UNIVERSITY OF CINCINNATI

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1 UNIVERSITY OF CINCINNATI Date: I,, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair:

2 FEASIBILITY OF EX VIVO EXPANSION, TRANSDUCTION AND TRANSPLANTATION OF MURINE BONE MARROW MESENCHYMAL PROGENITOR/STEM CELLS A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Department of Transfusion and Transplantation Sciences of the College of Allied Health Sciences Committee Chair: Dr. J. A. Cancelas 2004 by Faiqa M. Sadique B.S. George Mason University, 1990

3 ABSTRACT Adult bone marrow mesenchymal stem cells and progenitors (BM MSC/P) constitute an attractive source of potentially transplantable cells for therapy of skeletal diseases. A murine model of BM MSC/P might be helpful in the design of approaches to correct specific genetic and skeletal diseases by gene delivery after MSC/P transplantation. Feasibility of isolation, ex vivo expansion, transduction and transplantation of BM MSC/P in a murine experimental model were investigated. For isolation and ex vivo expansion, BM MSC/P isolation based on plastic adherence and culture under defined conditions followed by macrophage depletion (method A) was compared with a hematopoietic/endothelial-depleted (CD45-/TER119-/CD11b-/CD34-/CD31-) bone marrow population (method B). Multilineage differentiation of MSC/P into osteoblasts, chondrocytes and adipocytes was also assayed in tissue-specific, medium-defined conditions. Transduction of MSC/P was performed with the retroviral MIEG3 vector expressing Enhanced Green Fluorescent Protein (EGFP). For analysis of skeletal tissue engraftment, 1) whole BM cells from smooth muscle "-actin/eyfp (SMAA8-EYFP) or smooth muscle (-actin/egfp (SMGA13.7-EGFP) transgenic mice, or 2) ex vivo expanded MSC/P expressing either an "-actin-driven EYFP or EGFP driven by a constitutively active retrovirus promoter (MIEG3-transduced) were transplanted. A total of either 10 7 BM cells or 2 x 10 5 MSC/P were injected intravenously via the tail vein of 7 Gy-irradiated syngeneic recipients. Method A yielded higher colony-forming-units of fibroblast type (CFU-F) content. MSC/P isolated and expanded by method A were easily transduced using retroviral vectors and grew exponentially until reaching greater than fold and 471-fold cell and CFU-F expansion, respectively. Exponentially growing MSC/P could be differentiated into adipocytes, osteoblasts and chondrocytes and we observed that transplantation of SMAA8-EYFP but not SMGA13.7-EGFP transgenic BM cells resulted in patches of spindled cells surrounding pre- or post-sinusoidal vessels in the BM of non-transgenic recipient mice. The feasibility of the expansion and transduction of MSC/P in a murine model and BM stroma transplantation by systemic infusion was demonstrated. These data confirm that MSC/P comply with the major requirements of a cell vector for gene therapy: efficient propagation and transduction ex vivo, together with the ability to be genetically loaded and transplanted.

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5 ACKNOWLEDGEMENTS I would like to extend my heartfelt thanks to Dr. Cancelas my mentor, advisor and co-adventurer in this journey towards the elusive MSC/P. To Dr. Leemhuis for your valued guidance. To Dr. Williams and the staff at the department of Experimental Hematology at the research facilities of Cincinnati Children s Hospital Medical Center especially Jeff and Andy; thank you all for the chance of a lifetime. Dr Lewis, for all your help. Margarette for rescuing my e-files. Nithya, for moral support and for being my friend, good luck with your baby girl. Ondrej for the possibilities. Cathy and Jan, for making me feel at home and for all your help and support throughout my stay at UC-Hoxworth, and for the endless revisions. Many grey hairs to be shared amongst us as a result of it all. Noni, thanks for sharing this adventure with me. My family, especially my mom and my sister Sadi for being there for me as always. Hesam, for sharing the burden. And Michael, for listening to me when I was up or down, love always. Faiqa June 2004.

6 1 TABLE OF CONTENTS Abbreviations 2 List of Figures and Tables 5 List of Appendices 6 Introduction 7 Research Objective/Plan 17 Materials and Methods 21 Facilities and Resources 40 Results 41 Discussion 61 Conclusions 66 References 74

7 2 ABBREVIATIONS 2-ME 7-AAD APC bfgf BM bmsc BSA C57BL/6J beta-mercaptoethanol 7-Amino-actinomycin-D Allophycocyanin basic Fibroblastic Growth Factor Bone Marrow bovine derived MSC/P Bovine Serum Albumin inbred mouse strain C57, black in color from 6 th line of substrains obtained from the Jackson Laboratory Ca CaCl 2 CCHMC CFU-F CHO DME DNA EDTA EGFP Env EtOH FCS FITC Calcium Calcium Chloride Cincinnati Children s Hospital Medical Center Colony Forming Unit, type Fibroblasts Chinese Hamster Ovary Dulbecco s Modified Eagle s Medium Deoxyribonucleic Acid Ethlyenediaminetetracetic Acid Enhanced Green Fluorescent Protein retroviral Envelope protein Ethanol Fetal Calf Serum Flourescein Isothiocyanate

8 3 FVB/N inbred albino mouse strain of Swiss origin established at NIH in 1935 Gag Gy hmsc hpdgf-bb HCl HEPES HLA ILGF-1 IMDM IU I.V. LIF MAPC Mg MHC MIEG3 Group Antigens (core poly-protein) Gray (unit of absorbed radiation energy) human derived MSC/P human Platelet Derived Growth Factor-BB Hydrochloric acid 4-2-hydroxyethyl-1-piperazineethanesulfonic acid Human Leukocyte Antigen Insulin Like Growth Factor-1 Iscove s Modified Dulbecco s Medium International Units Intravenous Leukemia Inhibitory Factor Multipotential Adult Progenitor Cells Magnesium Major Histocompatibility Complex retroviral plasmid driven by a Murine Stem Cell virus promoter and containing an Internal Ribosome Entry Site and the Enhanced Green Fluorescent Protein mmsc mrna MSC/P murine derived MSC/P messenger Ribonucleic Acid Mesenchymal Stem Cells and Progenitors

