, the following experimental approaches will be used: lentiviral vectors of AddGene enterprise (EUA) will be inserted into 293 cells by transient cotransfection with vectors; frozen mesenchymal cells of the bone marrow of patients who have type 1 diabetes and leukemia PMA cells will be expanded in culture for viral transduction; standardization of line establishment of adequate cells for induction from human peripheral blood; transduction of human cells with lentiviral vectors and selection of ips cells in culture medium for ESCs; postimmunofluorescence characterization of ips and FACS using pluripotent cell markers (OCT4, NANOG, SSEA-1,2,3,4); differentiation into embryoid bodies and characterization by immunofluorescence; teratoma formation in SCID mice; characterization of pluripotency of ips through immunofluorescence, perform differentiation into embryoid bodies in vitro, and teratoma formation assays in vivo. Although techniques for the induction of pluripotency involving the incorporation of transcription factors in the genome of somatic cells (Takahashi et al., 2006) constitute a possible approach to autologous cell therapy, it is important to stress that the practical application of cells induced to differentiation by genetic modification still has to be studied further and evaluated (Liu, 2008). On the other hand, the technique of nuclear transfer (NT) is well-established and has been capable of producing healthy animals, confirming the efficient capacity of reprogramming of a differentiated cell (Wilmut et al., 1997; Keefer et al., 2000). Additionally, when used as a receptor, the cytoplastic bovine cell has been capable of reprogramming differentiated cells from various species (Chen et al., 2002, Ilmensee et al., 2006), supporting the initial view that embryonary development is necessary for the isolation of embryonary stem cells. Together with the efficient reprogramming of the cytoplasm, the abundance and the ease in obtaining biological material makes the bovine the perfect model for the establishment of efficient methods for obtaining pluripotent cells of embryonic origin through the 50

51 reprogramming of differentiated somatic cells of various species (Cibelli et al, 1998). By keeping that in sight, the group coordinated by Dr. Flavio Vieira Meirelles (from now on called group) of USP in Pirassununga (USP) through subproject 8, entitled Autologous pluripotent cells generated from differentiated somatic cells, will have as a general goal to characterize methodologies to obtain pluri/totipotent cells from somatic cells, in term of morphology, genetic expression and epigenetic studies. For that matter, a bovine somatic cell line derived from the in vitro cultivation of adult fibroblasts will be initially established. Part of these cells will be used in the lentiviral transduction of transcription factors responsible for cellular dedifferentiation (Takahashi et al., 2006). Such cells will be characterized in the bovine model and, along with the non-modified cells; they will be used as nuclei donors in the process of nuclear transfer. Embryonary cells derived from those processes will be compared among themselves and with those somatic undifferentiated cells in terms of morphology as well as genetic expression and epigenetic expression. They will also be compared to the embryonary cells obtained by the natural fertilization processes. The one methodology that provides characteristics closes to the ones of the control group will be considered the most appropriate for the production of stem cells from differentiated somatic cells and, so, it will be utilized in the interspecific model where pluripotency cells of primates will be produced. The reprogramming of the nucleus of somatic cells by the cytoplasm of an oocyte implies in the combination between the nuclear genome of an individual and the mitochondrial of another. Considering the great potential of this kind of approach in the development of the future of cell therapy, the control of the inherited mitochondrial is very important to make possible the following steps: i) the production of human stem cells by the reprogramming of the nucleus donor cell in non-human cytoplasm and, ii) the production of autologous human stem cells (Illmensse et al., 2006). For that matter, the bovine model is interesting because of the abundance of material available and for the great quantity of available knowledge on the mechanisms that regulate the mitochondrial DNA (mtdna) during embryogenesis. In bovine embryos produced by nucleus transfer of somatic cells, the percentage of mtdna of the nucleus donor cell rises between the third and fourth cellular cycle in relation to the mtdna that comes from the oocyte (Ferreira 51

52 et al., 2007). On the other hand, the centrifugation of bovine zygotes makes the mechanical depletion of part of the mitochondria possible without compromising the embryonic development because the embryo is capable of replacing the same content of mtdna observed in undepleted blastocysts (Chiaratti et al., 2008). Furthermore, through mitochondrial depletion it is possible to introduce a greater quantity of exogenous mitochondrial zygotes (Ferreira et al., submitted). After what has been exposed, the group coordinated by Dr. Flavio Vieira Meirelles (from now on called group) of USP in Pirassununga (USP) through subproject 9, entitled Animal models to study mitochondrial inheritance intraand interspecies, will have as a general goal, to evaluate the viability of production of embryos having mtdna of inner and interspecific somatic origin and to increase the percentage of such mtdna inherited by the blastocysts. More specifically, they intent to produce bovine blastocysts (Bos taurus) that present mtdna from inner or interspecific somatic origin (B. indicus or Homo sapiens, respectively), in heteroplasmia with mtdna from embryonic origin (inherited from the oocyte) and; additionally develop a method to raise the inherited percentage in blastocysts of somatic mtdna (B. indicus or H. sapiens). For that, the following technological approaches will be used the establishment of mesenchymal cell lines originated from the B. indicus and H. sapiens; enucleation of mesenchymal cells by centrifugation and cytoplasm fusion for utilization as a cytoplasm donor (Marchington et al., 1999; Shay et al., 1975); in vitro production of bovine zygotes (B. taurus) parthenogenetic oocytes aspirated from ovaries collected on the slaughterhouse and matured in vitro (Méo et al., 2007); centrifugation of zygote to concentrate the mitochondria in one of the poles of the embryo and the removal of part of the mitochondria through micromanipulation (Ferreira et al., submitted); cytoplasts fusion (B. indicus or H. sapiens) to deplete zygote (B. taurus) and in vitro cultivation (Inoue et al., 2000); determination of the percentage of mtdna (somatic mtdna in relation to the total quantity of mtdna) by PCR in real time immediately after fusion, at 72 hours (embryos with 5 or more cells) and at 168 hours (blastocysts) after parthenogenic activation (Ferreira et al. submitted; Ferreira et al., 2007). The percentage of mtdna will be analyzed considering the following as effect - the cytoplasm used (B. indicus and H. sapiens), the moment of the analysis (0, 72 and 168 hours) and the interaction both factors. 52

53 Somatic stem cells In the adult individual, different tissues have the capacity of renewal or, at least of partial or total repair, the blood and the epidermis for instance, are constantly renewing themselves, while other tissues, such as the liver, can be repaired totally. Tissues, like muscles or neural present a reduced potential of repair. Such potential is related to the existence of stem cells that are able of proliferating and differentiating into different cellular types, the so called adult stem cells. Among those, we can highlight the hematopoietic stem cells (HSCs) and the mesenchymal stem cells (MSCs). Both can be found in the bone marrow (BM). The MSC differentiate in vitro and in vivo into the four main cellular types that form the micro-architecture of the bone marrow: adipocyte, chondrocyte, osteocyto and stromal cells. The interaction of the HSCs with this microenvironment allows the constant renewal of blood tissue. Opposed to the ESC, therapies based on HSC have been used for over 30 years, in the reconstruction of the hematopoietic system through bone marrow transplantation. In the transplant, the incompatibility between donor and receptor can result in graft-versus-host disease, or GVHD (graft versus host disease), when the transplanted cells recognize the receptor as nonself. The mesenchymal stem cells can be used in the reduction of GVHD in transplants, once there is strong evidence that they reduce the immune response for their effect and T and dendritic cells. In addition to their use in reconstructing hematopoietic system, mesenchymal and hematopoietic stem cells have been used to repair or replace damage or sick tissues. These approaches are based on the observation that those cells, originated in the mesoderm, could differentiate themselves (or transdifferentiate) into cells of tissues originated from other embryonic layers, such as hepatocyte (endoderm) or neuron (ectoderm) a process called plasticity. In fact, these cells have been used in the attempt to repair myocardial tissue after infarct, injured nervous tissues, or even in a field called organ engineering, with the intention of generating tissues (and eventually organs) that can be implanted in humans, eliminating tissue rejection, once stem cells could be originated from the 53

54 patient himself. Within that framework, mesenchymal stem cells apparently have a wider potential of differentiation (and transdifferentiation) than the hematopoietics, for they may be isolated from several tissues, besides the bone marrow. Despite the plethora of paperwork in this area, the potential of application of those cells will only be reached after deeper knowledge of their functional and molecular biology is achieved. Basic Research: As previously mentioned, the MSC stand out for their great capacity of in vitro differentiation, being comparable to the ESCs (reviewed in da Silva Meirelles et al., 2008). Interestingly, it was demonstrated that one type of cell related to MSC, which is called multipotency progenitor adult cell (MPAC), is capable of contributing to several rodent tissue when injected in the blastocyst (Jiang et al., 2002). The great criticism to the MAPCs experiments is that these cells could be mere artifacts of the cellular cultivation process used for their isolation. Currently, it is known that cells having the characteristics of MSC are distributed all over the post-natal organism. This wide distribution was assigned due to their association with blood vessels in a model that proposes pericytes are stem cells present in vessels (da Silva Meirelles et al., 2006). Pericytes are cells which embrace endothelial cells in the blood vessels. Specialized forms occur in the liver and in glomeruli. Pericytes are defined based on their relation to endothelial cells, especially capillaries. There is evidence, however, indicating that they form a sub-endothelial net all over the vasculature, in big and small caliber vessels. Pericytes present a close relation with endothelial cells during the formation of blood vessels and play an important role in the maintenance of their structure. The pericytes markers were reviewed in a recent publication (da Silva Meirelles et al., 2008) Pericytes present overlapping features with MSCs such as their potential of differentiation in vitro and in vivo, and also regarding the expression of markers. Besides, there are studies that stress the performance of pericytes as MSCs cartilage, bone, periodontal ligament, endometrium and fat tissue. Data like that 54

55 led to conception of a model where pericytes are stem cells present all over the vasculature and act like MSCs in mesenchymal tissues (da Silva Meirelles et al., 2006). The performance of pericytes/mscs as active components in the tissue repair/ regeneration process, and also in the maintenance of immune selftolerance was also postulated (da Silva Meirelles et al., 2008). According to that, the tissue lesion would lead to the migration of pericytes from their niche, which would change to an activated state and would proliferate and through the production of extracellular matrix and molecules regulating trophic effects and immune response, thus leading to repair of the site of the lesion. One consequence of that is the pre-assumption that cultivated cells defined as MSCs correspond to activated pericytes and are not, thus, equivalent to MSCs under physiological conditions in vivo. The low homing efficiency of MSCs cultured for some passages as compared to MSCs in primary culture (Rombouts and Ploemacher, 2003), argues favorably to this idea. With that in sight, the group coordinated by Dr. Dimas Tadeu Covas through subproject 10, entitled Basic knowledge of MSC biology by means of comparison with pericytes, will have as primary goal, to generate basic knowledge about MSC biology through the comparative analysis between pericytes and MSC. The following experimental approaches will be used: Isolation and cultivation of human MSCs from fat tissue, umbilical cord and/or placenta, as well as from mice tissue. Using magnetic columns or cellular separation by fluorescence 3G5 antibody; adipogenic, chondrogenic and osteogenic differentiation in vitro; the in vivo differentiation of MSCs may be tested by introducing them in porous ceramic cubes, and subcutaneously implanting them into immunocompromised mice; the injection of freshly isolated pericytes, cultured pericytes and MSCs from Rosa26 mice into blastocysts will be performed in collaboration with Dr. Irina Kerkis at Instituto Butantan (São Paulo, SP); the contribution of the injected cells to the embryo will be assessed by incubation of cryosections with the substrate of the - galactosidase enzyme (X-gal), which will allow for the visualization of the injected cells in blue; comparison of the surface molecules expressed in pericytes, cultured pericytes and MSC (molecules typically associated or not with MSCs, such as CD14, CD31, CD44, CD45, CD90, CD29, CD105 and CD117, will be evaluated by flow cytometry); cytokine profile study with immunomodulatory potential (hepatocyte 55

56 growth factor, transforming growth factor-β, interleukins 6, 10, 11, 15 and 27 in fresh or cultured pericytes and MSC by flow cytometry, pericytes (fresh or cultures) and MSC application obtained from Rose26 mice in animal model of induced muscular lesion by cardiotoxin (the presence of muscle fibers containing the infused cells will be observed after incubation of transversal cryosections with the substrate for the X-gal enzyme). The greater understanding of similarities between mesenchymal stem cells and pericytes (Covas et al., 2008) and their close contact to endothelial cells open important avenues about the interactions among these cell types during blood vessel formation in response to angiogenic factor stimulation. Vasculogenesis is the de novo formation of vessels from progenitor endothelial cells (angioblasts), whereas angiogenesis refers to the sprounting of neovessels form a preexisting vasculature. Angiogenic sprouting in the adult was initially considered an exclusive feature of terminally differentiated endothelial cells, which have the capacity to form vascular tubes and recruit pericytes. Previous studies conducted by our group gave us the hypothesis that immature mesenchymal stem cells reside in several peripheral tissues where they participate in the angiogenic response to injury (Covas et al., 2008). Moreover, we conducted studies of generation mature endothelial cells from endothelial progenitor cells (EPC) derived from bone marrow (BM) and cord blood (CB) after selection of specific CD133 marker expression. These CD133 positive cells from BM and CB were induced to differentiate into endothelial cells using three different protocols. Altogether, these studies demonstrated that vasculogenesis can be reproduced ex-vivo by culturing BM and UCV progenitor endothelial cells with specific cytokines and in matrigel system. Also, the role of several transcriptional factors involved in this process could be characterized (Covas, personal communication). In order to continue these studies we propose (subproject 11) to investigate the molecular interactions between bone marrow derived-mesenchymal stem cells (BM-MSC), umbilical cord vein derived-msc (UCV-MSC) and central nervous system microvascular pericytes (CNS-P) after co-culture with human umbilical vein endothelial cell (HUVEC) and ex-vivo derived endothelial cells from BM and CB. The study of these interactions will provide relevant information concerning physiological or pathological angiogenic and vasculogenic responses as well as 56

57 provide important information about vascularization process which remains one of the major challenges in cell therapy protocols. With this in mind, the group coordinated by Dr Dimas Tadeu Covas will carry out the subproject 11, entitled Investigation of angiogenic process and molecular interactions between mesenchymal stem cells and pericytes with endothelial cells using developed in vitro and in vivo systems in order determine the molecular interactions of cross-talk between FACS-sorted BM-MSC, UCV-MSC and CNS-P with CD31 positive endothelial cells. To stimulate angiogenesis in vitro and in vivo we will develop three models using BM-MSC, UCV-MSC or CNS-P with endothelial cells: 1) in vitro three dimensional co-culture model of BM-MSC, UCV-MSC and CNS-P with endothelial cells (HUVEC and BM and UCV-derived endothelial cells); 2) the matrigel plug assay co-infused with MSC or pericytes plus endothelial cells followed by subcutaneous injection in NODSCID mice and 3) the treatment of acute ischemic injury in a mouse hind limb model with endothelial progenitor or mature endothelial cells, as well as, BM-MSC, UCV-MSC and CNS-P. For in vitro analyses of three dimensional co-culture model all cultures will be grown for 5 to 15 days and then cells will be harvested for: a) characterization of morphology by conventional microscopy; b) characterization of immunophenotypic profile by flow cytometry; c) capacity to take up DiI-labeled acetylated low-density lipoprotein (ac-ldl); d) gene expression analysis by real time PCR; e) assessment of in vitro angiogenesis by the formation of vascular-like structures on a matrigel system and f) characterization of new blood vessels formed by confocal microscopy as well as by conventional microscopy after staining the tissue formed with HE in order to determine the presence of eritrocytes indicating functional capillaries. For in vivo analysis two angiogenesis models will be studied: 1) the Matrigel plug assay containing mesenchymal stem cells or pericytes plus endothelial cells followed by subcutaneous injection in NODSCID mice and 2) NODSCID mouse hindlimb ischemic injury produced by removal of the femoral artery. For these in vivo models MSC or pericytes will be genetically modified with fluorescent and bioluminescent proteins using transposon system which will allow the characterization of new capillaries by confocal microscopy and the analysis of new blood vessel formation in live animals using Xenogen IVIS system. 57

58 Moreover, the interactions between these two cell types during the angiogenesis process will be investigated using MSC and endothelial cells from large animal models such as canine model in collaboration with Professor Maria Angelica Miglino from Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. MSC from yolk sac canine will be isolated and characterizated because it is the first site of blood-cell production during embryogenesis. Recent studies conducted by Maria Angelica group have demonstrated that the canine yolk sac contains three preferential sites for binding MSC and endothelial cells (Miglino et al 2008). Therefore, after isolation, culture and characterization of MSC and endothelial cells from canine yolk sac, will be developed to a three dimensional co-culture model assess the cross-talk between these two cell types. It should provide molecular insights into vasculogenesis and angiogenesis process during development and tissue repair. Similarly, the same analysis will be carried out after 15 days of culture. The MSCs present in bone marrow and umbilical cord vein are very similar from the phenotypic and genotypic point of view. However, a previous study performed at Centro de Terapia Celular (CEPID - FAPESP), comparing the mrna expression profile by SAGE, showed small differences, indicating that the MSCs derived from bone marrow would be more compromised to osteoblastic e adipocytic lineages, while the ones derived from the umbilical cord vein would be compromised to functions related to angiogenesis or vasculogenesis. If confirmed at protein level, these differences of expression can contribute to a better comprehension of differences between MSC of different origin and its implications for the adequate choice for different applications in cell therapy. Based on that exposed above, the group coordinated by Dr. Lewis Joel Greene through subproject 12, entitled Comparison of proteins expression of human mesenchymal stem cells (MSC) obtained from bone marrow and umbilical vein cord, will have as main objective to compare the protein profile of MSCs isolated from the vein of the umbilical Cord and bone marrow using proteomic approach with 2D DIGE, followed by mass spectrometry, for the identification of pre-selected proteins. Humans mesenchymal stem cells (MSC) will be obtained from the bone marrow, as described by Silva et al. (2003) and from the human umbilical cord vein 58

59 as described by Covas et.al. (2003) and Panepucci et al. (2004). The protein will be extracted, quantified and utilized for 2D eletrophoresis. The comparison of the spots profile between the different samples will be performed through the image acquisition system Image Scanner and the program ImageMaster 4.0 (Amersham Biosciences). The spots from each sample, will be cut out from the gels and the proteins will be identified by MALDI-TOF mass spectrometry after digestion with trypsin as described by Pereira et al. (2005). The identification will be done automatically in the data banks of NCBI. Additionally, the list peptides mass for each sample will be manually submitted to MS-Fit ( for confirmation of the identifications done automatically. The functional classification of the identified proteins will be performed in the Gene Ontology database (Ashburner et al., 2000) through the FatiGO tool ( Al-Shahrour et al., 2004) in an attempt to standardize the terms used to describe classes and functions of proteins. An additional step will be the use of 2D DIGE system (two-dimensional difference gel electrophoresis). The derivatization of proteins with fluorescent markers will be done according to the manufacturer instructions (GE Healthcare) and the acquisition of the gels image will be done without the removal of the gels scanned with 3 fluorescence channels (Typhoon Trio). This step will be performed at the Laboratório de Imunologia do Instituto do Coração (INCOR) da Faculdade de Medicina da USP - São Paulo. The analysis of the gels will be made with the utilization of the software DeCyder 6.0.version In addition to hematopoietic and mesenchymal stem cells, other cells have been shown to be potentially useful for therapeutic purposes. Amniotic epithelial cells (AEC) can undergo in vitro adipogenic, chondrogenic, osteogenic, skeletal, myogenic, cardiomyogenic, neurogenic, pancreatic and hepatic differentiation. These cells express embryonic stem cell markers (SSEA-4, TRA 1-60, TRA 1-80, Oct- 4, FGF-4, SOX-2, Rex-1), unlike amniotic mesenchymal cells, which do not express those markers. Furthermore, amniotic epithelial cells are strong candidates for allogenic transplantations because they possess low immunogenic and immunomodulatory properties. The great differentiation potential and the easy access and isolation suggest that AEC can be useful and non-controversial source of stem cells for transplantation and regenerative medicine. AEC are often cultured in 59

60 serum, but their clinical use requires their isolation and culture without animal components. Thus, the group coordinated by Dr. Wilson Araújo da Silva-Jr. in subproject 13, entitled Differences in gene expression and immunophenotypic profile of amniotic epithelial cells obtained with and without animal substances will compare amniotic epithelial cells obtained with fetal calf serum and a subpopulation of amniotic epithelial cells, called amniotic multipotent progenitor cells (AMC), isolated and cultured in animal substances-free conditions. Term placentas will be obtained after delivery and amniotic membrane will be peeled from the chorion and digested with trypsin to detach them from the adjacent mesenchyma. Epithelial cells obtained are isolated with two different protocols: the so called AEC are separated with a Percoll density gradient and cultivated with fetal calf serum and epidermal growth factor (EGF). The subpopulation named amniotic multipotent progenitors (AMP) is obtained in serum and EGF-free conditions. Both cultivated cell are used for RNA extraction (TRIzol LS Invitrogen) and to perform Microarray analysis of gene expression. Flow cytometry will be performed to determine the expression of embryonic/epithelial markers (SSEA-1, SSEA-3 e SSEA-4, TRA1-60, TRA1-81, NANOG e OCT-4, CD9, CD10, CD29, CD49f, CD90, CD104, CD34, CD45, CD117 and CD140b) and immunologic surface markers (HLA-ABC, HLA-DR, HLA-G, CD80, CD86, CD40, CD40L, PD-1, PD-L1, PDL-2 e HLA-G). The ability to differentiate into all three germ layers will also be compared. According to Miki and colleagues (2005), RNA will be extracted from both types of cells and gene expression profiles will be evaluated by microarray analysis (Agilent Technologies). Although, stem cells are isolated on the basis of the expression of cellsurface markers together with the well known plastic adherence property, more primitive stem cells can be identified on the basis of the their ability to effluxe Hoechst dye. As a consequence, these cells acquire the profile of low side scatter and bright Hoechst staining cells, which is termed side population (Goodell et al.,1996). Originally, side population phenotype was described in murine bone marrow preparations which are present at frequency of 0,05%. Moreover, the SP cells have a greater ability to reconstitute hematopoietic system compared to HSC-CD34+ obtained from bone marrow. The presence of SP cells was 60

