Induced Pluripotent Stem Cell

Transcription

1 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 a 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 histocompatible 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 the genetic change of mesenchymal stem cell and endothelial progenitor cells with lentiviral vectors carrying the stemness genes, 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 afterwards four vectors at the same time will be used to induce de ips; c) Evaluation of the

2 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 in São Paulo (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 in 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 co-transfection 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 with genetic diseases of interest to the group, particularly patients who have

3 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

4 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 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 et al., 2007). On the other hand,

5 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 intra- and 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 mitochondrias 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.