9 4 NaOH OM PBS PE Phoenix-GP Sodium hydroxide Optical Magnification Phosphate Buffered Saline Phycoerythrin Phoenix Gag-Pol retrovirus producing cells. Second generation cell lines based on 293T cell line (human embryonic kidney) constructs capable of producing retroviral gag and pol proteins. Pol rmegf SMAA8-EYFP Polymerase (reverse transcriptase) recombinant mouse Endothelial Growth Factor Smooth Muscle Alpha Actin promoter 8 th construct driving the expression of Enhanced Yellow Fluorescent Protein SMGA13.7-EGFP Smooth Muscle Gamma Actin promoter 13.7 construct driving the expression of Enhanced Green Fluorescent Protein TGF-ß3 Transforming Growth Factor ßeta-3

10 5 LIST OF FIGURES AND TABLES Figure 1 Marrow Stromal Cultures 50 Figure 2 CFU-F Morphology 50 Figure 3 MSC/P Morphology 51 Figure 4 Fold Expansion over time in culture 52 Figure 5 Clonogenic Efficiency Over Time in Culture 53 Figure 6 Flow Cytometry Analysis of Transduced Cells 54 Figure 7 Whole BM Engraftment of Donor Cells in BM Vesseles 55 Figure 8 Whole BM Engraftment of Donor Cells in Periosteum 56 Figure 9 Adipocyte Differentiation (Phase contrast) 57 Figure 10 Adipocyte Differentiation (Oil Red O Staining) 57 Figure 11 Osteoblast Differentiation (von Kossa staining) 58 Figure 12 Osteoblast Differentiation (Alkaline Phosphatase Content) 58 Figure 13 Chondrocyte Differentiation (Alcian Blue Staining) 59 Table 1 Hemtopoietic Depletion Analysis, Method A 60 Table 2 Hematopoietic/Endothelial Depletion Analysis, Method B 60 Table 3 Phenotype Analysis of Isolated Cells, Method B 60

11 6 LIST OF APPENDICES Appendix A Cellular Surface Markers 68 Appendix B Supplies and Manufacturers 70 Appendix C Instruments 72

12 7 INTRODUCTION Stem cells are characterized by their ability for self renewal and differentiation into multiple, yet distinct cell lineages. 1 Three different types of stem cells have been defined: totipotential (able to differentiate into all the somatic and germinal lineages), multipotential (able to differentiate into all the somatic lineages) and pluripotential (able to differentiate into all the lineages of any ectodermic, endodermic or mesodermic origin). 2 Historically, postnatal bone marrow has been seen as an organ composed of two main components based on the two distinct lineages found in it, the hematopoietic tissue and the supporting stroma. 3,4 The sizable body of evidence addressing the presence of a pluripotential progenitor cell responsible for all the diverse cell lineages comprising the bone marrow stromal system has made the bone marrow the only known organ in which two separate and distinct stem cells, along with their dependent tissue systems, coexist and cooperate. In fact, bone marrow stromal cells which were generally of interest because of their presumptive role in hematopoietic support (Dexter-type 5 and Witte-Whitlock type cultures 6 ) have been shown to generate skeletal tissues in an adequate environment. Following ectopic transplantation of a colony of the marrow stromal cells under the kidney capsule, and the subsequent differentiation of these cells into a broad spectrum of connective tissues including bone, cartilage, adipose and hematopoietic-supportive stroma, it was proposed that all of these connective tissues might have originated from a common precursor residing in

13 8 the bone marrow. 7,8 Subsequent observations have confirmed their ability to differentiate into the many different lineages compromising the bone marrow stromal environment. 9 These cells which generally had been referred to as marrow stromal cells are now also called mesenchymal stem cells and progenitors (MSC/P) depending upon their ability to differentiate ex vivo and in vivo. 4 More recent data pointing to the unexpected differentiation potential of MSC/P into ectodermic, mesodermic and endodermic-derived lineages have granted these cells membership in the diverse family of somatic stem cells. 4,10,11 The increasing recognition of the unexpected properties of marrow stromal cells has spawned a major change in the perception of their nature, and potential therapeutic applications in both cell and gene therapy have been envisioned. 4 The ethical controversy about the use of embryonic stem cells has favored the development of multiple research projects for the isolation, expansion and manipulation of transplantable adult mesenchymal stem cells with pluripotential capability of differentiation. 1,2,10,12,13 Despite the advances in the knowledge about and applications of MSC/P, many aspects of MSC/P biology remain in question. 3,14,15,16,17 These include, the identity, nature, cell proliferation and differentiation abilities, in vivo function of MSC/P, and their potential ex vivo manipulation and transplantability for cell/gene therapy. 16,18 Although MSC/P have been studied in many species of animals including mouse, guinea pig, rat, rabbit, dog, horse, baboon and man, the murine model appears the most promising as it can easily be challenged in gene targeting strategies. 19

14 9 Friedenstein et al in the 1960 s and 1970 s 20,21 were the first researchers to assay MSC/P by the CFU-F assay (colony-forming-unit-fibroblast). In order to optimize their growth, selected serum batches and irradiated marrow feeder layers were proposed. 22 The mitogenic factors that are required to stimulate the proliferation of CFU-F are not completely known at this time, but include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), basic fibroblast growth factor (bfgf), transforming growth factor-ß (TGF- ß), and insulin-like growth factor-1 (ILGF-1). 10,23,24 Other authors have pointed out that leukemia inhibitory factor and fibronectin to which stromal cells bind are important factors for maintenance of Bone Marrow (BM) MSC/P in their pluripotential differentiation state. 25 In addition, it has been proposed that grown ex vivo, the rate of expansion and the yield of multipotential progenitors are inversely related to plating density and incubation times of each passage. 24 Under optimal conditions, multi-colonyderived strains (where all colonies are combined by trypsinization) are present at a frequency of 1/10 4-1/10 5 total BM cells and undergo over 25 passages in vitro, demonstrating a high capacity for self-replication. The in vitro definition of MSC/P is that they are rapidly adherent, clonogenic, and capable of extended proliferation and differentiation into, at least, the classical skeletal lineages 26 such as osteocytes, chondrocytes, myotubes, adipocytes, marrow stromal cells, tendon/ligament fibroblasts and other connective tissue cells. 16,27 Thus, this differentiation into cells from different mesenchymal lineages serves as a functional criteria commonly used to define MSC/P. 9