61 also demonstrated at human bone marrow, as well as, other tissues such as, liver, brain, mammary gland, kidney, skin, testis and retina (reviewed in Challen e Little, 2006). These data suggest that SP might be widely distributed into several tissues; although functional role and characterization are not well understood. Previous studies conducted by our group demonstrated the presence of SP cells in human bone marrow which express high level of pluripotent gene markers, such as, Nanog, Oct4, c-myc e b-catenina (Picanço-Castro, personal comm.). Moreover, since embryonic stem cells present two important application limitations: a) immunological rejection and b) tumorigenic potential, the use of SP cells can constitute an alternative safe cell source for autologous clinical applications. The group of researchers from the Center for Cell-based Therapy has been developing research projects with mesenchymal stem cells for the last eight years. Indeed, we were the first to demonstrated the presence of MSC in umbilical cord vein (Covas et al., 2003) and recently we showed that MSC are widely distributed into the organism and present extensive similarities with pericytes (Covas et al., 2008). In view of the importance of identifying and characterizing more primitive stem cells followed by the evaluation of their therapeutic potential, the group coordinated by Dr. Dimas Tadeu Covas will carry out the subproject 14, entitled Isolation and functional characterization of pluripotent stem cell - side population, whose aim is to isolate the side population from two human tissues (umbilical cord vein and central nervous system blood capillaries) and to compare then with SP isolated from bone marrow. The second aim of the subproject 14 is the isolation and characterization of human SP obtained from mesenchymal stem cells expanded in culture and isolated from bone marrow, umbilical cord vein, as well as, brain pericytes expanded in culture. In parallel, SP cells isolated from human bone marrow, umbilical cord vein and brain capillaries at day 0 will be submitted to expansion in culture using four different medium conditions: a) culture medium for MSC expansion (MSC-M); b) culture medium for expansion of hematopoietic stem cell (HSC-M); c) culture medium for expansion of endothelial progenitor cells (EPC-M) and d) culture medium for expansion of human embryonic stem cells. After expansion SP will be characterized in terms of morphology, immunophenotying and multipotentiality features. Also, the gene expression profile 61

62 will be determined by RT-PCR for the functional characterization of these different sources of SP cells. Finally, the third aim of the subproject 14 is to investigate the therapeutic potential of cell-derived SP cells using murine model for hepatic endothelial injury, as well as, for hind limb ischemia injury. In collaboration with the group of Professor Dra Maria Angélica Miglino the fourth aim objective of the subproject 14 consists of isolating SP from ovine bone marrow and brain capillaries, as well as, from canine yolk sac. The number of SP cells will be analyzed by flow cytometry and clonogenic assay. Next, these SP will be grown and expanded in three different mediums a) MSC-M; b) HSC-M and c) EPC- M and the therapeutic potential of some of these SP cells will be investigated in a model of canine bone marrow aplasia and ovine brain ischemia injury. A second collaboration with the group of Professor Dr. Flávio Vieira Meirelles to isolate SP from two bovine/gfp+ fetal tissues consists in the fifth objective of this subproject. The bovine fetal GFP tissues, the bone marrow (BM) and umbilical cord vein (UCV) will be collected in utero. After the isolation, the SP cells will be grown and expanded under three specific medium cultures: a) MSC-M; b) HSC-M and c) EPC-M and the therapeutic potential of some of these SP cells will be investigated in the NODSCID murine bone marrow transplantation model. In addition nuclear transfer experiments will be performed in bovine enucleated oocytes using BM or UCV-SP/GFP+ cells in order to evaluate the genetic reprogrammation ability of SP cells. Also, in collaboration with Prof Dr. Flávio the nucleus of human SP cells will be used for genetic reprogramming studies using a bovine cytoplast as receptor. The use of the entire potential of stem cells in therapies directed to repair of some tissue, depends on the deep understanding of the mechanisms that control the processes. Mesenchymal stem cells.(mscs) are capable of differentiating into multiple cell types, however,the knowledge of the paths and molecules that regulate the maintenance of the undifferentiated state, as well as the differentiation process, are not clear. In this last decade, special attention has been given to the noncoding RNAs, since they are considered to be important elements related to the endogenous regulation system. The mirnas compose a small group of these endogenous RNAs, containing 18 to 25 nucleotides. In humans, 500 mirnas have already been identified (GRIFFITHS-JONES et al., 2006). The 62

63 mirnas seem to have several biological functions, such as in the insuline secretion, hematopoiese, embryonic muscle development and maintenance and differentiation of stem cells (BARTEL, 2004; DU & ZAMORE, 2005; WIENHOLDS & PLASTERK, 2005). They also participate in the functional aspects of cells, such as proliferation, survival and apoptose (DI LEVA et al., 2006). The expression of the mirna in multicellular organisms is not just temporal, but also specific in some tissues, implicating that they have an essencial role (BARTEL, 2004; DU & ZAMORE, 2005; WIENHOLDS & PLASTERK, 2005). Some author investigations have already demonstrated that micrornas (mirnas) are unregulated in situations like cancer, in viral and genetic diseases, in the maintenance and differentiation of adult and embryonic stem cells, among others. Recently, Mizuno e cols. (2008) demonstrated the reduction of the mir-125b expression during the osteoblastic differentiation process in murine MSCs. Up to the present moment, no mirna has been reported to be an important factor in the differentiation of human MSCs. Thus, the group coordinated by Dr. Dimas Tadeu Covas in subproject 15, entitled Functional evaluation of micrornas in mesenchymal stem cells differentiation, will have as general objective, the evaluation of the profile of the expression, as well as the role of mirnas during the differentiation of MSCs into osteocyte. The following experimental approaches will be taken: to isolate and cultivate bone marrow MSC; to promote the differentiation into osteocyte and collect samples in different times: day zero, 24 hours, 72 hours, 7 days, 14 days and 21 days; to quantify the level of expression of mature mirnas by using the TaqMan microrna assays Human method and select those potentially involved; analise the functional effect of selected mirnas by reducing their levels making use of the Anti-miR mirna Inhibitor (Ambion) system; to analise the functional effect of selected mirnas selecionados by redicing their levels through the PremiR mirna Precursor Molecule (Ambion) system; to detect potential target genes for mirna-125b using algorithms such as miranda and RNAhybrid. The capacity of self-renewal, as well as the potential of differentiation in different cellular types of different tissues, make the adult stem cells a promising tool for the study of certain diseases. Moreover, the ease of cultivation of the CTM and their capacity of differentiation into osteoblasts allow that those cells be used as model of study for bone disease. The osteogenesis imperfecta (OI), one of the most frequent dysplasia, occurs equally among all ethnic groups and is charcterized 63

64 as a genetic disorder of mesenchymal cells, in which a generalized osteopenia leads to bone deformity, excessive bone fragility and short stature. The main characteristics of the disease are from the insufficient or defective production of colagen molecules. Some studies have been conducted with the objective of identifying genes with differentiated expression during the osteogenic differentiation process. However, there are few functional studies of differentially expressed genes in cells from patients presenting OI. This way, the group coordinated by Dr. Wilson Araújo da Silva Jr, through subproject 16, entitled Gene expression profile of mesenchymal stem cells of patients with Osteogenesis Imperfecta during in vitro osteogenic differentiation, will analize the profile of genic expression in mesenchymal stem cells during the differentiation process in osteoblasts of bearers of Osteogenesis Imperfecta and of control individuals. For that objective, we propose: the cultivation and expansion of mesenchymal stem cells from patients and normal donors; analysis of genic expression in large scale making use of microarrays; analiysis of bioinformatic for the identification of gene the networks, which are fundamental for the osteogenese; analysis of the expression by Real-Time PCR; functional studies of gene interference using RNAi. The systematic localization of CTMs in the walls of all blood vessels associated with their imunnossuppress e imunoregulator properties raise questions about the eventual participation of these cells in inflamatory auto-imune diseases (AID).There are few studies about the genetic and functional characteristics of the CTMs of patients presenting AID s and no data about the animal model CTMs and the genetically determined AID s. Furthermore, it has not been established if the CTMs of patients having AIDs or genetic animal models of AIDs are normal or defective cells. The diabetes mellitus type 1 (DM-1) and the multiple sclerosis (MS) are mediated by self-reactive T cells, and characterized by the pancreatic β cells, which produce insulin and the central nervous system, respectively. In addition the susceptibility to DM-1 and to MS is determined by genetic and environmental factors. Thus, the molecular characterization of the CTM obtained from these patients may reveal both altered genes and sinalization path ways and/orfunctional efects, possibly related to the initiation of autoimmune and or the pathogenesis of the diseases which, in the future, can be throroughly studied will facilitate the development of new therapeutic reassures for the treatment of AIDs. On the other 64

65 hand, if the CTMs from patients with MS to be genetically and functionally normal, it will be possible to use them as source autologous cells for the treatment of patients with DM-1 and MS. The group coordinated by Dr. Júlio César Voltarelli, through subproject 17, entitled Genic, proteic and functional analysis of mesenchymal stem cell (MSC) in patients with auto-immune disease, will evaluate the functional characteristic and the profile of gene and protein expression on large scale of CveTMs of patients with DM-1 e EM. For that, 15 patients with DM-1 and 15 patients with MS will be studied and submitted to the treatment with HDI/AHSCT, at the Unidade de Transplante de Medula Óssea do Hospital das Clínicas da FMRP-USP. Bone marrow samples of the patients with DM-1 or EM before the HDI/AHSCT and six months after the transplantation. Samples of BM of healthy individulas will be collected (bone marrow volunteer donors) for isolation of MSCs or control of the experiments. The mononuclear cells of the BM of the patients will be isolated by density gravity using centrifugation for isolation and culture of MSCs, and at the third step, samples of MSCs will be separated for: 1, biological characterization of MSCs (CFU-F, immunophenotyping, proliferative capacity); 2, evaluation of the immunosuppressant capacity of the MSCs in vitro (lymphocyte proliferation inhbitions assays by the co-culture with MSCs); 3, adipocytes and osteocytes differentiation; 4, RNA extration to the gene expression analysis by large scale microarray and RT-PCR validation; 5, protein extration to the protein expression analysis by 2D eletrophoresis. Subproject 18: Determination of some proteins related to apoptotic process and cell cycle control of hematopoietic and leukemic progenitors The new chemtherapy schemes developed for acute myeloid leukemia (AML) have induced the remission of the disease and increased the survival of leukemic patients. However, relapses are a very common especially among older patients and/or patients having a bad prognosis. The relapses were attributed to the existence of quiescent leukemic stem cells resistent to chemiotherapy. The leukemic cells, the origin of normal precursor cells are similar to those though, not identical, because genetic events such as mutations and chromossomic translocations were responsible for their neoplasic properties. Several studies have demonstrated that differences in the level of RNA and proteins involved in 65

66 apoptosis and control of the cellular cycle are demonstrable when leukemic and normal cells are compared. Differences in the protein levels were also demonstrated when leukemic cells were treated with the trans-retinoic (ATRA) and As 2 O 3. The purpose of this research is to identify the differences in the level of proteins of apoptótico interest in normal hematopoietic stem cells (CTHs)(CD34+CD38-) and in leukemic stem cells (CTLs) (CD34+CD38-CD123+). The comparison between the expression level of non or treated blasts with As2O3 will also be made. The proteins to be studied are: Electron transfer flavoprotein betasubunit, antioxidant 2 protein, HMGB-1, c-h-ras, Anexin I, Peroxirredoxin-2, Rho- GDI alfa, Rho-GDI beta and also three proteins described in the apoptose pathways: caspase-3, caspase-9 and BCL2. The nucleofosmina-1 protein, of major interest in the control of the cellular cycle will also be monitored at the present study. A selective proteomic approach for proteins related to the apoptosis will be used in this work, which does not represent an inventory of proteins of leukemic cells. The proteins involved in the apoptotic processes and control of the cellular cycle described in the literature, whenever possible, will be identified by immuneological methods and confirmed by when possible by mass spectrometry of tryptic peptides. The proteins will be separated by 2D electrophoresis. We hope to obtain a profile of apoptotic proteins in leukemic stem cells and also in the leukemic blastocytes treated with As2O3. Dr. Eduardo Magalhães Rego s group in the subproject 19 entitled Evaluation of the function of dyskerin in early hematopoietic differentiation will study the mechanisms involved in the malignant transformation of the hematopoetic stem cells using two animal models: a) mutant mice for the DKC1 gene and b) an acute leukemia model using vervet monkeys transplanted with hematopoetic stem cells retrovirally transduced with the CALM-AF10 fusion gene (subproject 20). The DKC1 gene encodes dyskerin (Heiss et al., 1998), and is characterized by multiple pathological features, including increased susceptibility to cancer and bone marrow failure (Dokal. 2000). Pancytopenia associated with hypocellularity of the bone marrow is the hallmark of severe aplastic anemia, a life threatening disease. The associated DKC1 mutations X-DC is one of the major inherited aplastic anemia syndromes. However the cellular and molecular mechanisms leading to bone marrow failure in dyskeratosis congenita are unknown. The DKC1 gene encodes for a pseudouridine synthase that modifies ribosomal RNA 66

67 (rrna) and is essential to ribosome biogenesis. Therefore, if the mechanism leading to aplastic anemia and cancer in X-DC is linked to the impairment of ribosomal function, it would be the first demonstration of the role of this organelle in the fate of immature undifferentiated progenitors. The specific objectives are: 1) To examine the role of dyskerin in hematopoietic stem cells. In order to determine whether the abnormalities in Dkc1 m mice is due to a autonomous cell defect, we will perform competitive repopulating assays. In these experiments, hematopoietic stem cells from Dkc1 mutants will be tested against their wild-type counterparts in their capacity for repopulating lethally irradiated recipients. If Dkc1 function was essential to the hematopoietic stem cell, one should expected the reduction in number of mature cells from all the tested cell lines (lymphoid, granulocytic/monocytic and megakaryocytic) of Dkc m mice. 2) To examine the role of dyskerin in early lymphoid differentiation. Based on our preliminary results, the best candidate to be the hematopoietic progenitors targeted by Dkc mutations are the Common Lymphoid Progenitors (CLPs). We will isolate CLPs from Dkc1 m and wild type controls, and perform competitive repopulating assays as described above, but as recipients we will use rag -/- mice, which do not present mature T or B cells in the peripheral blood. 3) To quantify the expression of the Dkc1 gene in isolated progenitors. We plan to isolate HSCs, CLPs, Fractions A and B of lymphopoieses, common myeloid progenitors (CMPs), Granulocyte-Monocyte Progenitors (GMPs), and Myeloid-Erythroid Progenitors (MEPs) from the bone marrow of Dkc1m and wild-type mice by Fluorescence-activated cell sorter (FACS), and to measure the expression of the Dkc gene by Real-time PCR. 4) To analyze apoptosis and cell growth of Dkc m lymphoid progenitors. Once the specific hematopoietic progenitor in which Dkc is qualitatively and quantitatively defective is identified. We will test whether it is more susceptible to apoptosis and/or may harbor defects in cell growth regulation compared to their wild-type counterparts. 67

68 Subproject 20: Acute Leukemia Model in Chlorocebus aethiops (Cercopithecus aethiops) using haematopoetic stem cells transduced with retroviral vector containing the hibrid gene CALM-AF10 In acute leukemia research, the animal model provides essential subsides for the understanding of molecular and physiological mechanisms involved in leukemia development. In subproject 20, we propose to generate a model of human acute leukemia using the vervet monkey. Despite the fact that the mouse model is the most commonly used, because of its relatively low costs and easy housing and manipulation, it has some limitations. From the experimental point of view, some techniques are not feasible due to the size of the mouse, making the serial sampling difficult. Genetically, the murine model differs from the human in many aspects, leading to the need of result validation in a phylogenetical closely related organism for pre-clinical studies. The vervet monkey (Chlorocebus aethiops) is one of the most used non-human primates in biomedical research (1). The Chlorocebus aethiops became an important model for the HIV study, once it is easily infected by the simian immunodefficiency virus (SIV) but differently than the Rhesus monkey, the Chlorocebus aethiops does not develop the disease (2, 3). The t(10;11)(p13;q14), involving the CALM e AF10 genes has been reported in acute lymphoid, acute myeloid leukemia and malignant lymphoma (7, 8, 9). It is involved with a bad prognosis disease, similar to the leukemias with involvement of the MLL gene (10). Leukemic cells from CALM-AF10 patients and mice co-express myeloid and lymphoid antigens, making these cells an important for the study of leukemia stem cells (11). The need for an animal model close to the human for translational and preclinical studies for leukemia and stem cell research, the availability of Chlorocebus aethiops at the National Primates Center (CENP) and the presence of a group of investigators with experience in the proposed models makes the proposed project feasible. The aims of the subproject 20 are: 1) Standardization of blood and bone marrow sampling at the CENP laboratory. Evaluation of hemogram, myelogram and cytogenetical parameters of Chlorocebus aethiops, establish reference values for the species in the maintainance facility. The extent of genetic homology 68

69 between human and Chlorocebus aethiops will be researched in parallel for relevant genes using bioinformatic tolls. 2) Immunosuppressive regimen determination with myelosuppressive drugs or irradiation at the CENP laboratory. The bone marrow stem cells transduction protocol will be established using the conditioned medium with viral supernatant (VCM) containing the retroviral construct with the CALM-AF10 fusion gene and the reporter gene GFP or YFP, and the empty vector containing only the reporter gene. The transduced cells will be analyzed in vitro for clonogeneicity and transformation. In case of transformation, the effect of CAPE on the cell growth will be analyzed. The transduced bone marrow cells will be transplanted back to the donor after myeloablative treatment. 3) Cytogenetic and gene expression profile of the leukemic cells. The receptors will be monitorized for any clinical/hematological/cytogenetic alteration. The gene expression profile of these cells will be analyzed. 4) Treatment with experimental drugs. Leukemic animals, as well as leukemic cells from the animals, will be treated with experimental treated with experimental drugs, such as cafeic ester (CAPE). The group coordinated by Dr. Júlio César Voltarelli will carry out subproject 21,entitled Evaluation of the therapeutic potential of mesenchymal stem cell (MSCs) from skin lesions caused by extensive thermal burns in animal model, will study the therapeutic potential of xenogenic and alogenic MSCs in the treatment of ulcers caused by extensive thermal burns in rats. Since Gal-1 takes part in the process of tissue regeneration and is highly espressed in CTMs, we also intend to study the impact of Gal-1 over the therapeutic potential xenogenic MSCs in the treatment of ulcers caused by burnt. MSCs will be isolated from the bone marrow of mice C57BL/6 wild (Gal-1+/+) or without the gene Gal-1 (Gal-1-/-) or from the rats and in vitro expanded. The MSCs (alogenics, xenogenic, wild or Gal-1+/+) and/or murine recombinant galectin- 1(Gal-1rm) wil be topci or systemic administratedc, animals previously submitted to extensive burning of their skin. After the treatment of the burns with thectms, or not, the following will be evaluated: the gravity of the burn, the process of 69

70 tissue regeneration, the state of systemic immunosuppression, the expression of angiogenic factors, growth factors, chemocinas and cytocinas in byopies of the ulcer and differential protein expression by proteomic methods. The action of mechanisms CTMs in the regeneration process of ulcers caused by extensive burning will be studied in this project. In addition to that, a comparison between the therapeutic potential of CTMs topically or systematically via and a comparison between the therapeutic potential of CTMs. The ability of mesenchymal stem cells (MSC) to differentiate into several cell types in vitro, their relative ease of expansion in culture, their immunologic characteristics, and their ability to be genetically modified maintaining the multipotent features, clearly make MSCs a promising source of stem cells for tissue repair and gene therapy. In this sense, the group coordinated by Dr. Dimas Tadeu Covas will carry out subproject 22,entitled Use of mesenchymal stem cells genetically modified with human recombinant coagulation factor IX in Hemophilic B mice with the aim to determine the suitability of developing a hepatic MSC cell line to produce the recombinant human factor IX for the treatment of hemophilia B in a mouse model. Hemophilia B is a genetic disease caused by the mutation in the gene encoding blood coagulation IX, which is located on the X chromosome. Hemophilia B occurs in about 1 in every 20,000 males, and the current therapy consists on intravenous administration of the clotting factor concentrates obtained from blood donors. The two major limitations to present day hemophilia therapy are: 1) viral contamination risks and 2) the cost, which limit their use to a small group of the global haemophilia population (Lillicrap et al., 2006). On the other hand, two facts: 1) the pathology involves a single protein and the replacement of this protein through cellular or genetic substitution should and does indeed eliminate the disease manifestation and 2) relatively small increments of clotting factor levels (1-5% of normal) have been shown to result in significant clinical benefits in the longterm have stimulated researchers to develop several gene therapy strategies for treatment of Hemophilia B. Among pre-clinical studies it can be cited: 1) gene therapy protocols which consist on the infusion of recombinant viral vector producing human factor IX and 2) cell therapy protocols which consist on retroviral transduction of autologous cells (skin fibroblasts) followed by infusion into haemophilic models (reviewied in Lillicrap et al., 2006). 70