15 10 The heterogeneous nature of the MSC/P population is apparent upon examination of individual CFU-F colonies. 4,23,,27, Typically, this is exemplified by a broad range of colony sizes that represent varying growth rates and different cell morphologies. 4 The cell morphologies range from fibroblast-like spindle-shaped cells to large flat cells. Moreover, if such cultures are allowed to develop for up to 20 days, phenotypic heterogeneity is also noted. Some colonies are highly positive for alkaline phosphatase, while others are negative. Some colonies form nodules (the initiation of matrix mineralization) while others accumulate fat, and, in adequate conditions of low-oxygen tension, some colonies form cartilage. The ability to isolate the subset of marrow stromal cells with the most extensive replication and differentiation potential is very important, for both theoretical and applicative reasons. This requires definitive linkage of the multipotency displayed in transplantation assays with a phenotypic trait that can be assessed prior to, and independently of, any subsequent assays. 4 Several laboratories have developed monoclonal antibodies using MSC/P as an immunogen in order to identify one or more markers appropriate for identifying and sorting human stromal cell preparations. Antibodies against a number of antigens representing several classes of surface molecules that are found on the MSC/P such as growth factor receptors, cell adhesion molecules, integrins and cytokine receptors are the result of such efforts. 19 Unfortunately, these antigens are not unique to MSC/P. It has been claimed that the human MSC/P can be isolated using rather standard procedures and characterized using a long list of indeterminate markers. 10,27,-28 However, the isolation of a "pure" population of

16 11 murine multipotent or lineage-committed marrow stromal stem cells remains elusive. Generally, the expression of markers such as CD44 (refer to appendix A for definitions), CD59, CD105, CD106 and MHC class I molecules are recognized on all MSC/P. 29,30 In their study of marrow stromal cells, Vogel et al 27 have found that MSC/P express CD10, CD13, CD61, CD90 (Thy-1), CD105, CD109, CD140b, CD164, and CD172a, but lack expression of CD45, CD34 and CD133. A distinct subpopulation of MSC/P was observed to preferentially express Nestin and react with the antibody W8B2. Lee et al, in their studies using human bone marrow to isolate and expand MSC/P, were able to phenotype the culture-expanded cells. 25 Their results indicate that the MSC/P expressed CD29, but did not express CD34, CD45, CDw49c, CD56, CD58, CD90 and CD105. The expression of CD71, CD106, and CD117 (c-kit) was variable among the expanded MSC/P. Lee et al demonstrated the ex vivo expansion of isolated cells for 20 population doublings, plus the chondrogenic and osteogenic differentiation potential of these cells. Pittenger et al 28 have described the putative MSC/P as expressing CD105 and CD106 in addition to other markers, but lacking expression of any hematopoietic markers such as CD14, CD34 and CD45. These morphological findings were consistent in many isolated MSC/P cultures that they studied, and the cells were able to consistently differentiate along the osteogenic, chondrogenic and adipogenic lineages. Jiang et al 10 have documented that a subset of MSC/P called multipotent adult progenitor cells (MAPC) does not express CD44, CD45, MHC class I and II,

17 12 c-kit (CD117), CD34, CD3, Gr-1 (Ly6G), Mac-1 (CD11b/CD18) or CD19. They found that undifferentiated MAPC are also negative for CD31 and CD62e, but express surface markers such as Flk-1, Sca-1, Thy-1 (CD90), and high levels of CD13 and SSEA-1. These cells were expanded in culture for more than 80 population doublings and the isolated cells were induced to differentiate into mesoderm-derived cells (endothelium), neuroectoderm-derived cells (astrocytes, oligodendrocytes and neurons), and endoderm-derived cells (hepatocytes). Despite the characterization of the MAPC in this study, others have had difficulty in reproducing these results. It is apparent from these observations, that there is not a consensus as to which markers are exclusive for MSC/P and their subpopulations, and at what level of maturation in the life cycle of the MSC/P these markers are expressed. Tremain et al have reported that a single-cell derived colony of undifferentiated human MSC/P represents more than 2000 unique gene expressions. 30 They found that a single-cell derived colony of human MSC/P simultaneously expressed mrna of numerous cell lineages. These observations may provide additional evidence for the stem cell-like nature of these cells, and also, may clarify why attempts to categorize MSC/P have resulted in confusion. 10,11,12,14,28 The accumulating evidence for the systemic and local transplantation capabilities of MSC/P, and their proliferation and differentiation ex vivo and in vivo in more than one organ has important implications for cell and gene therapy. 10,26,29,31 32,33,34 Somatic mammalian cells are putative gene delivery

18 13 vehicles with the potential for overcoming physiological barriers to conventional viral vectors, linking cell therapy with gene delivery. It has been suggested that the ideal cellular vector system would have to comply with stringent criteria, such as easy and non-invasive access to desired cell population, simple isolation procedures and the ability to efficiently propagate in culture. 35 Cellular vehicles must also possess the ability to be genetically loaded and to deliver gene expression after systemic or local infusion into target organs. A variety of cell types have been tested in this regard, confirming that the ideal cellular vector system for gene therapy has yet to be found. 36 Given that the intrinsic properties of MSC/P satisfy most of the abovementioned criteria, MSC/P might be the ideal candidates as cellular vehicles for cell and gene therapy. Theoretically, stem cells have the capability to proliferate and propagate in vivo and their engraftment is long term as compared to somatic cells. Although stem cells may be present in many organs, their relative small numbers and their location in these organs is a limiting factor for their usefulness in cell and gene therapy. Bone marrow, on the other hand, has a seemingly limitless supply of stem cells that can be used for these purposes. Bone marrow derived MSC/P, in particular, have been shown to have the potential to proliferate and differentiate ex vivo into various tissue types including smooth muscle cells, 34 myotubes 37 and neural 1 cells, in addition to the classical mesenchymal lineages found in the marrow stroma. In vivo differentiation of MSC/P into bone 32 and the regeneration of the non-hematopoietic environment of the bone marrow by MSC/P have been demonstrated in both human and

19 14 animal studies. 9, 35 The purported in vivo differentiation of MSC/P into other lineages remains questionable. For instance, Toma et al in their studies with mice, injected human derived MSC/P (hmsc/p) directly into the heart and observed that over time a number of the injected cells morphologically resembled the surrounding host cardiomyocytes. 38 Although unlikely, the possibility remains that the hmsc/p may have converted to skeletal muscle phenotype and then adopted the cardiac environment. In addition, it has been shown that MSC/P may be manipulated and propagated ex vivo without apparent loss of differentiation potential which would make them ideal cellular vehicles for cell/gene therapy. 10,29,31,35 However, the in vivo behavior of the MSC/P after these manipulations is unclear. Rombouts and Ploemacher 39 concluded that MSC/P lose their bone marrow homing capabilities following culture, but show highly efficient homing to the bone marrow when injected systemically as primary cells into mice. Others have not observed this. 10,40 In fact, Prockop et al, following the ex vivo expansion of MSC/P and subsequent systemic infusion of these cells into irradiated transgenic mice, found that human collagen type I marked cells engrafted into a variety of organs when analyzed one to five months after transplantation, albeit in small numbers 29. The studies differed in that one assessed CFU-F homing (short-term study), while the other studied overall engraftment (long-term study). Devine et al have also demonstrated that culture propagated baboon MSC/P (bmsc/p) preferentially home to the baboon BM following systemic infusion. 36

20 15 Furthermore, MSC/P appear to have absent or minimal immunogenicity or toxicity. 26,36 Liechty et al reported the persistence and engraftment of hmsc/p transplanted at 85 days of gestation in a sheep fetus for up to 13 months after transplantation. 33 Devine et al confirmed this in their studies of adult baboons that were systematically infused with ex vivo expanded HLA (histocomaptibility antigens) mismatched bmsc/p as well as autologous bmsc/p. 40 Recently, Krampera et al 41 demonstrated MSC/P s inhibition of response by antigenspecific T cells to their cognate peptides, by an as yet unknown mechanism, thereby limiting graft versus host disease. In addition, Chamberlain et al 42 have shown that transfection of a neocassette containing adenovirus-associated vector into MSC/P, which knocks down the expression of the mutant allele of MSC/P obtained from osteogenesis imperfecta patients, reverts this phenotype in a xenogenic tranplantation model. This study gives further credence to the notion that MSC/P can be genetically loaded and manipulated. The above-mentioned observations support the view that the MSC/P are ideal candidates for cell/gene therapy. However, there are several issues regarding their potential therapeutic application that remain unresolved. These include their isolation and in vivo characterization, transduction efficiency, silencing of transgene expression, side effects associated with virus-mediated gene transfer, and possible immune suppression of the recipient (suppression of T-cell activation, especially in tumor growth) mediated by MSC/P

21 16 In an attempt to shed light on some of these issues, this study was undertaken to establish methods for isolation and ex vivo expansion of MSC/P in a mouse model. Additionally, using gene-marking tools, this study was conducted to determine whether the isolated cells are indeed progenitor/stem cells of marrow stromal origin capable of transduction and engraftment in syngeneic recipients after systemic infusion.

22 17 RESEARCH OBJECTIVE/PLAN After establishing protocols for isolation of MSC/P, the purpose of this project was 1) to compare two methods of isolation of MSC/P for ex vivo expansion of the murine bone marrow mesenchymal progenitors/stem cells, 2) to explore the ex vivo differentiation potential of the isolated fibroblast-like cells of the bone marrow that were shown to be plastic adherent, clonogenic and capable of extended proliferation and replication, 3) to transduce MSC/P with a reporter gene (EGFP- Enhanced Green Fluorescent Protein) and subsequently, 4) to transplant the MSC/P into syngeneic recipients. Bone marrow (BM) and MSC/P from transgenic mice (FVB/N) expressing the Green or Yellow Fluorescent Protein genes whose in vivo expression is driven by the Smooth Muscle (-Actin (SMGA13.7-EGFP) or "-Actin promoter construct (SMAA8-EYFP), respectively, were used as an uncultured control for stromal transplantibility, and for analysis of MSC/P engraftment, respectively. Engraftment of fluorescent MSC/P (transduced or transgenic) in target tissues (bone and BM) was analyzed to prove whether the isolation method was inclusive of engraftable MSC/P. Read out was planned as the engraftment of fluorescent CFU-F and stromal cells in the recipient s bone marrow and femur.

23 18 Specific Aims: 1. Comparison of two methods for the isolation and ex vivo expansion of MSC/P a. The first method was based on plastic adherence and in vitro expansion of BM cells followed by immunomagnetic depletion of leukocytes and erythroid cells. b. The second method was based primarily on selection of putative MSC/P by direct immunodepletion of hematopoietic and endothelial cells followed by expansion ex vivo. Assessment of ex vivo expansion for both methods was to be measured by total cell count and by CFU-F assay at each passage. 2. Engraftment of non-hematopoietic cells after transplantation of whole BM Whole BM cells from FVB/N transgenic mice expressing either SMGA13.7-EGFP or SMAA8-EYFP were to be transplanted intravenously (i.v.) into 3-12 week-old, sublethally irradiated (7.0 Gy) FVB/N non-transgenic littermates. Five weeks later, bone marrow cells and femur sections from transplanted mice were to be analyzed for presence of florescence and assayed for their content of chimeric CFU-F and ex vivo cultured BM stromal cells. 3. Feasibility of transduction of MSC/P In order to assess whether the MSC/P could be genetically loaded, MSC/P were to be isolated from whole BM, and transduced using a retroviral plasmid expressing a reporter gene. Transduction efficiency was to be assessed by flow cytometry.