71 Recently, two clinical trials have been approved by FDA. Both include the use recombinant adenovirus producing hfix. The major limitations of this strategy are the adverse host immune responses to these vectors and the high demand of viral particles due low cell production level. The research group of Hemocentro de Ribeirão Preto has large experience with gene modification of stem cells and mammalian cell lines using retrovirus system. Moreover, the group develop research projects in this field since 11 years ago which resulted in the generation of a recombinant cell library containing 43 transgenic cells producting VIII and IX coagulation factors. Three of these cell lines produce high level of recombinant factor IX. Also, the group develops research projects with mesenchymal stem cells since 8 years ago and recently designed a clinical protocol to treat patients who developed the graft versus host disease after bone marrow transplantion. The clinical trial has been approved by the Research Comite and the program has been started. With this in mind, the proposal of this project consists on generating a cell therapy strategy for hemophilia B in a murine model. The increasing number of clinical applications has stimulated a need for alternative to rapidly expanding MSCs. The actual methodology for mesenchymal stem cell culture presents four main limitations: 1) excessive manipulation which can interfere with functional cell proprieties due to successive passages employing enzymatic treatments; 2) high contamination risk recurrent from extensive manipulation; 3) absence of culture control parameters and consequently absence the control of cell physiology control and 4) high cost and time-consuming procedures for generation of sufficient number of undifferentiated cells of adequate quality. This proposal involves the technology development for optimization of MSC culture system in bioreactors aiming to reduce one of the limitations cited above and to promote the development of a scale-up with tighter control of culture conditions in order to provide a clinical grade product in an efficient, economic and safe manner. The stem cell culture in bioreactors using microcarriers, to be efficient in terms of the expansion and maintenance of original phenotype (Schop et al., 2008; Frauenschuh et al., 2007; Malda et al., 2006). Futhermore, some types of microcarriers provide an available cell growth area of about 20 cm 2 /ml compared with conventional monolayer cultures are 4 cm 2 /ml. 71

72 With that in mind, the group coordinated by Dr. Dimas Tadeu Covas in subproject 23 entitled Bioprocess development for mesenchymal stem cell expansion on microcarriers, will have as its main objective, the development of a bioprocess for culture and expansion of mesenchymal stem cells from bone marrow and umbilical cord in microcarriers and the evaluation of the biological and functional proprieties post-culture. The methods involved in this project include mononuclear cells separation and culture and expansion of mesenchymal stem cells from bone marrow blood and umbilical cord; MSC culture in spinner bioreactor on commercial microcarriers (Cytodex 3 - GE Healthcare and Cultispher S - Percell Biolytica) with dissolved oxygen, ph and stirring control; morphological analysis employing phase contrast and conventional microscope after coloration by Leishman; analysis of imunophenotypic profile by flow cytometry using 18 monoclonal antibodies (CD105, CD73, Stro-1,; CD90-PE,CD29, CD13, HLA ABC, CD49e, CD54, CD44, CD51/61, CD106, CD34, CD14, CD45, HLA-DR, CD31, KDR). The cytogenetic profile obtained using classic cariotyping protocol using GTG banding, evaluation of differentiation potential in vitro (stain by SudanII/Scarlat for adipocyte, von kossa and alkaline phosphatase for osteocyte, hematoxilin-eosin for chrondocyte and immunostain with anti-type II collagen; and quantitation of gene expression by Real-Time RT-PCR using SYBR green chemistry and TaqMan PCR assay. Thus, the group coodinated by Dr. Jose Cesar Rosa with the subproject 24, called Functional Proteomic: the role of nucleophosmin (NPM) in gliomagenesis, has as main objective study, through proteomics methodology, the NPM role in T98G and U87MG cell lines, derived from multiform glioblastoma (grade IV. To do that, the changes in proteome will be relacionated with proliferation, cell migration, and apoptosis apoptose under EGF stimulation and its effect in these protein level, as well, as the NPM expression silencing by interference RNA (irna). Consequently, we will be able to infer, by the proteomic methdology, other proteins up or down-regulated that will be directly related to the NPM expression. We also propose an extension of our proteomic studies of glioma to additional 5 patient sample of each grade using 2D gel electrophoresis/maldi-tof/tof-ms and complementation by shotgun peptide sequencing (SPS) which should contribute to increase the number of proteins candidates for new biomarkers or targets for therapeutic intervention. 72

73 Chronic lymphocytic leukemia is characterized by progressive lymphocytosis of small mature B cells, imunophenotype CD5+/CD19+ and accumulation of cells in blood, bone marrow and lymphoid organs (Caligaris-Cappio e Hamblin, 1999). CLL represents 40% of all leukemias in adults over 65 years old. Only rare cases occur in patients below 30 years old. In addition to the mutational status of variable region of the immunoglobulin heavy chain (IGHV), and expression of ZAP70 and CD38, DNA copies number variation are found in 50-80% of CLL patients and are considered important prognostic markers of the disease (Doneda, Montillo et al., 2003). The most common chromosomal abnormalities seen in CLL are the trisomy 12 and deletions in 13q14, 11q22-q23 and 17p13. Among them, the del (13)(q14) is associated with a favorable prognosis and the other chromosome losses are associated to a unfavorable prognosis (Dohner, Stilgenbauer et al., 2000). Although several genomic regions involved in chromosomal changes have been described in CLL, relatively few onco and tumor suppressor genes have been involved (Tyybakinoja, Vilpo et al., 2007). The combination of cytogenomic methods, (spectral karyotyping and arraycgh) will allow the identification and characterization of the break points, associated with chromosomal changes, detection of changes in the number of DNA copies and identification of possible involved genes. The analysis in high resolution will identify submicroscopy deletions, which will be informative, especially in patients with normal karyotype. Thus, the group coordinated by Dr. Roberto Passetto Falcão with the subproject 25, named Cytogenomic tools applied to the investigation of chromosomal instability in chronic lymphocytic leukemia (CLL), this Project has the aims: to evaluate the genomic profile of the LLC, through cytogenetics molecular tools, as the spectral karyotyping (SKY) and arraycgh to better define and characterize genomic regions and possible genes involved. Additionally, the project aim to create a bank of samples from patients with CLL (cells, DNA and RNA) to be used for the development of other research projects. For that reason, the following methods will be employed: obtaining cells for SKY technique application; metaphasic cells hybridization for spectral analysis (Spectral Applied Imaging); extraction of DNA from blood of patients and controls and changes evaluation in the number of genomic copies regions (duplications or deletions) by the arraycgh method using Agilent platform. 73

74 Estabishment of Animal Models and Pre-clinical trials Embryonic and adult stem cells research described in this Project cover the methodology establishment and questions of important studies, in a conceptual and practical order. Together, forms the development base ot technical-scientifically know how necessary for the stem cell therapy establishment, as a future reality in Brazilian medicine. However the effective practical application of these potential cells demonstrated in vitro or in laboratory animal s trials, will must depends of new clinical trial with animals with more close characteristics of human organism. In this way, we describe a propose of effective implantation of a Centre capable to base studies in this nature. In front of the establishment necessity of expertise on the applicable biotechnology field of cell and gene therapy in animal models, the infrastructure adaption for new technologies development and training program of students is fundamental to guarantee the quality of the results of this project. Then, last years ago, different initiative must be done to create a Center of Pre-clinical trials and a Stem Cell bank of animal models, centered locate at Faculdade de Medicina Veterinária e Zootecnia of USP centered with the establishing of a core facility system located at USP and wide-range throughout the Satellite Laboratories Net associated to Pre-clinical Studies Center. With great importance, this Center open new venue to establish pré-clinical trials in animal with high disease incidence common in human, neurodegenerative disease, spinal cord lesions and bone defects, and also, disease of the eyes. The group coordinate by Dra. Maria Angélica Miglino related to subproject 26, titled Pre-clinical studies center and animal stem cells banking, Will have the main objective to structured a bank of animal stem cells with high level competence related to Regenerative Veterinary Medicine. Different protocol will be used in various pathology and the procedures collection of stem cells in na idela medium for expansation, analyses of potential differentiation, protocols, transfer of pre differentiated cell to a surgical area, byopsie and biodistribuition and engraftment of stem cell inside the damage tissue Follow the list of animal models that will be used and cell types related to each disease, in a partner work between groups. 74

75 Acquired and/or induced-diseases in animal models like: Species: canine, suine, ovine, equine, rabbits, mouse, rats, hystricomorphs and monkey; Diseases: bone marrow aplasia, acute kidney lesions, head femur osteonecrosis, corneal and retina processes and, cardiac and pulmonary diseases; Criate by animal stem cells banking (culture and differentiation); Canine mesenchymal cells from yock sac, boné narrow na fetal liver Equine mesenchymal cells from umbilical cord e amnion; Progenitors cells by canina olphactory epitelium Stem cells from amniotic flui, yolk sac e alanttois Experimental topics Produce positive LACZ mesencymal cells from umbilical cords and bone narrow to autologous transplantation Intra-uterine injection and biodistribution of the canine mesenchymal stem cells; Biodistribution of mesenchymal stem cells and GFP+ precocities in ovine embryos; Culture and differentiation from monkey mesenchymal stem cells - Callitrix (collaboration to Maria de Fátima Messias and Penelope Nayund, Germany). The large animal models definitely contribute for the cell therapy protocols establishment, permitting that clinical procedures near to the utilized in humans are developed. However, the real transference for humans, can depend on behavior and biological characteristics that are human exclusivities or. at least, of animal near from them on evolutive process. Primates not-humans are considered good experimental models in biomedical researches because they present anatomy and immunologycal system with very similar answers them found in humans (CCAC, 1993). Despite of this characteristics, researches related to primates, not human, using cells are still in smaller number compared to those developed in others animal models as rat and mice, this fact is because it is more difficult to access 75

76 and handle primates not humans in researches (Jin et Al., 2002; Hodge Jr., 2002; Takagi et Al., 2005; Vrana et Al., 2002). Because of this, the availability of a plantel of primates not humans and maintained in captivity for experimentation biomedical researches, would be able to contribute hugely for the advancement of the researches that utilize stem cells for treatment of diverse illnesses. In this sense, the National Center of Primates - Evandro Chagas Institute (CENP- IEC/SVS/MS), Ananindeua, Pará, is going to dispose the experience already established in management, clinical and surgery of primates not humans maintained in captivity, for the utilization in biomedical researches using stem cells. Like this supracited group, the group coordinated by Dra. Klena Sarges Marruaz da Silva with the subproject 27, titled " To make Neotropical primates (new world) and Chlorocebus aetiops (old world) to stem cell research as animal model for cellular therapies ", will have as principal objective, to dispose the plantel of neotropical primates neotropicais and Chlorocebus aethiops, in adult ages, born in the National Center of Primates - Evandro Chagas Institute (CENP- IEC/SVS/MS), to use adult and embryonic stem cells with purpose of cell therapy. For that, adult males of Chlorocebus aethiops specie (or of other neotropical species) will be selected by the veterinarians of the CENP-IEC/SVS/MS through clinical selection where will be carried out clinical and laboratory exams (blood count, serum biochemical, urinalisys, parasitologic analyses of excrements and X ray). Only the clinically healthy animals will participate of the experimental groups. The animals selected will be separate. Them the cellular cultive prepared for transplant will be transported from the university or partner center to the laboratory of cell cultive of the Section of Environment/IEC/ SVS/MS, where will be stored, and, to the epoch of the transplants, caused to the CENP-IEC/SVS/MS, them these cells will be achieveted until the experimental phase with the animals. The transplant of the cultive will be done according to experimental protocol established for each experiment in anesthetized animals, by a veterinarian of the CENP-IEC/SVS/MS according to the protocol already established. The procedures will be done in aseptic conditions, and the trichotomized region, cleared with antiseptics, will receive the cells or placebo. For the clinical accompaniment aftertransplant, the animals will be evaluated through complete blood counts (CBC), with white blood count (WBC) and also by clinical evaluations for corporal 76

77 temperature, weight or any another exam suggested according to the kind of transplant used. For histopathologic evaluation of the main organs (heart, lung, kidneys, liver, spleen, linfonodes, etc) and bone marrow, the euthanasia will be done in one animal of each group participant of the experiment. Clinical Studies In spite of the small number of clinical studies aimed at the evaluation of stem cell based therapeutic approaches, the use of adult stem cells to treat some diseases is a reality. Thus, the group coordinated by Dr. Júlio César Voltarelli is in the vanguard of autoimmune diseases treatment in Brazil and worldwide. In addition, the group led by Dr. Dimas Tadeu Covas is in the forefront of the use of autologous mesenchymal stem cells for the treatment for graft-versus-host disease (GVHD). Due to their importance, they will be discussed in details: Subproject 28: Treatment of type 1 diabetes mellitus by infusion of mesenchymal stem cells Abstract The type 1 diabetes mellitus (DM-1) is an inflammatory autoimmune disease (AID), mediated by autoreactive T cells and characterized by the selective destruction of insulin-producing β cells. The susceptibility to DM-1 is determined by genetic and environmental factors. The clinical disease expresses itself only after the destruction of approximately 80-90% of the β cells. The destructive process of β cells leads to the lack of insulin, which results in hyperglycemia and its acute and chronic complications. If not treated, the metabolic consequences progressively lead to depression of the central nervous system, coma and death. Thus, patients need permanent treatment with exogenous insulin for survival. Among other serious chronic complications are the vascular problems that lead to renal failure, blindness, heart disease and chronic ulcers. DM-1 is treated by conventional or intensive insulin therapy. Clinical tests with immunosuppressive drugs, monoclonal antibodies (anti-cd3) and islet transplantation have had significant results, but they have been insufficient for routine clinical application. This scenario stimulated new studies concerning to therapeutic alternatives, such as in vitro differentiation of human stem cells into 77

78 pancreatic β cells for transplantation and treatment. In this context, mesenchymal stem cells (MSCs) represent an ideal source for cell therapy due to their ease of isolation and in vitro expansion, and also by their immunsuppressor, immunoregulators and regenerative capacities. Recent studies have demonstrated a suitable therapeutic effect of MSCs infusion into animal models of autoimmune diabetes and in patients with manifestation of GVHD, whose immunologic manifestations are similar to those in autoimmune diseases. On the basis of published studies, our hypothesis is that the autoimmune aggression over the pancreas can be controlled through the MSCs because after infusion in diabetic patients, they preferentially migrate to the inflamed pancreatic tissue, promote local immunosupression and stimulate the local regeneration through paracrine mechanisms that involve the production of anti-inflammatory molecules, imunorregulators, growth factors and angiogenic molecules. Thus, our objective is to evaluate the safety, the therapeutic effect and the mode of action of the infusion of allogenic MSCs in the treatment of recently diagnosed DM-1 in humans and animal models. We will evaluate the immune response (frequency of T cell subpopulations, naïve T cells, memory T cells, NK cells and CD4 and CD8 T cells; cytokines measure at serum, T cell repertoire, autoreactive T cells, and anti- β cells) in patients at different time after the infusion of the MSCs. Parallel to the studies in humans, we propose to evaluate the response of experimental autoimmune diabetes (a genetically determined model and a chemically induced model) to the infusion of the human MSCs. The extent of the pathogenic autoimmune response will be evaluated in these models, as well as the role of the MSCs in the regeneration of the pancreatic tissue damaged by the autoimmune aggression, during the early stage of the disease. The results obtained from this project will demonstrate the therapeutic effect of the infusion of MSCs in autoimmune diabetes treatment in humans and will also elucidate the immune and regenerative mechanisms of action involved in this therapeutic response through the studies in animal models presenting autoimmune diabetes. If a good quality therapeutic efficacy is demonstrated, the infusion of MSCs could become a therapeutic alternative for the treatment of DM-1, which is an autoimmune disease associated with a low quality of life and requires chronic treatment. 78

79 General Objective This project has, as a general goal, to evaluate the safety, the therapeutic efficacy and the mechanisms of action of the human mesenchymal stem cells infusion for the treatment of recently diagnosed type 1 diabetes mellitus. We have established the following specific goals: Methodology Fourteen patients having recently diagnosed DM-1 (for less than 4 weeks; antibody anti-gad65 positive) will be receive an infusion of allogenic MSCs at Unidade de Transplante de Medula Óssea do Hospital das Clínicas da FMRP-USP. This research project and its informed consent form have been approved at Local and National Ethical Committeee for Humans Research. The bone marrow donor (BM) will be a healthy member of the family of the diabetic patient selected on the basis of HLA typing. Preferentially a donor who does not present diabetes-related HLA (DRB1*03 and/or *04 and DQB1*0201 and/or *0302) will be used. The donor of the BM will be submitted to the collection on iliac crest of, approximately, 100 ml of bone marrow (BM). The BM samples from the donors will be processed at Laboratório de Terapia Celular do Centro de Terapia Celular (CEPID-FAPESP) of Centro Regional de Hemoterapia do HC-FMRP-USP. The MSCs will be cultivated in sterile conditions within the strict requirements of good manufacturing practice (GMP) for the production of cellular components for infusion in humans. Samples of BM of healthy individuals (volunteers BM donors) will be collected for the isolation of MSCs for infusion in animal models of diabetes. The PBMCs will be isolated from BM by density gradient centrifugation (Ficoll Hypaque TM, d=1,077), then 4x10 7 BM mononuclear cells will be ressuspended in α-mem medium supplemented with 15% of fetal bovine serum (FBS) and cultured at 37ºC in 5% CO 2. After three days, the non-adherent cells will be removed, and 50 ml of medium will be added. The medium will be changed every 3-4 days and when the cells are semi confluent, they will be dissociated using tripsin and expanded (first passage). The MSCs will be submitted to successive passages (3-5) until a sufficient number of cells are acquired for the procedure (1-2 x10 6 /kg of the patient in each infusion). Three days before the infusion of the MSCs in the patient, the FBS (medium) will be replaced by autologous serum (at 5-10%). A second infusion of MSCs will be carried out, both with the same number of cells (1-2 x10 6 /kg) and separated by 30 days. Samples of the MSCs will be separated before the infusion 79

80 for: 1) morphologic, immunophenotypic and cytogenetic characterization; 2) adipogenic and osteocytic differentiation; 3) evaluation of the in vitro immunosuppressant capacity. After the infusion of the MSCs, the patient will be monitored weekly until D+60 post-infusion and, then, every 3 months. The monitoring will be done by clinical and laboratory exams (which include glycemic fasting, C-peptide and glycosated hemoglobin levels). Peripheral blood of the patients will be collected for the study of the immune response on days +1,+2, +7, +14, +30, +60, +180, +270, +360 and, then, every 6 months after the second infusion of the MSCs. Focusing on the study of the regenerative and immune systems of the action of infusion of human MSCs (isolated from BM) for the treatment of experimental autoimmune diabetes, we will use a genetically determined model of diabetes (NOD mice, Non-Obese diabetic mouse) and a chemically induced autoimmune model of diabetes induced by administering streptozotocin (STZ) in mice C57BL/6. The C57BL/6 mice (male, 8 weeks old) will be submitted to 5 consecutive daily doses of STZ. Five days after the last dose of STZ (initial phase of the disease) the C57BL/6 mice will endogenously receive the infusion of human MSCs. The NOD female mice, 12 weeks old (initial phase of the disease) will be infused with human MSCs. After the infusion of the MSCs, the mice will be monitored twice a week in relation to glycemia and weight. After 10, 17, 24, 32 and 60 days from the infusion, the animals will be sacrificed and the pancreas, spleen and serum of these animals will be collected for immunologic analysis. We will evaluate the occurrence of fusion between human MSC cells and pancreatic murine β cells through FISH analysis of X and Y chromosome for murine and human. Immunohistochemistry will be performed to evaluate the capacity of human MSC cells to differentiate into insulin-producing β cells. In addition, MSCs will be infected with recombinant retrovirus that carries the LacZ gene for studies of migration and stability of MSCs in the injured tissue. 80

81 Subproject 29: Treatment of multiple sclerosis with hematopoietic stem cells: evaluation of the clinical response and immune mechanisms of action Abstract In the past years, clinical studies have demonstrated that high doses immunossuppression (HDI), followed by the autologous transplant of hematopoietic stem cells (AHSCT) is able to suppress the inflammatory activity in patients who have autoimmune diseases (AID) and can induce long clinical remissions. The AHSCT in AIDs is based on the idea that intense immune ablation may eliminate the autoreactive cells. The immune system reconstituted from hematopoietic precursors may reestablish immune tolerance. Over 1,000 transplants in clinical trials phases I/II have already been performed. Recently, randomized phase III clinical trials comparing the AHSCT with conventional cell therapy for AIDs diseases, were initiated in the United States and Europe. In Brazil, the treatment of AID with HDI followed by AHSCT, has began at the Bone Marrow Transplant Unit in the Hospital das Clínicas da Faculdade de Medicina in Ribeirão Preto Universidade de São Paulo(USP) in September 2001, it focused on severe rheumatic (Systemic lupus erythematosus and multiple sclerosis {MS}) and neurologic diseases (secondary progressive multiple sclerosis), which do not respond to conventional treatments. HDI therapy, followed by AHSCT led to the remission of the disease in 75% of the MS patients (secondary or primary progressive forms) transplanted at hospitals which participate in the multicenter study (N=41), confirming the in the literature. In these patients, the value of EDSS (Expanded Disability Status Score) improved or stabilized after transplant. In addition to clinical studies our group has studied the action mechanisms of HDI/AHSCT for the treatment of organ-specific AIDs, i.e., which are the induced changes in the immune system of the patient caused by HDI/AHSCT could explain the remission of the AID. Our results and recent reports in literature, suggest that the HDI/AHSCT induce a reprogramming of the immune system after the AHSCT, i.e., a regeneration of a new or different and self tolerance in patients with AIDs. However, additional molecular and cellular immune studies are necessary and important to better understand the mechanisms of action of HDI/AHSCT for the treatment of (Type-1 diabetes mellitus) DM-1 and MS. The knowledge of these 81