24 19 4. Feasibility of transplantation of cultured MSC/P MSC/P isolated from the FVB/N, SMAA8-EYFP transgenic mice and subsequent to a few passages in culture were to be transplanted i.v. into sub-lethally irradiated FVB/N non-transgenic mice. After five weeks, chimeric CFU-F content and stromal engraftment of transplanted mice were to be assayed. 5. Feasibility of transplantation of transduced MSC/P Cultured MSC/P from C57BL/6 mice were to be transduced with a retroviral plasmid expressing a reporter gene (MIEG3, a eukaryotic expression vector of the EGFP) and transplanted i.v. into 3-5 week-old, sublethally irradiated syngeneic mice. After five weeks, chimeric CFU-F content and stromal engraftment were to be assessed in the bone marrow and femorae of the recipient mice. 6. Ex vivo differentiation of MSC/P into different mesenchymal lineages BM MSC/P at or after passage five were to be grown to confluency, and under specific conditions, induction of differentiation into osteoblasts, adipocytes and chondrocytes was to be initiated. (Note: Passages are defined by the number of times a cell culture has been divided into other culture plates because it has covered 80-85% of the plastic surface, with the time between passages being anywhere from twenty four hours to two weeks.)

25 20 Null Hypothesis: Transduced and cultured cells are not transplantable. This may be due to various reasons: 1) Isolated cells may not contain MSC/P; 2) The MSC/P are not transducible due to lack of appropriate ecotropic receptor or due to silencing of the transgene expression; 3) The cells may lack homing ability to the BM or other tissue; 4) These cells may lack proliferation and differentiation ability under the conditions in which they were assayed.

26 21 MATERIALS AND METHODS 1. Bone Marrow Harvest (BMH) Murine BM was harvested as per approved protocol 2D1280 of the animal facility at Cincinnati Children s Hospital Medical Center (CCHMC) using ethical guidelines as established by CCHMC. The mice were sacrificed in a carbon dioxide (CO 2 ) chamber and then cervically dislocated. The sacrificed mice were dissected and the BM was obtained after removing muscles, tendons and ligaments from femorae, tibiae and iliac crest bones. The bones were crunched in a mortar until no visible cells could be discerned in the bones (indicated by color change, from red-pink to white). The BM cells were suspended and collected in a medium consisting of IMDM (Iscove s Modified Dulbecco s Medium, Gibco Invitrogen Corp. Carlsbad, CA) (Refer to appendix B) supplemented with 20% selected-batch FCS (Fetal calf serum, Hyclone, Tissue Culture Biologicals, Tulare, CA), 100 µm 2-ME (betamercaptoethanol, Fisher Scientific, Fairlawn, NJ), 100 IU/mL penicillin (Mediatech, Herndon, VA), 0.1 mg/ml streptomycin (Mediatech) and 2 mm L- glutamine (Mediatech). The crunched remains of the bones were discarded and the cell suspension filtered through a 100 µm filter (Fisher Scientific) to separate small pieces of bone from the BM cells. The filtered cells were concentrated by centrifugation at 500 x g for five minutes and the supernatant was discarded. The concentrated cell-button was re-suspended in 3 ml of the media described above and the cells were counted using a Burk s hemocytometer (Fisher Scientific).

27 22 2. MSC/P Isolation Method A Using the cells obtained at BMH (Materials and Methods #1) a total of 2 x 10 6 cells were cultured by CFU-F assay. The remaining cells were suspended in a medium (from here on referred to as complete medium) consisting of IMDM supplemented with 20% selected-batch FCS, 100 µm 2-ME, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, 2 mm L-glutamine, 10 ng/ml hpdgf-bb (human Platelet Derived Growth Factor, Peprotech Inc, Rocky Hill, NJ), 10 ng/ml rmegf (recombinant mouse Endothelial Growth Factor, Peprotech) and 0.2% LIF (Leukemia Inhibitory Factor, from transfected Chinese hamster ovary (CHO) cell line, kindly provided by Dr. David A. Williams at CCHMC), in order to be plated for isolation of MSC/P. The cells were plated at a density of approximately 6.5 x 10 5 cells/cm 2 on fibronectin treated (Sigma Aldrich Chemical Company, St. Louis, MO) (Materials and Methods #19), 75 cm 2 tissue culture plates (Corning Incorporated, NY). The final volume of the complete media in which the cells were suspended and subsequently plated was 12 ml per dish. The cultured plates were incubated at 37 C, in 5% CO 2 /95% air and 100% humidity for 72 hours. After 72 hours, the culture was hemidepleted by replacement of 50% of the media in each plate with complete medium supplemented with a full dose of cytokines. The plates were incubated for another 24 hours. At the end of this time, non-attached cells were eliminated from the culture by removal of the medium and by washing off the non adherent cells from the plate(s) with PBS (phosphate buffered saline without Ca and Mg, ph 6.5 ± 0.1, Media Tech). The

28 23 removed medium was spun down at 500 x g for five minutes, and filtered through a 0.2 µm filter (Fisher Scientific) in order to remove the hematopoietic cells. The filtered supernatant (minus the hematopoietic cells) was added back to the adherent cells remaining in the plates. The cells thus treated were reincubated at 37 C, in 5% CO 2 /95% air and 100% humidity. Thereafter, the culture was hemidepleted weekly, while the growth of adherent cells was monitored daily by visualization under a light microscope. After two weeks in culture, the adherent cells were detached by using CDB (Cell Dissociation Buffer, Gibco Invitrogen Corp.) in order to further deplete the hematopoietic cells. After detachment, the cells were counted using a hemocytometer. An aliquot consisting of 2 x 10 5 cells was cultured by CFU-F assay. The remaining cells, suspended in µl of PBS/1% BSA (Bovine serum albumin, Roche Diagnostics, Indianapolis, IN) buffer were incubated at room temperature (RT, C) with rat anti-mouse CD45-FITC (Flourescein isothiocyanate, Becton Dickinson, Pharmingen, San Jose, CA) and rat anti-mouse TER119-biotin (BD, Pharmingen) at a concentration of 10 µl of antibody per 10 8 cells (1:100 dilution). To reduce non-specific binding of the antibodies, mouse serum (Sigma) was added for a final concentration of 1-2% of total volume. This cocktail was incubated for 15 minutes at RT. Then the cells were washed with PBS/1% BSA buffer and incubated with streptavidin-fitc (BD, Pharmingen) for 10 minutes at RT at a concentration of 10 µl of antibody per 10 8 cells (1:100 dilution).

29 24 After washing off the excess streptavidin with PBS/1% BSA buffer, the cells were incubated with anti-isomer FITC beads (Miltenyi Biotech, Auburn, CA) (at the same concentration as for the other antibodies) for 15 minutes at 4 C. The cells were washed with copious amounts of PBS/1% BSA buffer. A small aliquot of washed cells (5,000-10,000 cells) was placed on ice for flow cytometry analysis and the remaining cells then underwent immunomagnetic depletion as follows: The labeled cells were suspended in 500 µl of PBS/1% BSA buffer and then passed through a magnetic activated cell sorter column (LD midimacs, Miltenyi Biotech) which was primed with 2 ml of PBS/1% BSA buffer. The column was washed twice with PBS/1% BSA buffer after the cells had gone through. Flow-through cells were passed through a second column as described above and then the collected cells were counted. A small aliquot of the collected cells (5,000-10,000 cells) was run through the flow cytometer along with the predepletion sample of cells (set aside earlier) to assess the depletion of hematopoietic cells. Of the remaining cells, a total of 1.2 x 10 5 cells were plated for ex vivo expansion, at a density of 2000 cells per cm 2 on a fibronectin treated, 6-well plate (Corning Inc.), in complete medium. The plate was incubated at 37 C, in 5% CO 2 /95% air and 100% humidity. In addition, an aliquot containing 1 x 10 4 cells was cultured by CFU-F assay. Any remaining cells were frozen.

30 25 3. MSC/P Isolation Method B Using cells obtained at BMH (Materials and Methods #1), a total of 2 x 10 6 cells were cultured for CFU-F assay. The remaining BM cells were suspended in µl PBS/1% BSA buffer and labeled with rat anti-cd45-fitc, rat anti- CD11b-FITC (BD, Pharmingen), rat anti-ter-119-biotin, rat anti-cd34-biotin (BD, Pharmingen), rat anti-cd31-biotin (BD, Pharmingen) and in a second step with Streptavidin-FITC at a concentration of 10 µl of antibody per 10 8 cells (1:100 dilution), in the same manner that the cells were labeled for immunodepletion in isolation Method A (Materials and Methods #2). The cells were then submitted to immunomagnetic depletion as explained in isolation Method A (Materials and Methods #2). Immunodepletion was assessed by flow cytometry analysis of pre- and post-immunomagnetic depleted aliquots of cells. Flow-through cells were plated at 2000 cells per cm 2 for ex vivo expansion, in the same medium and under identical conditions as the cells plated for ex vivo expansion in isolation Method A (Materials and Methods #2). An aliquot of flow-through cells that contained 1 x 10 4 cells was cultured by CFU-F assay. In addition, as the phenotype CD105+/ CD106+/CD45-/CD31-/CD33- /CD11b- has been reported by some authors to comprise the putative MSC/P, 28 a small portion of the isolated cells was further analyzed and phenotyped. 28 The cells were suspended in 100 µl of PBS/1% BSA buffer and labeled with rat-anti-cd105 at a concentration of 10 µl antibody/10 8 cells or 1:100 dilution and 1% mouse serum for 20 minutes at 4 C. After washing with PBS/1% BSA

31 26 buffer, the cells were incubated with APC-conjugated goat-anti-rat antibody (allophycocyanin, BD, Pharmingen) at a concentration of 10 µl antibody/10 8 cells, and 1% rat serum for 15 min at 4 C. The cells were washed and labeled with anti-cd106-biotin for 15 minutes at RT, and in a second step with Streptavidin- PE (phycoerythrin, BD, Pharmingen). Cells were washed with PBS/1% BSA buffer. Then 1-5 µg/ml of 7-AAD (7-amino-actinomycin-D, BD, Pharmingen) was added to the washed cells and the cells were analyzed by flow cytometry. 4. Ex vivo Expansion of Isolated MSC/P After isolation of MSC/P (refer to Materials and Methods #2 & #3) the plated cells were monitored daily for growth and the culture was hemidepleted weekly. At each passage (initially every two weeks, then after passage 3, whenever the cells reached approximately 80% confluency) the cells were detached using trypsin or CDB and the cells counted. A total of 1.2 x 10 5 cells at a density of 2000 cells per cm 2 were re-plated under the exact conditions that they were plated initially for ex vivo expansion. In addition, an aliquot of cells consisting of 1 x 10 4 cells was cultured for CFU-F assay. The remaining cells at each passage were frozen. The ex vivo expansion culture was monitored for growth and treated in this manner with periodic detachments and re-plating at each passage until there was no growth or until the cells had reached a plateau of growth at which time the cultured cells were detached and frozen, thereby terminating the ex vivo expansion of the cells.

32 27 5. Detachment of Adherent Cells From Plastic Trypsin (Gibco Invitrogen Corp.) or CDB (1-2 ml/well if 6-well plate or 4-6 ml/plate if 75 cm 2 plate) was placed directly onto the cells in each well or plate. After 5 minutes at RT or 10 minutes at 37 C, the detached cells were treated with FCS to stop the reaction. The detached cells were then transferred to a 50 ml conical tube (Becton Dickinson, Franklin Lakes, NJ) and the plate/well was washed with PBS to collect the remaining cells. This lavage was also transferred to the tube containing the detached cells. The collected cells were then centrifuged at 500 x g and the supernatant discarded. The cells were resuspended in the media (described in Methods and Materials #1) or in PBS/1% BSA buffer for further processing as needed. 6. Viral Transfection of Phoenix-GP cells and Retroviral Supernatant Collection A calcium phosphate transfection kit (Invitrogen Life Technologies, Carlsbad, CA) was used to transfect Phoenix-GP (Phoenix Gag-Pol, American Tissue and Cell Collection, obtained through Stanford University) cells using established protocols at CCHMC for retroviral transfection and as recommended by the manufacturer. The media used during transfection contained: 500 ml high glucose Dulbecco s Modified Eagle s medium (Cellgro, Media Tech), 55 ml FCS, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, 5 mm L-glutamine and 1% HEPES buffered saline (provided in the kit). The ph was adjusted to 7.9 by addition of NaOH (Fisher Scientific). Transfection was carried out as follows:

33 28 a. Phoenix GP cells were grown on gelatinized (Gibco Invitrogen Corp.) 75 cm 2 plates overnight at 37 C, in 5% CO 2 /95% air and 100% humidity. (If frozen cells were used, the cells were cultured for at least one passage and then transfected for 24 hours after the first passage of cells.) b. For each plate of PGP cells growing, 36 µl of 2 M CaCl 2 (provided in the Kit), 10 µg Gag and Pol DNA, 3 µg Env DNA and 8 µg of plasmid DNA, MIEG3 (all DNA provided by CCHMC) were mixed together 45,46. The final volume of this mixture was adjusted to 300 µl by addition of sterile H 2 O. c. Using a pipette, 300 µl of HEPES buffered saline was added to the above mixture, very slowly, drop-wise, while bubbling air through the solution by another pipette until a fine precipitate was formed (1-2 minutes). Once this was formed the solution was incubated at RT for minutes. d. For each plate of Phoenix-GP cells growing, 9 ml of the media described above plus 9 µl of chloroquine (Sigma) was mixed with the precipitate after incubation in Step c. e. The media was removed completely from the Phoenix-GP growing plates and replaced slowly with 10 ml of the mixture made in Step d. f. The plates were incubated overnight (minimum 12 hours) at 37 C, in 5% CO 2 /95% air and 100% humidity. g. In the morning (or after 12 hours), media was removed from the plates of growing Phoenix-GP cells and replaced with 10 ml of fresh media (without chloroquine) pre-warmed to 37 C. The cells were re-incubated for 12 hours at 37 C, in 5% CO 2 /95% air and 100% humidity.

34 29 h. After 12 hours, MIEG3 containing retroviral supernatant was removed from the Phoenix-GP growing plates and placed in 14 ml round bottom collection tube(s) (Becton Dickinson) after filtration through a 0.22 µm filter. The supernatant was stored at -80 C, if not used immediately. i. The cells were treated with fresh media (9 ml) and the plates were incubated for 12 hours at 37 C, in 5% CO 2 /95% air and 100% humidity for additional supernatant collection. j. At 12 hour intervals, the supernatant was collected in the same manner for several collections. k. The cells were discarded after the last collection on the evening of day five. 7. Transduction of MSC/P Four to six ml of the media was removed from the BM cells that were cultured on fibronectin (MSC/P isolation Method A; refer to Materials and Methods #2, #19) and replaced with 4-6 ml of the MIEG3 retroviral supernatant (Materials and Methods #6) in order to transduce the BM stromal cells with the EGFP reporter gene. This process was undertaken in one of two ways: 1) transduction was initiated on day 10 through day 14 after BMH, once per day followed by hematopoietc depletion procedure, and then the transduction was continued for four additional days post depletion starting on day 16 for 8 additional cycles of transduction (two experiments); 2) transduction was initiated after depletion of the hematopoietic cells for four days post depletion starting on

35 30 day 16 (one experiment). Negative non-transduced control cells were analyzed by flow cytometry as well as a sample of the transduced cells for assessment of transduction three days following the last transduction cycle. 8. CFU-F Assay A total of 2 x 10 6 cells at BMH, or 2 x 10 5 cells at pre-depletion stage, or 1 x 10 4 cells after depletion and at each passage, or 2 x 10 6 to 40 x 10 6 cells post transplant at BMH were plated onto three 35 mm grid-dishes (Nalge Nunc International, Apogent, USA) in a medium containing IMDM, 30% (by volume) FCS, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, 5 mm L-glutamine, 10 ng/ml rm-egf, 10 ng/ml h-pdgf-bb, and 40% by volume of 1% methylcellulose (Methocult M-3134, Stem Cell Technologies, Vancouver Canada), and cultured for ten days at 37 C, 5% CO 2 and 100% humidity. On the tenth day, the methylcellulose (containing the non-adherent cells) was removed by washing with ice-cold PBS. If the cells assayed were transgenic or transduced then the plastic adherent colonies were observed under UV light for fluorescence, and the fluorescent colonies were counted. Then the colonies were fixed in 100% methanol (AAPER, Shelbyville, KY) for 10 minutes. Otherwise, the colonies were fixed in 100% methanol directly without direct observation under UV light. The fixed colonies were then stained. (Diff-Quick, Dade Behring, Newark, NJ) according to manufacturer s instructions and the stained colonies were counted. Clones containing 50 or more cells that had a

36 31 fibroblast-like appearance (large cells with large nuclei) were counted as one colony under the light microscope. 9. Irradiation of Recipient Mice Mice were loaded evenly into a round divider box in order to equalize the dose of irradiation per mouse. A 137 cesium (Cs 137) irradiator (Model 68A, JL Shepherd and Associates, San Fernadeno, CA) was used and a dose of 7.0 Gy (dose rate = 60 cgy per minute) was administered to the mice. 10. Transplantation Seven Gy irradiated 3-12 week-old recipient mice were transplanted via the tail vein with either whole BM or the isolated and ex vivo expanded MSC/P (isolation Method A. Materials and Methods #2) from syngeneic mice that had undergone transduction with EGFP, as well as with cells from FVB/N transgenic (SMAA8-EYFP or SMGA13.7-GFP) mice. The dose of MSC/P for each mouse was 2 x 10 5 cells, whereas the dose for the whole bone marrow was 10 7 mononuclear cells per mouse. The mice were subsequently sacrificed after 4-6 weeks and organs harvested. The harvested organs were analyzed for engraftment of EGFP or EYFP positive cells in the recipient femur.