82 diseases remission mechanisms is fundamental for the establishment of the HDI/AHSCT therapy as an alternative way for the MS and DM-1. The success of these first experimental clinical protocols of phase I/II has opened perspectives to use the HDI/AHSCT therapy in other groups of patients. Thus, this project will evaluate if the AHSCT preceded by the HDI is able to induce long clinical remission in patients who have remissive MS (inflammatory, recurrent to the treatment with interferon), as well as investigate the molecular mechanisms of the therapeutic action of the HDI/AHSCT in this disease. General Objective This project has as a general objective, to determine if the autologous transplant of hematopoietic stem cells, preceded by immunossuppression in high doses is able to induce long clinical remission in patients who have relapsingremitting MS, as well as investigate the molecular and immune mechanisms of the therapeutic action of the HDI/AHSCT in these diseases. Methodology Patients with MS relapsing-remitting (N=15) will be submitted to the treatment with HDI/AHSCT at the Bone Marrow Transplant Unit in the Hospital das Clínicas da Faculdade de Medicina in Ribeirão Preto USP. The will be monitored, after the transplant, in regards to clinical and laboratorial response. The clinical project of the HDI/AHSCT for the treatment of MS was approved by the Ethical Committee of the Hospital das Clínicas of FMRP-USP (Proc /05) and by the National Ethical Comitte for Research ( / ; RG 12908). This clinical project is part of a phase III clinical trial, multicenter and randomized (MIST, Multiple Sclerosis International Stem Cell Transplant Trial) to evaluate the efficacy of the HDI/AHSCT in comparison with the conventional treatment (Interferon, Copaxone ou Mitoxantrone). Other participant centers of the MIST are: Northwestern University (Chicago, USA), University of California (San Diego-CA, USA), Baylor College of Medicine (Houston-TX, USA), Charite Hospital (Berlin, Germany), Singapore General Hospital (Singapore) and Hospital Israelita Albert Einstein (São Paulo-SP, Brazil). Peripheral blood samples will be prospectively collected from patients who had MS relapsing-remitting before (pre-mobilization) and after the transplant 82

83 (D+180, D+360, D+540, D+720), for isolation of the T CD4+ e CD8+ cells. The gene expression profile of the T CD4+ e CD8+ cells will be compared between: 1) pre/post-ahsct patients and healthy individuals; 2) pre-ahsct and post-ahsct patients. The analysis of the gene expression will be done using the cdna microarrays technique (platform Agilent, EUA), that is already standardized in the Hematolgy Laboratory- Centro de Terapia Celular (CEPID FAPESP) do Centro Regional de Hemoterapia do HC-FMRP-USP. The results of the gene expression profile studies will be analyzed by bioinformatics programs which are specific for the determination of differentially expressed genes. The results will be validated by the RT-PCR technique, using 7500 Real Time PCR System (Applied Biosystems), which is well standardized at our laboratory. Subproject 30: Mesenchymal stem cell (MSC) for treatment and prevention of graft versus host disease (GVHD) in patients transplanted with hematopoietic stem cells Abstract The allogenic transplantation of hematopoietic stem cells is considered to be the therapy of choice for the treatment of several diseases, among them hematological neoplasm and other hematological diseases. However, despite the immune suppression of the patients they may trigger a series of secondary effects, such as graft-versus-host disease (GVHD). GVHD is the main cause of morbidity and mortality in patients submitted to bone marrow transplant (BMT). This disease is due to the activation of T cells in response to the allogenic MHC after bone marrow transplantation. The T lymphocytes of the donor identify the antigens of the host as foreign bodies and trigger the immune response against them. The GVHD is normally classified as acute (generally occurring up to 100 days after the transplant, with skin, intestine and liver involvement) or chronic (generally occurring over 100 days after the transplant, characterized by lesions, dryness, constriction or several organs sclerosis). The mesenchymal stem cells (MSC) are rare and are present in the bone marrow in a frequency of 0,001% to 0,01% of all nucleated medullar cells and can be identified by the positivity of four markers: Stro-1+, CD73 (SH3/SH4), CD105 83

84 (SH2) and CD90 (Thy-1) and negativity for endothelial (CD31, KDR, VE-cadherin) and hematopoetic cells markers (CD34, CD14 and CD45). These cells adhere to plastic and can be cultivated in the presence of fetal bovine serum (FBS), with rapid multiplication of approximately 40 fold. Therefore, about one billion mesenchymal cells are obtained from the culture of one single cell. MSCs express intermediate levels of MHC class I, and do not express the MHC class II antigens on their surface. The expression of MHC class I is important and prevents the MSCs from being eliminated by the NK (natural killer) cells. On the other hand, the lack of MHC class II and of co-stimulatory molecules (CD40, CD40L, CD80 and CD86) permit to this cell to develop immune vigilance escape mechanisms, preventing the activation of T CD4+ and T-CD8+ lymphocytes. This immunossuppressor potential led several researchers to develop clinical studies using MSCs for the treatment of GVHD and, in many cases, successful results have been reported. Similarly, the present project aims to use MSCs for the treatment and prevention of the graftversus-host disease in patients submitted to the hematopoietic stem cells transplant. General Objectives: To contribute to the formation of human resources, nucleation of the staff and integration with multidisciplinary teams to develop clinical protocols for cell therapy. To determine the efficiency and safety in the use of allogeneic mesenchymal stem cells expanded in vitro for the treatment of patients who developed the graft-versus-host disease after allogeneic hematopoietic stem cells transplantation. Methodology: Isolation, selection and cell culture of MSCs from blood and the bone marrow. The PBMCs will be isolated by density gradient centrifugation (Ficoll Hypaque TM, d=1,077), followed by cell number counting in Neubauer chamber for further cell plating. After 72 hours of culture, non adherent cells will be removed and the attached MSCs will be expanded in culture. Determination of the number of colony forming unit (CFU). Mononuclear cells will be cultured on Petri dishes and the number of colonies counted from day 5 to 7. 84

85 Immunophenotypic Characterization. A panel of 18 monoclonal antibodies will be used: CD105-FITC (SH-2) BD; CD73-PE (SH3/SH4) Pharmigen; Stro-1 supernatant fluid ascite; CD90-PE (Thy-1) BD; CD29-PE (integrin b-1) BD; CD13-PE (aminopeptidase-n) BD; HLA ABC-PE Pharmigen; CD49e-PE (VLA-5) BD; CD54-PE (ICAM-1) BD; CD44-FITC (HCAM-1) BD; CD51/61-FITC (Avb5) Pharmigen; CD106-PE (VCAM-1) BD; CD34-PE (Sialomucyn) BD; CD14-PE (LPS-R) BD; CD-45-FITC (LCA) BD; HLA-DR BD; CD31-FITC (PECAM-1) BD; KDR (VEGFR-2) Sigma e Isotype Simultest (IgG1/IgG2) BD. Cells (5x10 5 cells) are analyzed after incubation with antibodies by flow cytometry as recommended by the manufacturer. Differentiation potential: The adipogenic, osteogenic and chondrogenic differentiation potentials will be evaluated as previously described (Covas et al., 2003) Cytogenetic profile: Samples from bioreactors and static cultures in exponential growth will be subjected to treatment with colcemid (0.15 mg/ml) for 4 hours at 37 C, followed by incubation with KCl (0.075M) for 20 minutes. Subsequently, the cells will be fixed with methanol and acetic acid, washed, and transferred to slides that will be kept at room temperature for a period of 24h before proceeding with the trypsin digestion. The remainder of the material will be stored in a freezer (-20 C) for the in situ fluorescence hybridization techniques (SKY and FISH). The chromosomal banding technique will be performed by incubation with a trypsin solution (1%), followed by washing in tap water and staining with Giemsa for 5 minutes. The slides will be ready for analysis after the staining. 85

86 INDICATORS The following indicators are numerical indicators for our estimates of the production of the Project for the next five years ( ). Bibliographic production Number of published papers specialized peer reviewed journals: 45 Number of thesis concluded (Master/Doctorate): 40 Number of deposited patents : 6 Number of substances or new articles propagated in the media printed or electronics Other products Number of courses offered: Courses of Post Graduation lato sensu for Professors of biology area: 2 Post Graduation Courses already offered: 9 Post Graduation Courses to be created : 12 Number of participating students: Pos Graduate: 120 Graduation (Scientific Initiation): 12 Junior and high school students: Number of hospital patients participating in the clinical studies: 60 Number of new developed animal models : 9 Number of derived cell lines: 20 Number of constructed vectors: 15 Number of concluded projects: 30 Number of expositions or presentations: 12 Number of accesses to the homepage of the INCTC: >

87 D) Program for High Qualified Human Resources Formation The proposal of this project aims in special at the formation of highly qualified human resources focused on capacity of interacting in all levels of Cell therapy process, since the generation of cells with the necessary characteristics up to the clinical studies. The complex methodologies employed in this project is a great opportunity for post graduation students and researchers to interact by sharing experiences through several laboratories of the Institutes and also with international collaborators. Running Courses COURSE INSTITUTE CODE HOURS CREDITS PLACES Topics in Cell Culture FMRP/USP/Ribeirão RCM hours 4 9 for Cancer Research Preto Molecular Methods UNESP/Araraquara 75 hours 5 10 applied to Hemotherapy Cellular and Molecular Mechanisms Related to the Physiopathology of Leukemias FCF/USP/ Ribeirão Preto hours 5 10 Immunotherapy: Fundamentals and Clinical Applications Cellular Therapy in Inflammatory, Autoimmune and Neoplasics Diseases: Basic and Clinical Aspects Stem cells and Animal Cloning Placenta Morphophysiology and Placentation of and Biology of Development FMRP/USP/Ribeirão Preto FMRP/USP/Ribeirão Preto RIM hours 2 10 RCM hours 2 10 FMVZ/USP/São Paulo VCI hours 4 20 FMVZ/USP/São Paulo VCI hours 4 20 FMVZ/USP/São Paulo VCI hours

88 NEW COURSES COURSE INSTITUTE HOURS PLACES Post Graduation Course latu senso in Biology UNIVESP Plataform for distant studies 360 hours 900 teachers students Post Graduation Course latu senso in Plataform for distant 360 hours 40 Biology - Pro-Reitoria de Cultura e Extensão studies Advances in Molecular Methods for FCF/USP/ Ribeirão 75 hours 15 Investigation in Biology Preto Use of animal models for leukemia FMRP/USP/Ribeirão 60 hours 6 studies. Preto Concepts in Biotechnology and FMRP/USP/Ribeirão 60 hours 15 Medical Field Applications Interdisciplinarity: A Tool for Socialization Knowledge and Citizenship Construction Preto Museums and Sciences Spaces 45 hours 40 Use of Museums and Sciences Centers Museums and Sciences 60 hours 40 Resources for Learning Dynamization Spaces Language, Writing and Scientific EAD platform 45 hours 40 Diffusion Moodle Scientific Diffusion Workshop EAD platform Moodle 30 hours 40 Research Methodology as Diffusion EAD platform Moodle 60 hours 40 Tools Midia and Scientific Diffusion EAD platform Moodle 30 hours 40 Mediation in Centers and Sciences EAD platform Moodle 30 hours 40 Museums Fundamentals in Scientific Education EAD platform Moodle 30 hours 40 Human Resources formation by the Coordinators Supervisor Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Student Júlio César Voltarelli Eduardo Antonio Donadi Silvia Beatriz Vieira Ramos Gil Cunha de Sanctis Raul Antonio de Moraes Year of concludion Actual position Institute Country Titular Professor, Coordinator of Transplant Unit of HCRP-USP Associate Professor Assistant Professor of Obstetrics & Gynecology 1993 Medical Director 1995 Adjunct Professor Research Coordinator of Fundação HEMOPE de Pernambuco Department of Clínica Médica, Faculty of Medicine of Ribeirão Preto - USP Department of Clínica Médica, Faculty of Medicine of Ribeirão Preto - USP Division of Reproductive Endocrinology & Infertility University of North Caroline Fundação Hemocentro de Ribeirão Preto Department of Clínica Médica, Faculty of Medicine of Pernambuco University Brazil Brazil Chapel Hill Brazil Brazil 88

89 Supervisor Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Roberto Passetto Falcão Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Student Eduardo Magalhães Rego Sergio Luiz Martins Cintia Machado Rodrigo Tocantins Calado Edgar Gil Rizzatti Nilce Marzola Ideriha Suzana Beatriz Veríssimo Melo Aureo Evangelista Santana José Orivaldo Mengele Augusto Cesar Cropanese Spadaro Leila Maria Beltramini Sabbag Silvia Nassif Del Lama Year of concludion Actual position Institute Country Associate Professor Clinical Analysis Division Coordinator Adjunct Professor Coordinator of Flow Citometry Laboratory of Fundação Hemope de Pernambuco Hematology Branch, National Heart, Lung, and Blood Institute Haematology Section Coordinator Associate Professor Associate Professor Associate Professor 1993 Titular Professor Titular Professor Associate Professor Adjunct Professor, Department of Clínica Médica, Faculty of Medicine of Ribeirão Preto - USP Laboratório Fleury Department of Clínica Médica, Faculty of Medicine of Pernambuco University National Institutes of Health Laboratório Fleury Faculty of Medicine of Universidade Estadual de Londrina Faculdade de Medicina da USP-SP Faculty of Veterinary Medicine of Jaboticabal, UNESP Centro de Pesquisas Gonçalo Moniz Osvaldo Cruz Foundation Fiocruz Salvador, Bahia Department of Physics and Chemistry School of Pharmaceutical Sciences of Ribeirão Preto, SP, Universidade de São Paulo Instituto de Física, Departamento de Física e Informática, São Carlos, SP, Universidade de São Paulo Department of Genetic and Evolution, Centro de Ciências Biológicas e da Saúde, Universidade Federal de São Carlos, SP. Brazil Brazil Brazil EUA Brazil Brazil Brazil Brazil Brazil Director Brazil Brazil 89

90 Supervisor Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Student Lusiane Maria Bendhack Year of concludion 1984 Clarice Izumi 1985 Vitor Marcel Faça Sandra Pereira Rodrigues Gustavo Antônio de Souza Fabíola Leslie A. C. Mestriner Augusto Cesar Cropanese Spadaro Almir de Souza Martins Julio César Padovan José César Rosa Actual position Institute Country Associate Professor Laboratory Specialist 1998 Pos doctoral 2000 Pos doctoral 2002 Professor Laboratory Specialist Titular Professor titular Director of School of Pharmaceutical Sciences of Ribeirão Preto, SP, Universidade de São Paulo 1996 Adjunct Professor 1996 Researcher 1998 Professor Department of Physics and Chemistry School of Pharmaceutical Sciences of Ribeirão Preto, SP, Universidade de São Paulo Centro de Química de Proteínas, Faculty of Medicine de Ribeirão Preto, Universidade de São Paulo Fred Hutchinson Cancer Research Center, Seattle Fred Hutchinson Cancer Research Center, Seattle The Gade Institute Section for Microbiology and Immunology, University of Bergen, Department of Farmacology, Faculty of Medicine of Ribeirão Preto, SP, Universidade de São Paulo Department of Physics and Chemistry School of Pharmaceutical Sciences of Ribeirão Preto, SP, Universidade de São Paulo Department of Phyisiology and Biophysics, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG. Rockefeller University Departamento de Biologia Celular, Molecular e Bioagentes Patogênicos, Faculty of Medicine of Ribeirão Preto, Universidade de São Paulo. Brazil Brazil USA USA Norway Brazil Brazil Brazil New York, USA Brazil 90

91 Supervisor Lewis Joel Greene Lewis Joel Greene Lewis Joel Greene Student Carolina Bosch Cabral Lyris Martins Franco de Godoy Maria Sumiko Arita Matsuura Year of concludion Actual position Institute Country 2004 Pos doctorate 2004 Pos doctorate professor retired Massachusetts General Hospital, Harvard Medical School, HMS, Max Plank Institute of Biochemistry, Department of Proteomics and Signal transduction Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, SP. USA German y Brazil Lewis Joel Greene José Godinho Neto 1991 a 1993 Assistant Professor Departamento de Química, Universidade Federal Rural do Rio de Janeiro Brazil Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Ana Cristina Silva Pinto Karen de Lima Prata Ana Paula Costa Nunes da Cunha Cozac Maria Ângela Pignata Ottoboni Eugenia Maria Amorim Ubiali Marina A Coutinho Benedito de Pina Almeida Prado Jr Luiz Paulo Cicogna Faggioni Simone Kashima Haddad Virgínia Proença Picanço Castro Silvana Maria Quintana 2007 Pos doctoral, Medical Doctor 2006 Medical Doctor 2004 Medical Doctor 2004 Supervisor 2003 Medical coordinator 2003 Medical Doctor 2002 Medical Doctor, Head of Transfusional Agency 2001 Medical Doctor 1996 Supervisor 2006 Pos doctoral 1997 Professor Unité Mixte du Recherch UMR 763 (INSERM) - Hospital Robert Debre - Paris Fundação Hemocentro de Ribeirão Preto Fundação Hemocentro de Ribeirão Preto Laboratory of Quality Control, Fundação Hemocentro de Ribeirão Preto Fundação Hemocentro de Ribeirão Preto Laboratorio Dr Coutinho Unidade de Emergência HCFMRP-USP Agência Nacional de Saúde Suplementar Molecular Biology Laboratory, Fundação Hemocentro de Ribeirão Preto Fundação Hemocentro de Ribeirão Preto Ginecology and Obstetrics Department- FMRP-USP France Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil 91

92 Supervisor Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Dimas Tadeu Covas Eduardo Magalhães Rego Eduardo Magalhães Rego Eduardo Magalhães Rego Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Julio César Voltarelli Student Elisa Maria Carbolante Antonio Adilton O Carneiro Evamberto Garcia Góes Rita de Cássia Viu Carrara Rodrigo Siqueira Abreu e Lima Gil Cunha de Santis Lorena Lobo Figueiredo Pontes Márcia Piuvesan José Hermênio C. Lima Filho Maria Angélica Watanabe Ana Beatriz Pereira Lima Stracieri Fabíola Attiê de Castro Kelen Cristina Malmegrim Luís Paulo Faggioni Year of concludion 2004 Professor 2003 Professor Actual position Institute Country Faculdade de Ciências Farmacêuticas de Ribeirão Preto, SP, Universidade de São Paulo Faculdade Filosofia Ciências e Letras de Ribeirão Preto USP Brazil Brazil 2001 PIPE fellowship FAPESP Brazil 2001 Pos Doctoral 2007 Head 2006 Medical Director 2008 Doctorate 1997 Pos doctoral Associate Professor Associate Professor Medical doctor Associate Professor 2006 Pos-doctoral 2006 Medical doctor Sulani S. Souza 1996 Medical doctor Márcia Cristina Furtado Nascimento Marta Maria L. Lemos 1999 Medical doctor 2000 Medical doctor Rejane Vieira 2001 Medical doctor Eduardo AJ Paton Maria Carolina O. Rodrigues Vivian Youssef Khouri Medical doctor, director 2006 Medical doctor 2007 Dentist Fundação Hemocentro de Ribeirão Preto Oncology Center of Brasília Fundação Hemocentro de Ribierão Preto Hospital das Clínicas da Faculdade de Medicina de Ribierão Preto Universidade Federal da Paraíba Universidade Federal do Paraná Universidade Estadual de Londrina Hospital das Clínicas FMRP-USP Faculdade de Ciências Farmacêuticas de Ribeirão Preto- USP Hemocentro de Ribeirão Preto Agência Nacional de Saúde Universidade Federal de Brasília Instituto Oswaldo Cruz, Salvador-BA Hospital AC Camargo-SP Hospital Geral de Fortaleza-CE Hospital do Câncer, Barretos-SP Hospital das Clínicas- FMRP-USP Hospital das Clínicas- FMRP-USP Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil 92

93 Supervisor Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Student Maria Rita Fernandes Marchado Francisco Solano Feitosa Antonio Augusto Coppi Maciel Ribeiro Cesar Augusto Water Francisco de Sales Rezende Carvalho Fernando Antônio Ferreira Adelmar A.Amorim Junior Miguel Ferreira Cavalcante Filho Rosa Helena dos Santos Ferraz Carlos Rosemberg Luis Rosana Marques e Silva Luciana Silveira Flores Schoenau Williams Gomes Neves Marilyne José Afonso A. Lins Amorim Maria Amélia Zogno Tatiana Carlesso dos Santos Moacir Franco de Oliveira Year of concludion Actual position Institute Country Assistant Professor Professor of Veterinary Surgery Livre Docente Professor in Animal Anatomy Professor Doctor in Animal Reproduction Professor Doctor in Equine Surgery Professor in Veterinary Clinical and Surgery Professor Doctor in Anatomy Professor Doctor in Animal Anatomy Professor Doctor in Animal Anatomy Professor Doctor in Animal Anatomy Professor in Animal Anatomy Professor Doctor in Animal Anatomy Professor Doctor in Animal Anatomy Professor Doctor in Animal Anatomy Technician Graduate Level Professor Doctor in Animal Anatomy Professor Doctor in Animal Anatomy and assistant Head of the Rector Departamento de Morfologia e Fisiologia, FCAV/UNESP Jaboticabal Universidade Federal do Piauí FMVZ/USP Universidade Estadual do Ceará Universidade Federal de Uberlandia Universidade Federal de Uberlandia Universidade Federal do Recife Universidade Federal do Piaui Universidade Federal do Mato Grosso Universidade Federal do Goias Universidade de Brasilia Universidade Federal de Santa Maria - RS Universidade Federal do Piaui Universidade Federal do Pernambuco FMVZ/USP Universidade Estadual de Maringá UFERSA Universidade do Semi-árido Nordestino Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil 93

94 Supervisor Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Maria Angelica Miglino Student Flávia Thomaz Verechia Pereira Carlos Eduardo Ambrosio Antonio Chaves de Assis Neto Lourivaldo Paz Landim Daniele dos Santos Martins Year of concludion Actual position Institute Country Professor Doctor in Histology Assistance Professor in Animal Anatomy Professor Doctor in Animal Anatomy 2005 Technician 2006 Erika Branco 2007 Researcher Professor Doctor in Animal Anatomy UNESP Dracena FMVZ/ USP UNESP/Dracena Empresa de Biotecnologia da Reprodução, BIOEMBRYO - Fertilização in vitro. Instituto Butantan Universidade Federal Rural da Amazonia Brazil Brazil Brazil Brazil Brazil Brazil 94

95 E) Detailed Description of the Actions for the Transfer of Knowledge to Society Science is a powerful enterprise that can substantially improve the life of people. Scientists have had and will continue to have a fundamental participation in the development of new medications and treatments that affect millions of persons and of new techniques that have improved the quality of life, such as the modern systems of communication and the computers, as well as the development of new technologies that will permit the sustainable development of civilization on this planet of limited resources. All of these aspects are and will continue to be fundamental for the survival of man, so that the development of an ample scientific culture is indispensable in order to guarantee a better future for the next generations. The maintenance and growth of this scientific culture requires training not only of professional scientists, but also the scientific education of a wide segment of society, including engineers, journalists, lawyers, health professionals, and mainly science teachers who, together with their students at the elementary and high school level, are at the basis of this immense system. In addition, scientific reasoning provides individuals with a secure basis for the development of language, of logic and of the ability to solve problems in the most varied and extensive fields of human activity, causing scientific education to be a necessary endeavor in order to face the new requirements of the 21st century. The objective of the present project is to reduce tne time between the production of knowledge and the acquisition of knowledge by society. In today s world, various spaces can be used for informal education, science education, and the dissemination of the knowledge produced. The various professionals involved in the INC&T intend to consolidate within a period of 36 months a program of informal science education with an ample capacity to train human resources (HR). During the first 12 months, the authors of the scientific projects will contribute topics of interest to society or to an information bank for the purpose of science education. The experiences originating from projects of informal education and dissemination of the knowledge of the team will form another data bank, which will be incorporated into the first. Between the 13 th and 24 th month, individuals able to produce materials, products or other mechanisms for the dissemination of knowledge on the basis of catalogued experiences associated with the available 95

96 knowledge will be identified among the HR of the IN. A lato and stricto sensu Postgraduate course in the dissemination of knowledge and in informal scientific education will be implemented, with 70% of the course load (CL) offered at a distance (distance education, DE) and 30% in the presence of the students. The latter CL will occur in spaces of scientific dissemination of the partner institutions or of the community and the CL at a distance will be structured in an ADE Program monitored by the Moodle Platform. Inviduals with the ability to disseminate science will be identified among the specialists and assigned to a group that will start a class of professionalizing master s education. Between the 24 th and 36 th month, the IN will assess the activities of informal education with emphasis on visits to museums in order to make available the validated experience to society. The research of the Master s students will have to focus on scientific topics of social interest for the dissemination of knowledge or for the production of teaching material, publications in the digital media or in print, and science kits among other possibilities. During the last six months the partners will assess the various phases of the process of construction of the group of specialists and master s graduates on the basis of group experience, in order to identify in each HR the ability to include young people in the actions and to create products and competence for itinerant education, among others. The experiences of the proposing team involve many ongoing projects, the actions of five museums or science spaces, as well as the participation of the investigators in numerous regional, national and international experiences of HR training and science education, which will give sustainability to this proposal. This project of dissemination of knowledge will be the responsibility of the following five partners: Casa da Ciência (House of Science) FMRP/USP, Interdisciplinary Dynamic Museum (MUDI)/UEM, Museum of Human Anatomy (MAH)/UnB, Museum of Comparative Anatomy Professor Plínio Matos Pessoa (MAC)/FMVZ/USP, and Interdisciplinary Science Museum (MIC)/UNIPAR. Their current activities permit the citizens to spend time in research environments in a ludic and interactive manner. The House of Science carries out educational projects with laboratory activities in schools, science fairs, in research groups and in courses for teachers; it publishes a science bulletin and is involved in the the innovative project called Talent Hunt. The collection of MAV FMVZ dates back to 1940 and the museum was opened to visitors in 1984, currently exhibiting the 96

97 largest comparative anatomy collection in Brazil, which is included in projects of social inclusion, itinerant activities, and science education. The MAH was founded in the 1970 s and was opened to visitors in the 1980 s. It is part of the tourist itinerary of the federal capital and has maintained since then an ample project of continued action in science education and in the support of science fairs in Brasília and surrounding area. The MUDI has 23 yars of experience in the training of human resources (elementary and high school teachers) and its team is currently involved in more than 30 projects of dissemination, participating in interactive exhibits and science fairs, with extensive experience in itinerant activities. The patrimony of the four museums involved has a great potential for the dissemination of knowledge and for scientific initiation. GENERAL OBJECTIVES To establish an information network among researchers that will permit them and the team of scientific dissemination of INC&T to discuss what part of research would be interesting to disseminate and when and how. To set up a well-qualified program of HR training in order to disseminate knowledge. 97

98 SPECIFIC OBJECTIVES To produce a data and/or scientific information bank covering various topics linked to the projects of the National C&t Institute for purposes of informal science education focusing on Brazilian children and adolescents and the population in general. To identify among peers initiatives already existing for the dissemination of information and to catalogue them by topic, type and specialty in order to use them during the execution of the mission of the Institute. To encourage young investigators to participate in projects or initiatives of dissemination of knowledge involving high school and university students. To identify human resources with the skill of translating the scientific language in order to disseminate and popularize the knowledge produced. To train human resources in postgraduate courses of Distance Education (DE) using the Moodle platform. To prepare teams with competence to receive scientific information and to identify ways of organizing it for dissemination, with emphasis on elementary education according to the mission of CAPES. To train HR with the skill and competence needed to elaborate different modalities (actions) of dissemination of knowledge based on trans-disciplinarity and on the educational level of the target public to be reached. To format actions of dissemination that will reach large groups of people. To have itinerancy as the guiding principle of the actions of dissemination and popularization of the knowledge produced. To select international, national and regional events for the dissemination of the knowledge produced such as National C&T Week, National Museum Week, IES Extension Weeks, Science Fairs, Agriculture and Animal Husbandry Fairs etc. The project of dissemination of knowledge and science education should reach the goals established within three years as follows: Goal 1: duration, 12 months 98

99 Stage 1: To organize a data bank compiled by the investigators of IN and of the partner museums in order to disseminate the knowledge produced in an interdisciplinary manner. Stage 2: To identify among partners the experiences in dissemination in which they participated, their forms and their results in order to set up a data bank consisting of the experiences of the group, with replication of viable procedures at the national level. Goal 2: duration, 18 months Stage 1: To produce material for dissemination generated from scientific data banks and from experiences of dissemination, catalogued in Stages 1 and 2 of Goal 1, with the addition of current literature for the construction of dissemination materials obtained in this phase. E.g.: science kits, sites, informative posters, newspaper and magazine articles, interviews, identification of lecturers, courses, science workshops, thematic exhibits, CDs, DVDs, films etc. Stage 2: To implement lato and stricto sensu postgraduate courses: specialization in the Dissemination and Popularization of Knowledge, with the following characteristics: 18 months duration, hour load of 360 hours, 24 credits (1 credit = 15 hours of programmed activities; conclusion of the course in the form of activity of scientific dissemination). Master s degree: 24 months duration; hour load of 450 hours; 30 credits and a thesis presented in the form of a method, process or product of dissemination of knowledge, which will correspond to an hour load equivalent to 15 credits. The courses will involve activities at a distance (70%) and in the presence of students and teachers (30%). Goal 3: 24 months duration Stage 1: 24 months duration To give continuity to the Interinstitutional Postgraduate course Stricto Sensu (professionalizing Master s degree) in scientific dissemination. A seminar of scientific orientation will be held during this phase for definition and discussionof the research topics between advisers and students. Stage 2: 6 months duration 99

100 To analyze the information of the data bank as a whole in order to format actions of scientific dissemination and popularization of knowledge. To identify HR with the skill and competence for the transfer of knowledge, production of materials for dissemination; ability to analyze the scientific discourse for communication with the lay public; setting up interactive exhibits, fairs and events; ability to produce scientific illustration, in addition to didactic and informative texts. Stage 3: 6 months duration To prepare a workshop in order to evaluate the activities of dissemination of knowledge catalogued in the information and research data bank. To make available to postgraduate students and recipients of fellowships the literature acquired in the project, permitting them to mix concepts having interdisciplinarity as a guide of the work for the conclusion of the course in formatting to be presented at the workshop. 100

101 F) Actions detailing for transfer of knowledge for the Business Sector or for the Public Policy formation In the past decades the global economy has fundamentally changed their characteristics. Previously based mainly on land, labor and capital, the global economy is based on current knowledge. The production and modern innovation systems have as its main component the information and knowledge, reflecting a society that has change deeply under the impact of the new technologies and it has been properly called the Knowledge Society. In this new Knowledge Society the public and private limits, between science and technology and between University and Industry are gradually diminishing, causing a system overlapped that did not exist before. In this scenario, it has appeared a new organizational context in which industry, government and academia tend to integrate their interests and goals. This new integrated environment University-Industry-Government was considered as the double-helix of DNA, a triple-helix in development (Etzkowitz, 1996; 2000; Gebhardt, Pohlmann et al., 2004) The CTC s proposal for transferring technology and innovation is located in this new context. We want to catalyze the development of a triple-helix which the academic and research interests are fully integrated to the objectives of government and the industry in line with modern times. The transfer of technology for government or business sector is one of the priorities of INCTC. The groups involved in this proposal have experience in this area and the prospect is that these activities can be expanded. The CTC-FAPESP supported the development of several projects which resulted in effective incorporation of technology by the public sector. Two examples illustrate this point: a) - the development of a system for irradiation of blood and blood components based on units of teletherapy installed in the hospital network(chen, Covas et al., 2001; Goes, Covas et al., 2004; Goes, Borges et al., 2006; Goes, Ottoboni et al., 2008). The knowledge generated was made available for the entire network of services and blood bank linked to the unified health system (SUS). 101

102 b) - the development of a superconducting susceptometre (SQUID) for noninvasive quantification of liver iron (Carneiro, 2003; Carneiro, Vilela et al., 2004; Carneiro, Fernandes et al., 2005). A prototype of this equipment was built. It is operating in the laboratory of medical physics at the Faculty of Philosophy, Science and Letters of Ribeirao Preto - USP, and soon, new equipment will be installed in the clinic dependencies of the Hemocentro of Ribeirao Preto. Another important process and that will be expanded in INCTC; it is the development of systems for heterologous proteins expression in human cells. The dominant practice in the biotechnology industry is the production of recombinant proteins in prokariotic systems or in non-human mammalian cells (CHO, BHK, etc.). However, the proteins produced by these systems generally differ from the native protein in terms of glycosylation and even in folding, although these proteins have the same primary sequence. In an original and successful way, we developed a system for efficient production of clotting factors VIII and IX of in human cell lines. In the developed system, the factor VIII production, for example, was 15 to 20 times more efficient than the production in the CHO conventional system which will allow a large gain in efficiency and economy. The developed system has been patented through the Agency for Innovation USP. The scale-up process to the industrial level has been done with funding by FINEP (Process 0927/07) and in partnership with the IPT (Technology Institute of São Paulo) and with Plantarium which is a private company. More recently, given the diversity of the group, we started a study in Pirassununga to change the expression vector with a specific promoter (b-lacto globulin) to use it in epithelial cells of bovine mammary gland. The cells expressing the protein of interest will serve as the nucleus donor for the animal production carrying this change. In the sequence we want to develop other protein expression processes of medical and biological interest, such as the clotting factor VII, growth factors such as VEGF and cellular IGF, recombinant viral proteins for the generation of diagnostic tests or to use it as antigens. Our competence in this area can be proven by publications related to the theme (Fontes, Davis et al., 2006; Penteado, Medeiros et al., 2006; Picanco, Heinz et al., 2007; Russo-Carbolante, Penteado et al., 2007; Picanco-Castro, Fontes et al., 2008; Picanco-Castro, Russo-Carbolante et al., 2008) 102

103 The development of modified human cells will allow the large-scale production of recombinant proteins and also it will allow the development of cell therapy. As described in the subproject 22, for example, want to test the treatment of hemophilic B mice through the liver transplant of modified stellate (star-shaped) cells with the gene for clotting factor IX. The development of these technologies is currently done in the research labs of the CTC-Hemocentro. It was planned a construction of a new building of 5,000 m2 (Laboratory of Biotechnology) specifically for this purpose and with allocated resources from the Health Department of the State of São Paulo. Another subproject related to the development of biotechnological processes is the scale-up production of stem-cells or progenitor cells in bioreactors. The goal is to amplify the number of cells, without cell differentiation, with the purpose of providing cells in sufficient numbers to the pre-clinical studies and clinical trials. The subprojects 2 and 23 are supposed to develop this technology for the cultivation of MSC and ESC respectively. These culture systems need to be developed under GMP conditions (good manufacturing practices). In this particular project, the institution CTC (Hemocentro de Ribeirão Preto) has a national prominence position. It was the first place in Brazil to develop a quality system to hemotherapy services based on the ISO standard. Hemocentro received the first national certification in this area and the developed model is considered as international standard. All cell therapy services done by Hemocentro are certified by NBR ISO 9001:2000. This ensures an excellence standard similar or even greater than those in many European or North American services. Moreover, the Hemocentro services are also accredited by the American Association of Blood Banks (AABB) since This whole experience in managing the quality of products and services was used in the implementation of structures for the stem cell culture and to make it available for cell therapy: the bank of umbilical cord blood and human stem cells, the cryobiology sector and the human cell culture laboratories for therapeutic use comprising four clean culture rooms equipped with HEPA filters and operating in GMP conditions since This structure has allowed the realization of important clinical work as one performed by Voltarelli et al. whose results were published in JAMA (Voltarelli, Couri et al., 2007). 103

104 Other clinical studies of phase I are in progress, including the treatment of DECHA post-transplant of bone marrow with mesenchymal stem cells. At the moment this study has included 13 patients and the treatment of one type I diabetes patients using the same type of cells. The next step in this project is to obtain the AABB accreditation for cell therapy with stem cells, it should happen next year. All this developed technology (cell culture methods in GMP conditions, quality control of products and processes, standard operating procedures, qualification systems for products and suppliers, specific rules for cell therapy that did not exist in this country before) are available to be transferred for both public and private sector. Moreover, the incubator companies linked to CTC-Hemocentro de Ribeirão Preto intend to develop the next set of projects that will integrate the transfer program of INCTC: Programs and Projects of the Business Incubator SUPERA will execute its actions and hold its events using of structured programs that are already working or that will be set up. The entire program content and the stages of execution have already been defined for all programs. The projects are briefly summarized below. a) Consultant ship and Capacitating Program: It will habilitate the businessmen linked to SUPERA, permitting them to acquire knowledge in the business area. It will support partnerships and will advise in the elaboration of proposals for obtaining funds from financing agencies. The practices of this action line involve: Guidance for the elaboration of Business Plans; Analysis and Discussion of Business Plans; Selection of Enterprises and/or companies and the signing of contracts; Monitoring of companies and of entrepreneurs who are in the preincubation phase by means of a monthly report and of an annual auditing; Provision of specific consultant ships for the incubated companies covering all the areas of management and of technological development, financial management, marketing management, juridical consultant ship and norms; Periodic Updating of Business 104

105 plans in the companies and elaboration of Business Plans by the entrepreneurs attended in the Project hotel modality; Qualification of businessmen and entrepreneurs by training for the development of business competence based on administrative and technological knowledge; To subsidize the participation of the members of the business incubator in business fairs with funds obtained from SEBRAE and FINEP; Support for a successful move of resident businesses into the market based on judicious evaluation of the Development Report and of the Business Plans; Permanent qualification of the managers of the incubator; Participation of the incubator in the Technological Park Project of Ribeirão Preto also with the objective of supporting the creation of an environment favorable to the permanence in the municipality of the businesses of technological basis that graduate from SUPERA. b) INCPAR Program Incubators of Technological Basis in Partnership for the Development of New Business: this is a project aiming at the prospection of new projects and at the qualification of the companies incubated. This project, which is financed by FINEP, is a partnership of 5 incubators with a technological basis in the state of São Paulo: SUPERA of Ribeirão Preto (coordinator of the project), CIE of São José do Rio Preto, INAGRO of Jaboticabal, IEJ of Jaboticabal, and the Technological Incubator of Botucatu. The project acts on two main fronts: In the prospection of research projects or of technologies with a market potential together with universities. In this respect, the presence of USP in Ribeirão Preto and of UNESP in Botucatu, Jaboticabal and São José do Rio Preto should be pointed out, in addition to several private universities. These public universities have in common research linked to biotechnology and health. The focus of this prospection will be in the areas of concentration of the investigations developed by these universities which, along general lines, are: Agronomy, Biological Sciences, Medical Equipment, Human Health, Animal Health, and Information Technology. Although these areas are a priority, other areas can also participate in the IncPAR. In actions aiming at the growth and strengthening of the businesses incubated, such as: the offer of qualification courses and of specialized services directed at increased competitiveness of these businesses and at the expansion of 105

106 their markets, and financial support for the participation in business fairs (Site c) National Program of support to the qualification and prospection of new biotechnology projects (Competition for the BioBusiness Plan): BioBusiness Brazil is a competition of business plans directed at the creation of companies in biotechnology and Health. SUPERA supports the participants in the elaboration of Business Plans and gives an award to the best. The 1st BioBusiness SUPERA involved 40 projects, the 2nd BioBusiness Brazil received support from nine technological incubators and involved 50 projects (from 8 different States) and received an award from ANPROTEC as the Best Project for the Promotion of Culture of Innovative Enterprise of The two editions involved a total of 94 projects registered for SUPERA and resulted in two incubated companies. The 3 rd edition will be launched in October of 2008 during the Biolatina and its goal will be the prospection of 100 projects (Site d) Program of support to the dissemination of Enterprising Culture on the Ribeirão Preto Campus of USP. The objective of these actions is to encourage the creation of new business by the transformation of the scientific knowledge generated in the university into new products, with the simultaneous generation of a working alternative for postgraduate students in the technological areas, and to expand in a sustainable manner the number of new projects in the selection process of SUPERA. These actions will have an effect on a medium-term basis and aim at the possible future expansion of the incubator by the transfer of companies to the Technological Park of Ribeirão Preto. The main actions are: To work with the postgraduate program of FMRP in order to offer the discipline Innovation and Intellectual Property on as semestral basis developed in partnership with FEA-RP, SUPERA and the Innovation Agency of USP; To replicate the discipline created by SUPERA in partnership with the Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, more specifically with the Department of Chemistry, entitled Technological Enterprise. This discipline was taught in the first semester of 2008 in the courses for the Baccalaureate in Chemistry, Forensic Chemistry and Habilitation in Technological Chemistry, Biotechnology and Agroindustry. The objective of the discipline is to present to the student the enterprising and innovative environment of the Ribeirão Preto USP 106

107 Campus, to encourage the transfer of the technologies produced at the university, and to train the students in the basic tools needed to plan and set up in practice a company based on technology in order to expand the number of new projects in the selection processes of SUPERA. e) National Program for the generation of new business (BRBiotec Brazilian Network of Biotechnology Companies). The general objective of BrBiotec is to act as a relationship network and to operate as a physical and virtual business location that will be located in strategic places in the country. It will promote the integration among Brazilian biotechology companies, investors, education and research institutions, investigators and the federal, state and municipal government. This network has already received funds of R$ 400 thousand from the Ministry of Health and is being executed by the incubators SUPERA, CIETEC and BIORIO, which are articulating a partnership with NISP NATIONAL INDUSTRIAL SYMBIOSIS PROGRAM OF THE UNITED KINGDOM, in order to exchange experience and the sustainable business development by transforming low value resources into added value which result most of the time in innovative products. f) First Enterprising Business Program (PRIME): SUPERA was one of the 18 incubators chosen by FINEP to act as a decentralized operator of this institution in the PRIME Program. The program intends to invest R$ 12 million in incipient businesses based on technology. The project of the company contemplated by the Prime will be supported by two granting modalities operated by Finep. The total financing amount will be of the order of R$ 240 thousand per company. These funds will be liberated over a period of two years, with the first parcel of R$ 120 thousand coming from the Program of Economic Support of Innovation. In this modality, the funds are not reimbursable. The second and last parcel will use money of the Zero Interest Program, which involves the return of the loan in twenty payments with no interest. The first R$ 120 thousand parcel will be disbursed in the form of non-reimbursable economic subvention and therefore will be tax-free. These funds can be used to hire technicians, administrators and consultants. SUPERA will publish the edict for the selection of businesses interested in receiving the subvention funds in November

108 G) Detailed Description of the Proposing Group The present proposal for constitution of the INCTC is a joint initiative of 112 professionals and students (researchers, postgraduate students, post-docs and research technicians) organized in eleven research groups represented by their respective coordinators. Figure 5 depicts this professional and academic universe. The Project will be coordinated by Prof. Dr. Roberto Passeto Falcão, CNPq researcher level 1A and full professor at University of São Paulo, who has the following qualifications: graduation in School Medicine at University of São Paulo, Ribeirão Preto (1966), doctorate in Medical Clinic at University of São Paulo, Ribeirão Preto (1972), post-doctorate at Oxford University ( ). Currently, Dr. Falcão is associate professor of the Faculty of Medicine, University of São Paulo, Ribeirão Preto. He has experience in Medicine area, with emphasis on Medical Clinic and Hematology and Immunology, and he is involved mainly in the following subjects: aplastic anemia, lymphocytes, myeloid and lymphoproliferative diseases. Outstanding points of his CV: 1. Vice-coordinator of Project CEPID-FAPESP Center for Research on Cell-Based Therapy, it started in 2000, with a budget of US$ Head of Medical Clinic Departament of Medicine Faculty of Ribeirão Preto from 1997 to This department is the largest at FMRP, and includes 60 professors. Dr. Falcão is one of the leaders in scientific production of this unit at USP. 3. President of Brazilian Hematology School from 1998 to Coordinator of Hematology sector of Clinical Medicine Departament of FMRP, and of Hematology Lab of Clinical Hospital of Ribeirão Preto since This division has been responsible for the education of approximately 80 residents and numerous professionals that currently develop professional or academic activities all around Brazil or abroad. 5. Founder (1993) and Coordinator of Brazilian Hematology School, it is an organization that offers continuing education courses in the field of Hematology. Twenty-one courses spanning different areas of Hematology have been offered. A partnership with the European School of Hematology was 108

109 established in 2000, and since then it has allowed lecturers visitations and the use of didactic material from that organization in tutorial courses. 6. Dr. Falcão has published 127 scientific articles listed in Lattes curriculum system, 100 are listed in PubMed (falcão rp; passetto falcão r; passeto falcão r; falcão r). He has been cited 666 times, and his h index is 14. His activities as advisor have resulted in 5 Master's dissertations and 9 PhD theses. He has also been the co-advisor in 3 Master's dissertations and 2 PhD theses. Currently, Dr. Falcão supervises one post-doc, one PhD student, and two Master s students. He has presented 181 communications in meetings with published abstracts. He has been granted 10 awards related to these works. 7. He has written 19 book chapters, and he is the editor, together with Marco Antonio Zago and Ricardo Pasquini, of the book Hematologia e Fundamentos e prática médica 1/1. ed. São Paulo: Atheneu, v which involves 50 collaborators. 8. Representative of SBPC at Conselho Nacional de Saúde 9. Member of the editorial board of the Periodical Brazilian Medic Association, Einstein, Journal of Brazilian Hematology and Hemotherapy, and Hematology/Oncology and Stem Cell Therapy The following principal researchers also take part in this proposal: 2. Maria Angélica Miglino - researcher level 1A of CNPq. Maria Angélica Miglino has received her PhD in Sciences (funcional anatomy: structure and ultra-structure) from Universidade de São Paulo in Currently, Dr. Miglino is a full professor at University of São Paulo. She has published 294 articles in specialized periodicals and 366 works in meeting proceedings. She has published 5 book chapters and 2 books. She has 1 technique process and other 10 technical production items. Dr. Miglino has participated in 23 meetings abroad and 59 in Brazil. Her activities as advisor resulted in 23 Master's dissertations, 27 PhD theses. She has been a coadvisor in 1 Master's dissertation, and in 2 PhD theses. In addition, she has advised scientific initiation works in the Veterinary Medicine and Morphology fields. She has been granted 8 awards and/or honors. She has coordinated 36 research projects 109

110 between 1993 and Currently, she is engaged in 24 research projects and coordinates 22 of these. Dr. Miglino develops activities in the Veterinary Medicine, with emphasis on animal reproduction. During her professional activities, she has interacted with 941 collaborators in co-authorships of scientific works. The most frequent terms used to contextualize her scientific, technological and artisticcultural in her Curriculum Lattes are: placenta, anatomy, morphology, bovines, study, ruminants, dogs, arterial, plaque, and vascularization Lewis Joel Greene researcher level 1A of CNPq. He is full professor at Medicine Faculty of Ribeirão Preto, University of São Paulo, in the Department Of Celluar Biology, Molecular and Pathogenic Bioagents. Dr. Greene is the Supervisor of Center of Chemic Proteins at Blood Bank of Ribeirão Preto, where he develops studies concerning the chemical, functional and structural characterization of proteins, using traditional protein chemistry approaches and proteomic analysis. The research lines developed by him comprise: a) Studies on the functional and structural characterization of proteins during the proliferation and cellular differentiation of hematopoietic progenitor cells and human tumor cells, using a proteomic approach; and b) Chemical and biological characterization of biologically active proteins using traditional protein chemistry methods. Dr. Greene has published 112 articles in specialized periodicals, 243 works in meeting proceedings including complete studies and abstracts, and 3 book chapters. His activities as advisor have resulted in 20 Master s dissertations and 21 PhD theses. In addition, he has advised 2 scientific initiation works Lygia V. Pereira, Ph.D., obtained her Bachelor of Physics from Pontifícia Catholic University of Rio de Janeiro in She received her Master s degree in Biological Sciences (Biophysics) from Federal University of Rio de Janeiro in 1990, and her Ph.D. in Biomedical Sciences from the Mount Sinai School of Medicine, City University of New York in Currently, she is Associate Professor MS5 (livre docente) at University of São Paulo, member of Science Academy of São Paulo State, and she is a head of Molecular Genetic Lab of Bioscience Institute, USP. She has experience in the field of Genetics, with emphasis in Human Molecular 110

111 Genetics, and she is involved in the following subjects: animal models of genetic disease, embryonic stem cells, epigenetic inheritance, and X chromosome inactivation. She is author of two scientific divulgation books: Seqüenciaram o Genoma Humano... E Agora? and Clonagem: da Ovelha Dolly às Células-Tronco, Editora Moderna. Dr. Pereira has published 39 articles in specialized periodicals, 30 works in meeting proceedings including complete studies and abstracts, 7 book chapters and 4 books. She has published 35 articles in magazines/newspapers. Her activities as advisor resulted in 4 Master s dissertations and 8 PhD theses. She has received 9 awards and/or honors Stevens Kastrup Rehen CNPq researcher level 2. He is a Biologist (UFRJ, 1994), Master of Biological Sciences (UFRJ, 1996), and Ph.D. in Sciences (UFRJ, 2000), and he did a post-doc at University of California at San Diego, USA ( ). He is an Associate Researcher at The Scripps Research Institute (California, USA) since 2005, Fellow of Pew Foundation, and Adjunct Professor at UFRJ. Dr. Rehen works with basic research. He investigates the mechanisms involved in the neural differentiation of embryonic stem cells and induced pluripotent stem cells, and also study the culture of these cells in large-scale. He has published 23 scientific articles in indexed scientific journals, 11 of these produced exclusively in Brazil, in addition to 8 book chapters, 16 scientific divulgation articles, 11 comments on science in digital magazines, and 1 scientific divulgation book. Dr. Rehen leads Laboratório de Neurogênese e Diferenciação Celular of Instituto de Ciências Biomédicas at UFRJ. He has advised 8 scientific initiation students, and two Master s students. Currently, he supervises 1 researcher (post-doc), 6 PhD students, 3 Master s students and 5 scientific initiation students. In addition, Dr. Rehen has acted as reviewer for 7 international periodicals, and is Affiliate Member of Brazilian Science Academy. More details can be found at: 6. Wilson Araújo da Silva Jr. - CNPq researcher level 1D. He graduated in Biology, Medical Modality (Biomedicine) at Pará Federal University 1989, and obtained his Master's degree in Genetics from Medicine Faculty of Ribeirão Preto, University of São Paulo in He received his Ph.D. in Genetics from Medicine Faculty of 111

112 Ribeirão Preto, University of São Paulo, in Dr. da Silva did a post-doc in Cancer Genetics at Ludwig Institute, Memorial Sloan-Kattering Cancer Center in New York City, NY, USA. He is Associate Professor of Genetic Department of Medicine Faculty, Ribeirão Preto USP. He develops his research in the field of Human and Medical Genetics, working mainly with the following subjects: DNA polymorphisms, Bioinformatics, and Cancer Genetics. Dr. da Silva has published 79 articles in specialized periodicals and 80 works in meeting proceedings including complete studies and abstracts. His activities as advisor have resulted in 9 Master s dissertations, 6 PhD theses, and he has advised 3 scientific initiation students. He has received 5 awards and/or honors. 7.Eduardo Magalhães Rego CNPq researcher level 2. He graduated in Medicine at Medicine Faculty of Ribeirão Preto, University of São Paulo, in He obtained his doctoral degree in Medical Clinic from the same institution (1997), and he did a post-doc at Memorial Sloan Kettering Cancer Center in New York, USA. Currently, Dr. Rego is Associate Professor at University of São Paulo, and he works in the field of Hematology. His research line focuses on acute leukemias, with special emphasis on Acute Promyelocytic Leukemia. He has published studies that demonstrate that the hybrid genes PML/RARa, PLZF/RARa, NPM/RARa and NuMA/RARa are able to induce leukemia in transgenic mice. These transgenic models have been used for the study of the leukemia molecular bases and for research on new therapeutic approaches. Dr. Rego also studies the relationship between ribosome function and cancer by analyzing mice that carry the mutations in the gene DKC1. His most important works are: 1. Yoon A, Peng G, Brandenburger Y, Zollo O, Xu W, Rego E, Ruggero D. Impaired control of IRES-mediated translation in X-linked dyskeratosis congenita. Science May 12;312(5775): Rego EM, Ruggero D, Tribioli C, Cattoretti G, Kogan S, Redner RL, Pandolfi PP. Leukemia with distinct phenotypes in transgenic mice expressing PML/RAR alpha, PLZF/RAR alpha or NPM/RAR alpha. Oncogene Mar 23;25(13): Rego EM, Wang ZG, Peruzzi D, He LZ, Cordon-Cardo C, Pandolfi PP. Role of promyelocytic leukemia (PML) protein in tumor suppression.j Exp Med Feb 19;193(4): Rego EM, He LZ, Warrell RP Jr, Wang ZG, Pandolfi PP. Retinoic acid (RA) and As2O3 treatment in transgenic models of acute promyelocytic leukemia (APL) unravel the distinct nature of the leukemogenic process induced by the PML-RARalpha and 112

113 PLZF-RARalpha oncoproteins. Proc Natl Acad Sci U S A Aug 29;97(18): Dr. Rego published 50 articles in specialized periodicals, 162 works in meeting proceedings including complete studies and abstracts, and 10 books. He has participated in 91 meetings in Brazil and abroad. His activities as advisor resulted in 5 Master's dissertations and 4 PhD theses. In addition, Dr. Rego has advised 4 scientific initiation works, and has received 10 awards and/or honors Júlio Cesar Voltarelli CNPq researcher level 2. He graduated in Medicine at Medicine Faculty of Ribeirão Preto, University of São Paulo (FMRP-USP) ( ). He completed his residency in Medical Clinic and Hematology at Clinical Hospital of FMRP-USP ( ), and obtained his Master s ( ) and doctoral ( ) degrees in Medical Clinic from FMRP-USP. Dr. Voltarelli engaged in post-doctoral activities in the following institutions: University of California at San Francisco, USA ( ), Fred Hutchinson Cancer Research Center in Seattle, USA ( ), and Scripps Research Institute in San Diego, USA ( ). Currently, he is full Professor of Clinical Medicine of FMRP-USP, and Coordinator of Divisão de Imunologia Clínica, Laboratório de Imunogenética (HLA), and Unidade de Transplante de Medula Óssea of HC-FMRP-USP. He is a researcher of Centro de Terapia Celular (CEPID-FAPESP), hosted at Centro Regional de Hemoterapia of HC-FMRP-USP. Dr. Voltarelli has published 99 articles in specialized periodicals, 41 works in meeting proceedings including complete studies and abstracts, and 13 book chapters. His activities as advisor have resulted in 13 Master's dissertations and 4 PhD theses. He has received 6 awards and/or honors. He has experience in the field of Clinical Immunology and Hematology, with emphasis on: hematopoietic stem cell transplantation for autoimmune diseases and malignant and benign hematological disorders; diagnosis and treatment of secondary immunedeficiencies and rheumatic disorders; selection of donors for renal and bone marrow transplantations Flávio Vieira Meirelles researcher level 2 of CNPq. He obtained his Doctor of Veterinary Science degree from Universidade Estadual Paulista Júlio de Mesquita Filho (1993), and his Master s degree in Sciences Vétérinaires Option Reproduction from Université de Montréal (1997). He received his Ph.D. in Genetics from 113

114 Universidade de São Paulo (1999). Currently, he is Associate Professor ms5 (livre docente) at Universidade de São Paulo; ad hoc consultant for Fundação de Amparo à Pesquisa do Estado de São Paulo, associate editor of Genetics and Molecular Research, Sociedade Brasileira de Tecnologia de Embriões, and consultant for the journals Heredity, Animal Genetics, and Genetics and Molecular Biology. Dr. Meirelles has experience in the field of Genetics, with emphasis on Cytoplasmic Inheritance, and is involved mainly with the following subjects: mtdna, Bos indicus, cattle, embryos and apoptosis Dimas Tadeu Covas CNPq researcher level 1D. He graduated in Medicine at Universidade de São Paulo (USP) in He received his Master s (1986) and doctoral (1993) degrees from USP, and he obtained livre-docência from the same University in Currently, Dr. Covas is Associate Professor of Faculdade de Medicina de Ribeirão Preto/USP, President-Director of Fundação Hemocentro de Ribeirão Preto, and Coordinator of Transference of Centro de Terapia Celular (CTC- CEPID-FAPESP). He is a hematologist and hemotherapist, and he develops research on the following subjects: molecular and cellular biology, stem cells, erythrocytic and platelet antigens, viruses (HIV and HTLV), protein heterologous expression in cellular systems in vitro. He is an active member of Sociedade Brasileira de Hematologia e Hemoterapia and of Colégio Brasileiro de Hematologia, and he is currently the treasurer of this organization. He is Associate Editor of Revista Brasileira de Hematologia e Hemoterapia, and is one of the founders of Escola Brasileira de Hematologia. In addition, Dr. Covas is member of Academia de Ciências de Ribeirão Preto. He has also promoted the dissemination of science and education, and organized postgraduate programs for teachers and elementary and high school students. He has edited two books, and one of these was awarded Prêmio Jabuti in Dr. Covas has published 75 articles in specialized periodicals, 380 works in meeting proceedings including complete studies and abstracts, 12 book chapters, and 10 books. He has participated in 121 meetings in Brazil and abroad. His activities as advisor resulted in 18 Master's dissertations and 5 PhD theses. In addition, Dr. Covas has advised 2 scientific initiation works, and has received 9 awards and/or honors

115 11. Lawrence Charles Smith received his Doctor of Veterinary Medicine from Universidade Estadual Paulista Júlio de Mesquita Filho at Jaboticabal in He obtained his Master s degree from Faculty of Sciences, Université d'édimbourg in 1984, and a Ph.D. in Physiology and Genetics from Faculty of Sciences, Institute of Animal Physiology and Genetics, in Currently, he is full Professor of Faculté de médecine vétérinaire, Centre de recherche en reproduction animale. Dr. Smith works mainly with the following subjects: Nucleus-cytoplasm interactions, embryos, cloning. He has been involved in the organization of postgraduate courses for teachers and elementary and high school students. He has edited two books, and one of these was awarded Prêmio Jabuti in Dr. Smith has published 59 articles in specialized periodicals, and 13 book chapters. His activities as advisor have resulted in 9 Master s dissertations and 9 PhD theses João Palermo Neto holds a productivity scholarship level 1B from CNPq. Currently, he is full Professor at Universidade de São Paulo, and consultant of Fundação de Amparo à Pesquisa do Estado de São Paulo, Food And Agricultural Organization of The United Nations, Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro, Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul and Conselho Nacional de Desenvolvimento Científico e Tecnológico. He has experience in the field of Pharmacology, with emphasis on Neuropsychopharmacology, and his work involves mainly the following subjects: CNS, diazepam, dopamine, neuroimmunomodulation, innate immunity and stress. Dr. Palermo Neto has published 137 articles in specialized periodicals, 304 works in meeting proceedings including complete studies and abstracts, 21 book chapters and 2 books. He has participated in 67 meetings in Brazil and abroad. His activities as advisor resulted in 36 Master's dissertations and 25 PhD theses. In addition, he has advised 18 scientific initiation works, and has received 14 awards and/or honors. 115

116 Researches Pos-docs PhD students Ms students Technicians (1) Researches Pos-docs (1) PhD students Ms students Technicians (1) Stevens Kastrup Rehen Roberto Passetto Dimas Tadeu Covas Researches Pos-doc (4) PhD students Ms students (12) Technicians (1) Researches Pos-doc (2) Eduardo Magalhães Rego Technicians (1) PhD students Ms students (1) Researches Pos-doc PhD students Ms students Technicians Lygia da Veiga Pereira INCTC (PI) Lewis Joel Greene Researches Técnicos (2) Pos-doc (2) PhD students Mestrandos (5) Researches Pos-doc PhD students Flávio Meirelles Researches Maria Angélica Miglino Pos-doc PhD students Ms students Ms students Technicians Technicians Researches Pos-doc (1) PhD students Júlio César Voltarelli Researches Wilson Araújo da Silva Junior Pos-doc (1) PhD students Ms students (5) Ms students (2) Technicians Technicians 116

117 H) Activities Specification that must be done by the Team members Researchers (PI) Roberto Passetto Falcão Dimas Tadeu Covas Eduardo Magalhães Rego Lewis Joel Greene Maria Angélica Miglino Wilson Araújo da Silva Junior Júlio César Voltarelli Flávio Vieira Meirelles Lawrence CharlesSmith Lygia da Veiga Pereira Stevens Kastrup Rehen Klena Sarges Marruaz da Silva Subprojects

118 I) Mechanisms that will be used to promote the Interaction of the Research Groups The interaction between research groups will be happen in different ways beginning in the researches projects since your conceptions, multilaterally and complementarily (item H). The INCTC s Coordination will be composed of five important research centers and universities (FMVZ-SP- USP, IB-USP, UFRJ, CTC, FZEA-USP). This collaboration will guaranty the viability and development of project. Another interaction mechanism that we will use it will be meetings each tree months and annual symposium with national and international investigators. The interactions day by day can be done using INCTC website ( and also meetings by phone, Internet and/or videoconference can be done. We must emphasize that there is already ongoing collaboration between the groups, independent of the beginning of this project. The groups will also interact in education programs and knowledge transfer to society. It is expected a creation of post-graduate courses intraunits (between USP units) and inter-units (between USP, UFRJ and the National Center for Primates). In the innovation project, the Enterprise incubator linked to the CTC can provide full logistic support and knowledge for the interaction of different groups with the productive sector (for example, studies about economic viabilities, business plans, micro-company, and intellectual property). Special attention will be give to the group of the National Center for Primate which needs intensive training in techniques of cultivation and updated characterization cell. The interaction mechanisms in this particular case include periods of Belém professionals training in other laboratories of the group and also the displacement of investigators to Belém for training in loco. 118

119 J) Forms of Interaction with Foreign Groups The development of an institute of this port must have an international collaboration. Our group has already collaboration with 5 international researches that are currently developing international activities in collaboration with our team. This institute aims, through face or non-face activities (video conference), to keep a strong contact with abroad institutions. The contact with the abroad Institution will be done meetings every three months where the participants will be invite to participate by videoconference. An annual meeting will be done we will organize a Symposium where the international researches will make a short presentation about their developed activities. In the other hand, the national group will present the results by poster or oral presentation. This annual symposium will be able to make the group close and to give an idea of what have been doing in the different labs. In some cases the visit of the international collaborator can be extended to promote the development of short or medium term internships. For each request of research visitation he will receive a member of our group in your Lab to be training or to develop our project. 119

120 K) Definition of specific tasks for each entity Institution Faculdade de Medicina de Ribeirão Preto/USP Hemocentro de Ribeirão Preto Centro Regional de Hemoterapia HCFMRP/USP Faculdade de Zootecnia e Engenharia de Alimentos Members involved 31 Subprojects Coordinator s Responsible 3 25 Roberto Passetto Falcão 3, 5, 10, 11, 14, 15, 22, 23 e 30 Dr Dimas Tadeu Covas 5 8 e 9 Flavio Vieira Meirelles Instituto de Biociências/USP 5 1, 6 e 7 Lygia da Veiga Pereira Faculdade de Medicina Veterinária e Zootecnia/USP Maria Angélica Miglino Faculdade de Medicina de Ribeirão Preto/USP 6 13 e 16 Wilson Araújo da Silva Jr Instituto de Ciências Biomédicas da UFRJ 13 2 Stevens Kastrup Rehen Faculdade de Medicina de Ribeirão Preto/USP 4 19 e 20 Eduardo Magalhães Rego Faculdade de Medicina de Ribeirão Preto/USP Faculdade de Medicina 18 17, 21, 28 e 29 Júlio Cesar Voltarelli João Palermo Neto Veterinária e Zootecnia/USP Faculdade de Medicina de Ribeirão Preto/USP 12 12, 18 e 24 Lewis Joel Greene Centro Nacional de Primatas 8 4 e 27 Klena Sarges Marruaz da Silva 120

121 L) Comparative Analysis between the Current and Expected Statuses The teams involved in this proposal have a large experience in the subject chosen, as demonstrated by their publications, patent deposits, and activities aimed at diffusing knowledge to the community. The ways with which this proposal intends to improve the current status are: effectively exchange previous experience; support Centro Nacional de Primatas (National Primate Center) effectively, enabling it to became an important center for preclinical studies involving nonhuman primates; structure two stem cell banks (one for animal cells, and another for human cells); establish a Preclinical Studies Center; develop methodologies to culture embryonic and mesenchymal stem cells in large scale and under clinicalgrade conditions; develop methodologies and products that may generate patents; perform two phase I and II clinical studies; diffuse the knowledge obtained to the community; train human resources; and contribute to the improvement of public education. Many of these activities are already performed, but the differential is that their range and scale will be increased. The most significant advancement, however, will be the establishment of the Institute itself, which will connect the participant teams and provide a powerful stimulus for the development of this area in the country. 121

122 M) Justified Budget General Datadescription TotalValue(R$) ConsumableMaterials 3,899, PermanentMaterials 3,547, TravelTickets 292, Scholarships 1,007, BibliographicMaterials 2, Travelsupport 152, ,903, Consumable Materials Item Value(R$) Antibodies 96, OfficeMaterials 54, BloodCollectionMaterials 416, LaboratoryPlasticware 502, Reagents 63, ElectrophoresisReagents 450, CytogeneticsReagents 142, CellCultureReagents 1,039, Reagentsforexperimentswithanimalmodels 46, ProteomicsReagents 900, Solvents 176, LaboratoryGlassware 12, ,899,

123 Permanent Materials Item Quant Description 1 3 Refrigerated microcentrifuge 2 7 Refrigerated bench centrifuge 3 1 Refrigerated floor-standing centrifuge 4 11 Laminar-flow Hood 5 1 work station 6 1 Network server 7 2 Transfection equipments 8 2 Thermal cycler 9 1 Shaking incubator 10 4 Water bath 11 2 Freezer -20 C 12 1 Refrigerator 4 C 13 3 Horizontal electrophoresis system 14 2 Notebooks 15 2 PC 16 3 Printers 17 3 Vertical Electrophoresis system and accessories 18 1 Helical computerized tomography equipment 19 3 Magnetic stirrer for spinner flasks Spinner flasks 21 2 Bioreactors 22 1 Automated cell counter 23 1 Filtration system 24 1 MILLI-Q BIOCEL System and accessories Liquid nitrogen containers and accessories digital ph meter Automatic micropipettes and automatic repeating pipettes 28 2 Real time PCR machine 29 3 Nucleic acid quantification systems 30 2 Analytical balances 31 6 Mixers (stirrers, hotplate stirrers and vortexes) 32 1 GFP goggles 33 4 Inverted trinocular microscope 123

124 Item Quant Description 34 1 Optical microscope with polarized light with image capture system 35 1 Digital camera for inverted microscope with USB connector 36 1 Inverted microscope Axio Observer D1 with accessories 37 2 Analogical and digital video camera 38 2 Trinocular microscope 39 1 Articulated arm base for stereomicroscope 40 1 Digital camera 1.4 MB with sensitivity to detect fluorescent light 41 1 Trinocular stereomicroscope 42 1 Simple binocular microbiological microscope 43 1 Light source for GFP (Green Fluorescent Protein) excitation 44 1 Fluorescence Stereomicroscope 45 1 C-mount 1X adapter for camera/microscope 46 1 Dapi/Hoescht/Amca/HiSky filter and cubes for all types of microscope 47 1 Software Multi-Species for use with BandView 48 1 Complete workstation for cytogenetics: HiSKY, G band and FISH 49 1 Light microscope 50 1 Image acquisition system for inverted microscope 51 1 Stereomicroscope L autoclave system Flow cytometer Accessories for cell separation columns CO2 Water-Jacketed Incubator Shaker Incubator Bacteriological incubator Tri-gas incubator C Freezers Microarray Scanner Microarray Hybridization oven Rotational chamber for hybridization oven SureHyb Hybridization chamber Femtojet microinjector Femtojet Microinjetor Pedal 124

125 Item Quant Narashige Air Microinjector Piezo Drill Description Fyrite CO 2 /O Automatic gas exchange system GFP excitation light source DAKO hybridizer Photographic documentation system for agarose gels Microplate luminescence reader Cryostat Automated pipetting system for PCR reactions Multimidia projector Digital video camera LCD TV Display cases 80 5 Showcase JUSTIFICATIONS 1) Refrigerated microcentrifuge The refrigerated microcentrifuges are designed for processing of DNA and RNA samples that will be utilized in the cloning and analysis of expression of target genes. 02 equipments will be installed at Laboratório de Hematologia of Hospital das Clínicas de Ribeirão Preto USP under the responsibility of Prof. Eduardo Magalhães Rego, and 01 equipment will be installed at Laboratório de Biologia Molecular of Hemocentro de Ribeirão Preto, under the responsibility of Dr. Simone Kashima Haddad. Subprojects: 3, 7, 11, 15, 18, 19, 20, 25, 26, 27, 30. 2) 07 Refrigerated bench centrifugue The refrigerated bench centrifuges are designed for processing samples of leukemic cell lines and retroviral packing cells, and also for Ficoll density 125

126 separation of bone marrow stem cells to be used in the protocols related to transplantation in the animal models involved in this project. 01 unit will be installed at Laboratório de Hematologia of Hospital das Clínicas de Ribeirão Preto USP under the responsibility of Prof. Eduardo Magalhães Rego; 02 units will be installed at Prof. Maria Angelica Miglino s laboratory (Faculdade de Medicina Veterinária-USP); 02 units will be installed in Dr. Passeto s laboratory (Hematologia, FMRP); 01 unit will be allocated to Laboratório de Biologia Molecular of Hemocentro de Ribeirão Preto and 01 unit to Dr Julio Voltarelli s laboratory. Subprojects: 3, 4, 6, 7, 8, 9, 10, 11, 13, 15, 17, 18, 19, 20, 21, 22, 25, 26, 27, 28, 29, 30. 3) 03 Refrigerated floor-standing centrifuge This equipment is intended for processing bone marrow samples before and after retroviral transduction that will be used in the transplantation protocols involving monkeys. This equipment will be installed in the Specific Pathogen Free Animal House under the responsibility of Prof. Eduardo Magalhães Rego (Hematologia, FMRP). Subprojects: 3, 7, 11, 18, 19, 20, 25, 26, 27, 30. 4) 11 Laminar-flow Hood The laminar-flow hood is designed for the protection of the product and operator in the manipulations of biological materials and toxic substances in the fields of medicine, pharmaceutical industry and microbiology (for manipulation of non-pathogenic materials). This equipment is proper for the manipulation of cell and biological materials in aseptic conditions. 01 unit will be installed at Laboratorio de Hematologia of Hospital das Clínicas de Ribeirão Preto USP and another one in the Specific Pathogen Free Animal House, both under the responsibility of Prof. Eduardo Magalhães Rego; 01 unit will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Wilson Araújo da Silva Júnior; 01 unit will be allocated to Centro de Quimica de Proteínas under the responsibiliy of Prof. Lewis Joel Greene; 05 units to Faculdade de Medicina Veterinária- USP under the responsibility of Prof. Maria Angélica Miglino; 01 unit to 126

127 Laboratório de Hematologia under the responsibility of Prof. Roberto Passeto Falcão and one to Laboratório de Morfofisologia Molecular e Desenvolvimento under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30. 5) 1 work station This hardware avoids PCR contamination, and will be installed at Centro de Química de Proteínas of Hemocentro de Ribeirão Preto under the responsibility of Prof. Lewis Joel Greene. Subprojects: 8, 12, 17, 18, 21, 23, 24. 6) 1 Network server The network server will be installed at Hemocentro de Ribeirão Preto for better connectivity between laboratories. Subprojects: 2, 3, 4, 5, 6, 8, 10, 11, 12, 14, 15, 16, 17, 20, 21, 22, 23, 26, 27, 28, 29, 30. 7) 2 Transfection equipments This equipment enables transfection of human cells with vectors and double-stranded RNA. This equipment enables the transfection without cell content destabilization, and allows the obtainment of higher cell viabilities after transfection. 01 unit will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Wilson Araújo da Silva Júnior, and the other will be allocated to Laboratório de Morfofisologia Molecular e Desenvolvimento under the responsibility of Prof. Flavio Meirelles. Subprojects: 1, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 16, 17, 19, 20, 22, 24, 26, 27. 8) 2 Thermal cycler Used to DNA and cdna amplification. 01 unit will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Covas, and the other will be allotted to Faculdade de Medicina Veterinária-USP under 127

128 the responsibility of Prof. Maria Angélica Miglino. Subprojects: 1, 4, 5, 6, 8, 9, 11, 19, 26, 27. 9) 1 Shaking incubator The incubator will be used for growth of bacterial cultures. This equipment will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas tadeu Covas. Subproject: 5. 10) 4 Water bath This equipment will be used for thawing cryopreserved cells, warming up reagents, and incubating samples with restriction and/or modification enzymes. 02 units will be installed in the Specific Pathogen Free Animal House under the responsibility of Prof. Eduardo Magalhães Rego; 01 unit will be allotted to Faculdade de Zootecnia e Engenharia de Alimentos, USP, under the responsibility of Prof. Flávio Meirelles, and one will be allocated to Centro Nacional de Primatas Evandro Chagas under the responsibility of Prof. Klena Sarges Marruaz da Silva Sarges Marruaz da Silva. Subprojects: 1, 3, 4, 5, 6, 7, 8, 9, 11, 18, 19, 20, 25, 26, 27, ) 2 Freezer -20 C For the cryopreservation of biological samples and reagents. Both equipments will be installed at Laboratório de Hematologia, FMRP under the responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 3, 7, 11, 18, 19, 20, 25, 26, 27, ) 1 Refrigerator 4 C Used to refrigerate biological samples and reagents. This equipment will be allotted to Laboratório de Hematologia da FMRP-USP under the responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 3, 7, 11, 18, 19, 20, 25, 26, 27,

129 13) 3 Horizontal electrophoresis system Equipment used to separate nucleic acids. 01 unit will be installed at Laboratório de Biotecnologia of Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas Tadeu Covas, and 02 units will be allocated to Laboratório de Hematologia of FMRP-USP under the responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 3, 5, 7, 11, 18, 19, 20, 25, 26, 27, ) 2 Notebooks In the itinerant actions and in courses conducted outside of museums, the notebooks are fundamental to multimedia projections, performance of Physics shows, and also to organization of databases and secretary services. 01 notebook will be used by Prof. Jussara R. Ferreira at Universidade de Brasilia, and the other will be used by Prof. Eduardo Magalhaes Rego at Labotarório de Hematologia of FMRP-USP. Subprojects: 3, 7, 11, 18, 19, 20, 25, 26, 27, ) 2 PCs These equipments are fundamental to diagramming, web design, image treatment, and also to production of didactic material and databases that use images. 01 unit will be used by Prof. Jussara R. Ferreira at Universidade de Brasília, and 01 unit will be used by Prof. Eduardo Magalhães Rego at Laboratório de Hematologia of FMRP-USP. Subprojects: 3, 7, 11, 18, 19, 20, 25, 26, 27, ) 3 Printers Multifunctional printers are necessary for printing high-quality photographs, and also to supply the graphic needs of the projects. 01 unit will be installed at Laboratório de Biotecnologia of Hemocentro de Ribeirão Preto, under the responsibility of Prof. Dimas Tadeu Covas; 01 unit will be allotted to Centro Nacional de Primatas Evandro Chagas, under the responsibility of Prof. Klena Sarges Marruaz da Silva, and 01 unit will be allocated to Laboratório de Hematologia of FMRP-USP under 129

130 the responsibility of Prof. Eduardo Magalhães Rego. Subprojects: 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 26, 27, 28, 29, ) 3 Vertical Electrophoresis system and accessories This equipment is necessary to separate protein extracts through mass/charge before their transference to nitrocellulose or PVDF membranes to do Western-blots. 02 units will be used by Prof. Stevens K. Rehen at Instituto de Biociências of UFRJ, and the other will be used by Prof. Eduardo Magalhães Rego at Laboratório de Hematologia of FMRP- USP. Subprojects: 2, 3, 5, 6, 7, 11, 18, 19, 20, 23, 24, 25, 26, 27, ) 1 Helical computerized tomography equipment This equipment would be important to support the diagnosis of diseases under study in subprojects related to stem cell therapy since it provides precise three-dimensional images, which allow for the examination of overlapping areas. This equipment also detects minor tissue alterations, allowing for the early diagnosis of several alterations such as hemorrhage, tumors, injuries, lung parenchymal alterations, etc. The aforementioned equipment would be, thus, an important tool for the detection of problems and, by extension, for the success of animal treatments involving cell therapy. This equipment will be allocated to Instituto Nacional de Primatas Evandro Chagas under the responsibility of Prof. Klena Sarges Marruaz da Silva. Subprojects: 4, 6, 20, 26, ) 03 Magnetic stirrer for spinner flasks This equipment is used to stir cells cultured in suspension in spinner flasks. The spinner flasks, as well as the stirrer, should be kept in a CO2 incubator. 2 units will be allocated to Instituto de Ciências Biomédicas of Universidade Federal do Rio de Janeiro under the responsibility of Dr K. Stevens K. Rehen, and 1 unit will be allotted to Hemocentro de Ribeirao Preto under the responsibility of Dr T. Dimas T. Covas. Subprojects: 2, 5, 6, 23,

131 20) 33 Spinner flasks Spinner flasks are small-scale flasks with different volumes where the stem cells can be cultured in suspension. The spinner flasks will be placed inside a CO 2 incubator and stirred by a magnetic stirrer also placed inside the incubator. 24 flasks will be allocated to Instituto de Ciências Biomédicas of Universidade Federal do Rio de Janeiro under the responsibility of Dr Stevens K. Rehen, and 9 of them will be allotted to Hemocentro of Ribeirao Preto under the responsibility of Dr T. Dimas Covas. Subprojects: 2, 5, 6, 23, ) 02 Bioreactors This equipment will be used for expanding stem cells in a higher scale and with precise control of culture parameters such as ph, dissolved oxygen concentration, and temperature. 1 unit will be allocated to Instituto de Ciências Biomédicas of Universidade Federal do Rio de Janeiro under the responsibility of Dr Stevens K. Rehen, and 1 unit will be allotted to Hemocentro de Ribeirão Preto under the responsibility of Dr Dimas T. Covas. Subprojects: 2, 5, 6, 23, ) 01 Automated cell counter For cell number determination of several samples in an automated and reproducible way. This equipment will be installed at Instituto de Ciências Biomédicas of Universidade Federal do Rio de Janeiro under the reponsibility of Prof. Stevens K. Rehen. Suproject: 2 23) 01 Filtration system This system will be used to filter samples containing cell and microcarriers for the determination of the respective dry weights. This system will be allocated to Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas T Covas. Subproject: 2,

132 24) 01 MILLI-Q BIOCEL System and accessories This Ultrapure water production system releases high quality water with minimum ionic and organic contaminants and pyrogens, which is mandatory for culture, expansion and maintenance of stem cells. This equipment will be allocated to Instituto Evandro Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects 4, 6, 20, 26, ) 02 Liquid nitrogen containers and accessories For cryopreservation of cell and biological samples at low temperatures (around -100ºC to -196ºC). One unit will be located at Instituto Evandro Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva, and the other at Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects 1, 4, 5, 6, 8, 9, 11, 19, 20, 26, ) 01 digital ph meter Physiological ph values range from 7.15 to 7.4 and, hence, keeping culture medium ph close to these values is important to maximize cell expansion in vitro. This equipment will be kept at Instituto Evandro Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects 4, 6,20, 26, ) 38 Automatic micropipettes and automatic repeating pipettes Pipettes are necessary to transfer various volumes of liquids in virtually all laboratory techniques (molecular biology, cell culture, flow cytometry). These equipments will be used by all groups, and they were requested by the following coordinators: Dr. Wilson Araújo da Silva Junior, Dr. Julio Voltarelli, Dr. Dimas Tadeu Covas, Dr. Roberto Passetto Falcão and Dr. Flávio Meirelles. Subprojects 1, 5, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25, 26, 28, 29,

133 28) 02 Real time PCR machine Real time quantitative RT-PCR is an essential technique to quantify gene expression. It is very useful whenever it is necessary to verify differences in gene expression among samples and/ or situations. Any model which needs to verify messenger or microrna expression will use quantitative RT-PCR. It is also needed to validate gene expression values obtained by microarrays, and to confirm the presence or absence of molecular markers on stem cells. These equipments will be allocated to Instituto de Biociências of UFRJ RJ and at Laboratório de Genética Molecular of Hemocentro de Ribeirão Preto. Subprojects: 2, 6, 7, 13, 16, 17, 20 e 24). 29) 03 Nucleic acid quantification systems The preparation of samples to be analyzed by RT-PCR consists in preparing cell samples, extracting their RNA, quantifying and verifying the quality of RNA, and synthesizing complementary DNA. Quantification and verification of the quality of this RNA are absolutely necessary to obtain correct results by quantitative RT-PCR. The equipments used to quantify and verify the quality of RNA samples also are also useful to perform these tasks on protein and DNA samples. The latest models of these equipments use very small sample amounts, which is very important to study rare cell populations such as stem cells. These machines can be used by all groups working with cells, DNA, RNA or proteins. These equipments will be allocated to Instituto de Biociências of UFRJ RJ, Laboratório de Biotecnologia, and to Laboratório de Genética Molecular of Hemocentro de Ribeirão Preto. Subprojects: 2, 5, 6, 13, 16, 17, 20, 23, ) 02 Analytical balances Analytical balances are essential to correctly prepare laboratory solutions, which should contain the precise amount of each reagent. This type of balance can be used by all groups, and was requested by Dr. Klena Sarges Marruaz da Silva from Instituto Evandro Chagas and Dr. Julio 133

134 Voltarelli from Faculdade de Medicina de Ribeirão Preto. Subprojects 4, 6, 7, 17, 20, 21, 23, 26, 27, 28, 29). 31) 06 Mixers (stirrers, hotplate stirrers and vortexes) Adequate mixing of reagents, solutions and samples is required in order to obtain correct results. These equipments will be allocated to Laboratório de Biologia Molecular and Centro de Químicas de Proteínas of Hemocentro de Ribeirão Preto, and to Laboratório de Hematologia of FMRP-USP. Subprojects: 5, 7, 8, 11, 12, 14, 15, 17, 18, 19, 20, 21, 22, 23, 24, 28, 29, 30, 12, 14, 15, 17, 20, 21, 22, 23, 28, 29 e ) 1 GFP goggles These goggles bear specific green filters for macroscopic observation of GFP animals. It will be allocated to Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 20, ) IInverted trinocular microscope For routine observation of cell cultures as well as the analysis of cell differentiation. They will be allocated to Departamento de Hematologia of FMRP-USP (1 unit) and Faculdade de Medicina Veterinária-USP (3 units). Subprojects: 3, 4, 6, 7, 8, 9, 10, 11, 13, 18, 19, 20, 22, 25, 26, 27, ) 1 Optical microscope with polarized light and image capture system For acquisition and analysis of high quality digital images. This equipment will be allocated to Faculdade de Medicina Veterinária-USP under the responsibility of Profa. Maria Angélica Miglino. Subprojects: 21, 23, 26,

135 35) 1 Digital camera for inverted microscope with USB device For capture of images obtained on inverted microscope. It will be allocated to Faculdade de Medicina Veterinária-USP under the responsibility of Prof. Maria Angélica Miglino. Subprojects: 21, 23, 26, ) IInverted microscope Axio Observer D1 plus accessories For observation, analysis and documentation of each step of cell development. It also allows for the exhibition of the images on the screen. It will be allocated to Instituto Evandro Chagas under the responsibility of Dr. Klena Sarges Marruaz da Silva. Subprojetos: 4 e ) 2 Analogical and digital video camera Image capture device for trinocular microscope. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira of Universidade de Brasília. 38) 2 Trinocular microscope Equipment necessary for acquisition of the images that will be used to build image databases; to prepare didactic materials for lectures, courses, and expositions; to prepare divulgation material (flyers, folders, games, CDs, etc) for the popularization of knowledge. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 39) 1 Articulated arm base for stereomicroscope This equipment is required for a trinocular stereoscope. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira of Universidade de Brasília. 40) 1 Digital camera 1.4 MB with sensitivity to detect fluorescent light This equipment is required for acquisition of the images used to build the image database; preparation of didactic material for lectures, classes, 135

136 and expositions; preparation of diffusion material (pamphlets, folders, handouts, games, CDs, etc.) for diffusion of knowledge. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 41) 1 Trinocular stereomicroscope This equipment is necessary to visualize biological material (plants, animal s cells), rocks, sand and other materials during lectures on the museum center in Ribeirão Preto, or even during itinerant actions. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 42) 1 Simple binocular microbiological microscope For cell counting using the Neubauer chamber during cell culture procedures. It will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas Tadeu Covas. Subprojects: 5, 8, 10, 11, 12, 14, 15, 20, 21, 22, ) 1 Light source for GFP (Green Fluorescent Protein) excitation This equipment will be used for gross analyses of expression of the GFP reporter gene by live organisms. This equipment will equip Laboratório de Morfofisiologia Molecular e Desenvolvimento, of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles, and will be used frequently in subprojects 1, 8, 9, 20, and 27 44) Fluorescence Stereomicroscope This equipment is necessary to visualize the GFP reporter protein in cultured cells or live organisms. This equipment will equip Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 20, ) C-mount 1X adapter for camera/microscope 136

137 It will be used to couple the CCD camera to the fluorescence microscope, for use with the softwares SKYView or SpectraView. It will be installed at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto under the responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20, ) Dapi/Hoescht/Amca/HiSky filter and cubes for all types of microscope These filters are necessary for the observation of fluorescent substances used in cytogenetic preparations. This equipment will be installed at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto under the responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20, ) Software Multi-Species for use with BandView This software will be useful for detailed analyses of cytogenetic findings that reflect genetic instability at the chromosome level in materials from humans and rodents, and also to build an image bank for future analyses and publication of the results. This equipment will be installed at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto under the responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20, ) Complete workstation for cytogenetics: HiSKY, G-banding and FISH This equipment will be used to investigate numeric and structural chromosomal abnormalities in metaphase cells of patients with chronic lymphoid leukemia (CLL), and will contribute to related projects that include cytogenetic analyses. It will be installed at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto under the responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20,

138 49) Light microscope This equipment will be used to investigate numeric and structural chromosomal abnormalities in metaphase cells of patients with CLL, and will contribute to related projects that include cytogenetic analyses. It will be installed at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto under the responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20, ) Image acquisition system for inverted microscope This equipment will be used for observation and documentation of cell culture conditions during the experiments. It will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas Tadeu Covas. Subprojects: 5, 8, 10, 11, 12, 14, 15, 20, 21, 22, ) Stereomicroscope This equipment is necessary for the selection of labeled cloned cells "in situ". It will be installed at Hemocentro de Ribeirão Preto under the responsibility of Prof. Dimas Tadeu Covas. Subprojects: 5, 8, 10, 11, 12, 14, 15, 20, 21, 22, ) 23 L autoclave system This equipment is necessary for sterilization and cleaning of materials used in cell culture. This equipment will be allocated to Faculdade de Medicina Veterinária-USP under the responsibility of Prof. Maria Angélica Miglino. Subprojects: 21, 23, 26, ) Flow cytometer Under any circumstances, such as after cell culture or sorting, adult or embryonic stem cells must be identified with the highest precision possible. Immunophenotypical identification using 4-color flow cytometry provides, in addition to precision, the possibility of simultaneous identification of 4 independent cell markers. This equipment will be 138

139 allotted to Instituto Nacional de Primatas Evandro Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects: 4, 6, 20, 26, ) Accessories for cell separation columns These accessories are required for assembly of the immunomagnetic cell separation system, enabling handling of four cell types at the same time. Cell isolation by MACS has been extensively used to obtain pure cell populations, and it yields cells that maintain their physiological status with high cell viability. The cells are immunomagnetically labeled and then passed through columns held in a magnetic field. This equipment will be allocated to Instituto Nacional de Primatas Evandro Chagas under the responsibility of Dra. Klena Sarges Marruaz da Silva. Subprojects: 4, 6, 20, 26, ) 09 CO 2 water-jacketed incubator The CO 2 incubators will be used for cell culture. Considering the projected volume of work necessary for the generation of the expected results, the acquisition of CO 2 incubators for stem cell culture will be indispensable. The incubators will be installed in laboratories of the following units/institutions: Ciências Biomédicas/UFRJ-RJ, Faculdade de Medicina de Ribeirão Preto/USP, Hemocentro de Ribeirão Preto, and Faculdade de Medicina Veterinaria e Zootecnia/USP. Subprojects: 2, 6, 7, 11, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, ) 01 Shaker Incubator This shaker incubator will be used for expansion of bacteria. It will be allocated to Laboratório de Biotecnologia of Hemocentro de Ribeirão Preto. Subproject: 5. 57) 01 Bacteriological incubator This equipment will be used to culture and select bacterial clones in solid medium, allowing for the obtainment of plasmids used for functional 139

140 analyses of micrornas. It will be installed at Laboratório de Biologia Molecular of Hemocentro de Ribeirão Preto. Subproject: ) 02 Tri-gas incubators These equipments will be used for cell culture in different types of cells in hypoxia conditions, to verify the cells behavior under these conditions. One of them will be installed at Faculdade de Zootecnia e Engenharia de Alimentos of Universidade de São Paulo Pirassununga, and the other will be allotted to Laboratório de Terapia Celular of Hemocentro of Ribeirão Preto. Subprojects: 1, 8, 9, 10, 19, ) C Freezers The -80 C freezer enables storage and maintenance of reagents and cell samples. They will be installed at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto and at Faculdade de Zootecnia e Engenharia de Alimentos of Universidade de São Paulo Pirassununga. Subprojects: 1, 7, 8, 9, 18, 19, 20, ) 01 Microarray Scanner * The acquisition of a scanner for DNA microarray (CGH array) analyses is necessary to read microarray slides, and for the evaluation and identification of genes. This equipment will be allocated at Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25, ) 01 Microarray Hybridization oven * This equipment is necessary to incubate microarray slides during the hybridization step. It will be allocated to Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25,

141 62) 01 Rotational chamber for hybridization oven * This equipment is necessary for the microarray hybridization oven. It will be allocated to Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25, ) 01 SureHyb Hybridization chamber * This equipment is necessary for the microarray hybridization oven. It will be allocated to Laboratório de Hematologia of Faculdade de Medicina de Ribeirão Preto. Subprojects: 3, 8, 13, 14, 15, 16, 17, 19, 20, 21, 25, 29. * These equipments will provide the infrastructure for establishment of the microarray methodology that will serve all subprojects that involve large-scale gene expression analyses. 64) 01 Femtojet microinjector This device is necessary to perform microinjection/micromanipulation of ova or embryos of different species. It will equip Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, ) 01 Femtojet Microinjetor Pedal This device is necessary to perform microinjection/micromanipulation of ova or embryos of different species. It will equip Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, ) 01 Narashige Air Microinjector This equipment will be used for aspiration of the cytoplasmatic content of ova. It will be allocated to Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos 141

142 under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, ) 01 Piezo Drill This device will be used in techniques involving microinjection and micromanipulation of embryos reconstructed in the project. It will be allocated at Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, ) 01 Fyrite CO 2 /O 2 This equipment is necessary for the maintenance of adequate gas concentrations inside the incubator, and it is essential for the adequate production of in vitro embryos. It will be allocated at Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, ) 01 Automatic gas exchange system This equipment is necessary for the uninterrupted supply of gas for incubators since it allows the use of two gas cylinders. It will be allocated to Laboratório de Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19, ) 01 GFP Model exciting power pack: FHS/LS-1B This equipment will be used for evaluation of macroscopic expression of reporter genes in living organisms. It will be allocated at Laboratório Morfofisiologia Molecular e Desenvolvimento of Faculdade de Zootecnia e Engenharia de Alimentos under the responsibility of Prof. Flávio Meirelles. Subprojects: 1, 8, 9, 19,

143 71) 01 DAKO hybridizer This system is necessary for hybridization of slides for molecular cytogenetics (FISH and SKY). It will be allocated to Laboratório de Hemtaologia of FMRP-USP under responsibility of Prof. Roberto Passeto Falcão. Subprojects 19, 20, ) 01 Photographic documentation system for agarose gels This system will be used for documentation of the initial tests for optimization of the conditions of the amplification reactions. In this case, the equipment requested will have 100% national technology, with excellent construction quality and easy maintenance. It will be allocated to Instituto de Biociências of UFRJ under responsibility of Prof. Stevens K. Rehen. Subprojects 2, 6, ) 01 Microplate luminescence reader This equipment will be used for the MycoAlert (Lonza) luminescent assay. Furthermore, a luminescence reader can be used for a large number of expression assays, in which the activation capacity of the promoter of a specific gene can be measured along the time, or upon exposure to some factor. It will be allocated to Instituto de Biociências of UFRJ under responsibility of Prof. Stevens K. Rehen. Subprojects 2, 6, ) 01 Cryostat A cryostat is necessary for the confection of sections of frozen tissue. It will be allocated to Instituto de Biociências of UFRJ under responsibility of Prof. Stevens K. Rehen. Subprojects 2, 6, ) 01 Automated pipetting system for PCR reactions This system will be used for large scale pipetting in classic and real-time PCR reactions. This equipment will also be used for nucleic acid extraction. It will be allocated to Laboratório de Biologia Molecular of Hemocentro de Ribeirão Preto, under responsability of Prof. Dimas Tadeu 143

144 Covas. Subprojects: 3, 5, 7, 8, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 25, 26, 28, 29, ) 5 Multimedia projectors These projectors are necessary for projections during itinerant museum activities, as well as in extramural courses. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 77) 5 Digital video camera These video cameras are necessary for documentation of lectures, courses, and itinerant activities. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 78) 03 LCD TVs These are necessary for projections during itinerant museum activities, as well as in extramural courses. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 79) 25 Display cases This demountable furniture will be used as showcases during the activities of the participant museums. It will be used in the educational projects under the responsibility of Prof. Jussara R. Ferreira from Universidade de Brasília. 80) Showcases - 5 This equipament will be used for traveling exhibition around the country. 144

145 N) Potential for the Generation of Patents Trials and Patents deposited/obtained by the proponent group Process PI PI PI PI Title Quimioluminescence diagnosis test for infections by T cruzi Method of animal cloning from apoptotic cells and its use Process of stem cells obtention from orbicular lip cells, composition and use. Process of isolation, purification and determination of crotamine aminoacid sequences and its use as vectors for condution of genetic material Process for obtaining stem cells, cell concentration, process for stem cell differentiation of cells, differentiated cells, different cells concentration, use of stem cells for treatment or prevention of diseases and stem cells bank Year of the deposit of the process 2007 PI Dimas T. Covas Luiz R Travassos Igor C Almeida 2008 Flávio Meirelles Total 5 Irina Kerkis Maria Rita Passos Bueno Daniela F Bueno Mayana Zatz Irina Kerkis Alexandre Kerkis E. B. Oliveira G-Radis Baptista M. A Hayashi Lygia da Veiga Pereira T. Yamana Irina Kerkis Alexandre Kerkis M. A Hayashi H. F Cerruti The INCTC project has a big potential to generate patents and products. Specifically in the areas of isolation, culture, establishment of new lineages, productive process based on GMP criteria (good manufacturing you practice), development of drugs use, tissue engineering and regenerative therapy both in animals as in humans. As showed in figure 4, the generation of products on gene therapy area should pass for distinct phases and the potential of generate patents is not the same in all of these phases. The phases with bigger potential of patentability are phases II and III. In Brazil, in general, the groups of research that are dedicated to the study of stem cells and cell therapy are still found in phase I. In this project, we are proposing activities that are distributed for phases I to IV and therefore we will be working in the streak of bigger patents generation 145

146 probability in process and products. Several participants of the project have experience in the development of patents and in the production of technological process that need intellectual protection. Dr. Dimas Covas, by example has 3 patients (one recorded and two in process of record), Dra. Lygia Pereira has one patent recorded, Dra. Irina Kerkis has another three recorded patents, Dr. Flávio Meirelles has several technological process developed, the same happens with Dra. Maria Angelical Miglino. Therefore, the group possess experience in the generation of technological products that will be able to originate patents. The question of the patents record of the products and process generated will be directed with the aid of the USP Innovation Agency, specialized in these questions, and also of the Foundation Hemocentro of Ribeirão Preto, directly, or by its Incubator of Companies. Another point that needs attention is the intellectual protection concerns of the copyright of the publications and educational stuff produced in the printed or electronic form (copyright) and to the protection of marks. The Hemocentro has a sector of his Legal Consultancy specially focused in these aspects and zealous of the record of the material produced. 146

147 O) List of the Projects Financed over the Last 5 years COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE(R$) VALUE (US$) PROF. DR. ROBERTO PASSETTO FALCÃO PROF. DR. DIMAS TADEU COVAS PROF. DR. DIMAS TADEU COVAS PROF. DR. DIMAS TADEU COVAS PROF. DR. DIMAS TADEU COVAS PROF. DR. DIMAS TADEU COVAS CNPQ FINEP FINEP FINEP FINEP FINEP MONOCLONAL B CELLS LYMPHOCYTOSIS IN FIRST-DEGREE RELATIVES OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA CLONING AND EXPRESSION OF HUMAN BLOOD CLOTTING FACTORS VIII AND IX IN MAMMALIAN CELLS CONSOLIDATION OF THE CELL CULTURE LABORATORY FOR MOLECULAR AND PROTEOMIC STUDIES OF DENDRITIC AND STEM CELLS SCALING OF THE PRODUCTION OF RECOMBINANT FACTORS VII AND IX IN BIOREACTORS AND PRECLINICAL ASSAYS IN HEMOPHILIC MICE BIOLOGICAL AND MOLECULAR ANALYSIS OF PLURIPOTENT SOMATIC STEM CELLS IN TISSUE REPAIR INFRASTRUCTURE FOR THE GENERATION AND MANIPULATION OF GENETICALLY MODIFIED ANIMAL MODELS IN THE STUDY OF HUMAN DISEASES , /11/ /11/ /08/ /08/ /12/ /12/ /12/ /12/ /09/ /09/ /05 370, /05 522, /07 2,384, /07 434, /06 742, PROF. DR. DIMAS TADEU COVAS FINEP DEVELOPMENT OF ANIMAL MODELS FOR THE UNDERSTANDING OF THE ROLE OF MESENCHYMAL STEM CELLS AND PERICYTES IN TISSUE REPAIR AND TUMOR VASCULARIZATION AWAITING AGREEMENT 343,

148 COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE (R$) PROF. DR. DIMAS TADEU COVAS CNPQ ISOLATION, CHARACTERIZATION, CULTURE, EXPANSION AND EVALUATION OF THE IN VIVO AND IN VITRO VASCULOGENIC POTENTIAL OF ADULT PLURIPOTENT STEM CELLS WITH CAPACITY FOR ENDOTHELIAL DIFFERENTIATION 01/03/2008 A 01/03/ / ,158,80 VALUE (US$) PROF. DR. DIMAS TADEU COVAS CNPQ EVALUATION OF THE GENE EXPRESSION OF ENDOTHELIAL PROGENITOR CELLS ISOLATED FROM UMBILICAL CORD BLOOD 26/12/2007 TO 25/12/ / , PROF. DR. DIMAS TADEU COVAS CNPQ EVALUATION OF GENE EXPRESSION IN ENDOTHELIAL PROGENITOR CELLS ISOLATED FROM UMBILICAL CORD BLOOD AND BONE MARROW 17/07/2008 TO 17/07/ / , PROF. DR. DIMAS TADEU COVAS BNDES UMBILICAL CORD BLOOD BANK AWAITING LIBERATION 872, PROF. DR. EDUARDO MAGALHÃES REGO FAPESP DETERMINATION OF THE EFFECT OF INACTIVATION OF THE GENE CAATα ENHANCER S LIGAND PROTEIN IN THE HEMATOPOIESIS OF IML-RARA TRANSGENIC ANIMALS 01/11/2001 TO 31/10/ / , PROF. DR. EDUARDO MAGALHÃES REGO CNPQ IN VITRO AND IN VIVO ANALYSIS OF THE ANTILEUKEMIC ACTIVITY OF TOCOPHEROLS IN ACUTE PROMYELOCYTIC LEUKEMIA 01/03/2005 TO 01/03/ / , PROF. DR. EDUARDO MAGALHÃES REGO PROF. DR. MARCO ANTONIO ZAGO CNPQ FAPESP ANALYSIS OF THE ROLE OF THE TGFβ PATHWAY AND OF THE THERAPEUTIC POTENTIAL OF ITS INHIBITOR HALOFUGINONE IN ACUTE PROMYELOCYTIC LEUKEMIA CELL THERAPY CENTER 01/02/2008 TO 31/01/ TO / , / ,022, ,830,

149 COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE (R$) PROF. DR. MARCO ANTONIO ZAGO PROF. DR. MARCO ANTONIO ZAGO PROF. DR. LEWIS JOEL GREENE PROF. DR. LEWIS JOEL GREENE PROF. DR. JULIO CÉSAR VOLTARELLI PROF. DR. JULIO CÉSAR VOLTARELLI FAPESP FAPESP FINEP FINEP AND FAPESP FINEP CNPQ TECHNICAL RESERVE FOR INFRASTRUCTURE TECHNICAL RESERVE FOR CONNECTION TO THE ANSP NETWORK STRUCTURAL AND FUNCTIONAL PROTEOMICS APPLIED TO THE BIOMEDICAL AREA SUPPORT TO THE PROTEOMIC NETWORK OF THE STATE OF SÃO PAULO HEMATOPOIETIC STEM CELL TRANSPLANTATION IN AUTOIMMUNE DISEASES HEMATOPOIETIC STEM CELL TRANSPLANTATION IN TYPE 1 DIABETES AND NEURODEGENERATIVE DISEASES 01/08/ /07/ /08/ /08/ /12/ /12/2009 October 2007 to October/ / , / , / , , , /10/ /10/ / , VALUE (US$) PROF. DR. MARIA ANGÉLICA MIGLINO FAPESP STUDY OF THE PLASTICITY OF IMMATURE MULTIPOTENT STEM CELLS DERIVED FROM HUMAN DENTAL PULP: AN ANIMAL MODEL. A BRIGE FOR CELL THERAPY 22/03/2007 to 21/03/ , PROF. DR. MARIA ANGÉLICA MIGLINO CNPQ RESEARCH SUPPORT, UNIVERSAL CNPQ PROJECT , PROF. DR. MARIA ANGÉLICA MIGLINO FAPESP EXPRESSION OF GREEN FLUORESCENT PROTEIN (GFP) AS A MARKER OF CELLS OF FETAL ORIGIN IN GESTATIONS OF BOVINE CLONES 2006 present date 05/ , PROF. DR. LYGIA DA VEIGA PEREIRA FAPESP ANALYSIS OF THE PATTERN OF CHROMOSOME X INACTIVATION IN HUMAN EXTRA-EMBRYONIC TISSUES 8/2005 2/ ,

150 COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE (R$) VALUE (US$) PROF. DR. LYGIA DA VEIGA PEREIRA FAPESP CLINICAL VARIABILITY IN AN ANIMAL MODEL OF MARFAN SYNDROME IDENTIFICATION OF PHENOTYPE-MODIFYING GENES. 11/ / , , PROF. DR. LYGIA DA VEIGA PEREIRA CNPQ ESTABLISHMENT OF NEW HUMAN EMBRYONIC STEM CELL LINES 1/2006 8/ , PROF.. DR. LYGIA DA VEIGA PEREIRA FAPESP CREATION OF AN ANIMAL MODEL OF MARFAN SYNDROME BY HOMOLOGOUS RECOMBINATION IN EMBRYONIC STEM CELLS 10/ / , , PROF. DR. STEVENS KASTRUP REHEN CNPQ CHARACTERIZATION OF THE IMPORTANCE OF LYSOPHOSPHATIDIC ACID FOR THE CULTURE OF NEURAL STEM CELLS DERIVED FROM THE CEREBRAL CORTEX OF MICE AND HUMANS 2005/ , PROF. DR. STEVENS KASTRUP REHEN CNPQ CHROMOSOMAL INSTABILITY DURING DIFFERENTIATION OF EMBRYONIC STEM CELLS TO MOTOR NEURONS 2008/ , PROF. DR. STEVENS KASTRUP REHEN CNPQ CONTROL OF ANEUPLOIDY AND CELL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS 2005/ , PROF. DR. STEVENS KASTRUP REHEN FAPERJ PROPAGATION OF EMBRYONIC STEM CELLS IN BIOREACTORS IN ORDER TO INCREASE THE SCALE FOR TRANSPLANTS IN NEURODEGENERATIVE DISEASES 2007/ , PROF. DR. STEVENS KASTRUP REHEN FAPERJ CONTROL OF CHROMOSOME INSTABILITY AND NEURAL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS 2007/ ,

151 COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE (R$) PROF. DR. STEVENS KASTRUP REHEN FAPERJ CHARACTERIZATION OF THE THERAPEUTIC POTENTIAL OF STEM CELLS IN A PRECLINICAL MODEL OF PARKINSON S DISEASE: MECHANISMS OF NEUROGENESIS AND FUNCTIONAL COMPARISON OF EMBRYONIC AND ADULT CELLS 2008/ , VALUE (US$) PROF. DR. WILSON ARAÚJO DA SILVA JUNIOR FAPESP IDENTIFICATION O AND FUNCTIONAL CHARACTERIZATION OF MOLECULAR MARKERS INVOLVED IN THE PROCESS OF FORMATION OF HEAD AND NECK TUMORS BY ANALYSIS OF THE DIFFERENTIAL PATTERN OF METHYLATION 464, PROF. DR. WILSON ARAÚJO DA SILVA JUNIOR FAPESP SEARCH FOR MARKERS OF AGGRESSIVENESS IN HEAD AND NECK TUMORS 451, ,85 DR.SIMONE KASHIMA HADDAD CNPQ QUANTITATIVE PROFILE OF MICRORNAs IN POPULATIONS OF T LYMPHOCYTES OF HTLV-I-INFECTED PATIENTS 01/10/ /09/ / , PROF. DR. JOSÉ CÉSAR ROSA FAPESP STUDY OF THE MICROSOME AND CYTOSOL FRACTIONS OF B LYMPHOCYTES OF PATIENTS WITH CHRONIC LYMPHOCYTIC LEUKEMIA A DIFFERENTIATED PROTEOMIC ANALYSIS , PROF. DR. JOSÉ CÉSAR ROSA FAPESP STUDIES OF POST-TRANSLATION MODIFICATIONS IN PROTEINS DIFFERENTIALLY EXPRESSED IN THE CELL LINES MELAN-A, TM1 AND TM5. A MURINE MODEL OF MELANOMA PROGRESSION , PROF. DR. JOSÉ CÉSAR ROSA FAPESP BIOLOGICAL EFFECTS AND PHARMACEUTICAL APPLICATIONS OF LECTINS ,114,

152 COORDINATOR SOURCE PROJECT VALIDITY AGREEMENT REFERENCE VALUE (R$) DR. KLENA SARGES MARRUAZ DA SILVA UNESCO/CEN P-IEC/SVS-MS IDENTIFICATION AND CONTROL OF ZOONOTIC ENDOPARASITOSES IN NON- HUMAN PRIMATES MAINTAINED AT THE NATIONAL PRIMATE CENTER SVS/MS February 2005 to April , VALUE (US$) DR. KLENA SARGES MARRUAZ DA SILVA OPAS/CENP- IEC/SVS-MS CLINICAL AND EPIDEMIOLOGICAL MONITORING OF NON-HUMAN PRIMATES SEROPOSITIVE FOR VIRAL HEPATITIS MAINTAINED AT THE NATIONAL PRIMATE CENTER SVS/MS July 2006 to 02 October , DR. KLENA SARGES MARRUAZ DA SILVA UNESCO/IEC/ SVS-MS IMPLANTATION OF A NEW METHOD FOR LABORATORY ANIMAL REARING AND RECOLONIZATION OF THE RODENT AND LAGOMORPH STOCK OF THE ANIMAL HOUSE OF THE INSTITUTO EVANDRO CHAGAS IEC- SVS/MS From February 2008 to the present date 47, ,960, ,125,

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176 Prof. Dr. Dimas Tadeu Covas Associate Professor FMRP/USP DD. President Director Fundação Hemocentro de Ribeirão Preto September 15, 2008 Dear Prof Dimas, I am delighted to accept your invitation to be a collaborator on the Project from CNPq Edital No 15/2008 entitled National Institute of Science and Technology, and thus continue our productive collaboration that began two years ago when one of your researchers visited my lab to learn the LM PCR technique. I am pleased to hear that a publication utilizing data generated with this technique is being written at the present time. Important to the current project, we have a great deal of experience with Mesenchymal and Hematopoietic Stem Cells, as well as gene modification of stem cells using murine retrovirus and lentivirus vector systems. Moreover, we have developed two large animal models which we are using to study the in vivo distribution and differentiative capacity of specific human stem cells: 1) a line of Hemophilia A sheep; and 2) the human/sheep xenograft model. Based on our prior collaborative productivity and our experience with in vivo models of stem cell transplantation/biology, I would be glad to collaborate with you and add my expertise to several of your sub projects. Sincerely, Graça Duarte de Almeida-Porada,MD,PhD Professor Director of Graduate Studies Department of Animal Biotechnology University of Nevada, Reno Mail Stop 202 Reno NV Phone:(775) (775) LAB Fax: (775) galmeida@cabnr.unr.edu 176

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