37 Whole Animal Tissue Fixation for Organ Harvest A dose of 30 mg/kg of pentobarbital was injected intraperitoneally into the mouse. After a few minutes, the chest was opened surgically and a fixative solution containing 4% paraformaldehyde (Fisher Scientific) and 0.5% gluteraldehyde (Electron Microscopy Sciences, Ft. Washington, PA) was pumped via the left ventricle to the entire body of the mouse using a peristaltic pump (Fisher Scientific) for minutes to fix the tissues. After the fixation procedure, the heart and bones were harvested. The harvested organs were placed in 10% formalin (Fisher Scientific) for hours. After this time, the bones were placed in 10% EDTA solution for two weeks for decalcification, and then paraffin embedded and sectioned (performed by the pathology department at CCHMC) for analysis. The heart was placed in PBS for further processing if indicated. 12. Induction of Ex vivo Differentiation Isolated MSC/P frozen at passages five, ten and eleven, respectively, were thawed. The cells were counted and cultured at a density of 2000 cells per cm 2 in a 6-well plate. The cells were grown to 100% confluencey in complete medium containing IMDM supplemented with 20% or 2% FCS plus 100 µm 2-ME, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, 2 mm L-glutamine, and cytokines (10 ng/ml rmpdgf-bb, 10 ng/ml rmegf and 0.2% LIF) at 37 C, 5% CO 2 /95%

38 33 air and 100% humidity for about 4 weeks. The medium was hemidepleted weekly. Once the cells were 100% confluent, the differentiation was initiated as follows: a. Osteoblasts: Ex vivo differentiation into osteoblasts was induced by replacing the media from the confluent plates with growth medium containing IMDM supplemented with 2% or 20% FCS, plus 100 µm 2-ME, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, and 2 mm L-glutamine, 0.1 µm dexamethasone (Sigma), 0.25 mm ascorbic acid (Sigma) and 10 mm 2-glycerolphosphate (Sigma). The cultures were incubated at 37 C, 5% CO 2 /95% air and 100% humidity. The cells were kept in culture for 1-6 weeks under these conditions. The media was replaced with fresh media three times per week. Differentiation was assessed by alkaline phosphatase expression using histochemical staining (Materials and Methods #17) and calcium deposition in the matrix using von Kossa s method 50 (silver nitrate staining under light emission, Materials and Methods #15) at the end of the culture period. b. Chondrocytes: Ex vivo differentiation into chondrocytes was induced by using microprecipitates (achieved by centrifugation of detached MSC/P when 100% confluent at 500 x g for 5 minutes) of MSC/P and growth in a medium containing IMDM supplemented with 20% or 2% FCS, plus 100 µm 2-ME, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, 2 mm L-glutamine, 0.1 µm dexamethasone and 10 ng/ml TGF-ß3 (Peprotech), in a low-oxygen tension incubator (lower than 10% O 2 ) for approximately 1-6 weeks. The media was replaced with fresh media three times per week.

39 34 Differentiation was assessed by Alcian Blue staining 50 (Materials and Methods #16) of glycosylaminoglycan in the matrix, indicative of chondrocyte differentiation at the end of the culture period. c. Adipocytes: Ex vivo differentiation into adipocytes was induced by replacement of the media in which the MSC/P were growing, with a medium containing IMDM supplemented with 20% FCS, 100 µm 2-ME, 100 IU/mL penicillin, 0.1 mg/ml streptomycin, 2 mm L-glutamine, 0.1 µm dexamethasone, 50 µm indomethacin (Sigma) and 5 µg/ml of insulin (Sigma). The cultures were incubated at 37 C, 5% CO 2 /95% air and 100% humidity for 3-4 weeks and the media was replaced with fresh medium three times per week. Differentiation was assessed by 0.5% Oil Red O staining 47 (Materials and Methods #14) of adipose tissue (indicative of adipocyte differentiation) at the end of the fourth week of culture. 13. Tissue Sectioning and Immunostaining Bones were embedded in paraffin wax and sectioned at 4 µm thickness according to standard protocols of the Pathology Department of CCHMC for direct observation of fluorescent cells in the tissue sections. Three to four sections were placed on each slide and observed under a fluorescent microscope (average excitation wavelength of 480 nm and average emission wavelength of 530 nm). If fluorescence was observed in any of the tissue sections, the slides were placed in a hot air oven heated to C for at least 30 minutes. They were then washed in three changes of 100% xylene (Fisher Scientific) (10 minutes per

40 35 wash), followed by two washes in each of 100% ethyl alcohol (EtOH) (AAPER), 90% EtOH, 80% EtOH, and then 70% EtOH (three minutes per wash), respectively. Slides were then placed in running tap water for 5 minutes and then transferred to PBS for 5 minutes. At this stage, tissue sections were de-paraffinized and ready for immunostaining. A modified Mouse-On-Mouse (M.O.M) VectaStain (Vector Laboratories, Burlingame, CA) kit for immunostaining tissue sections was used for immunostaining with a monoclonal antibody to smooth muscle alpha-actin, clone 1A4 (Sigma-ACC) as described below: a. A hydrophobic barrier was placed around each section of bone on the slide using a Pap pen, and the slides were placed in a wet chamber. Then, 150 µl of mouse Ig blocking reagent (supplied in the M.O.M kit) diluted in PBS according to the manufacturer s instructions was placed on each section for one hour at room temperature. At the end of the hour, the slides were washed twice with PBS for 2 minutes. b. After the slides were dry, 150 µl of working solution of M.O.M diluent was placed on each section for 5 minutes. c. At the end of 5 minutes excess diluent was removed from the slides and 150 µl of 1A4-anti-smooth muscle antibody diluted 1:400 in M.O.M diluent was added to each section for 30 minutes at room temperature. As a negative control, primary antibody was omitted from one section to determine the extent of non-specific staining.

41 36 d. 150 µl of M.O.M-supplied biotinylated anti-mouse antibody (1:250 in M.O.M diluent) was added to each tissue section and incubated for ten minutes. e. The slides were washed twice for 2-3 minutes with PBS. f. After the wash steps, 150 µl of a 1:20 dilution of Texas Red streptavidin (Vector Laboratories) was added onto each section in a wet chamber and incubated in the dark for 30 minutes at room temperature. g. The slides were washed twice for ten minutes with PBS. h. A drop of Vectashield (Vector Laboratories) was placed on the sections, which were then cover-slipped and sealed with nail polish to protect the immunostained tissue until sections were analyzed via the microscope under UV light using a rhodamine filter. 14. Oil Red O Staining for Adipocytes Staining of adipose in MSC/P cultures was carried out as follows. The media was removed from MSC/P cultures and the cells were fixed in 4% formaldehyde/1% calcium (Fisher Scientific) for one hour, after which time the fixative was removed and the cells were stained directly in the culture plates with a mixture made up of three parts Oil Red O (Sigma) dissolved in isopropyl alcohol (99%, Fisher Scientific), and two parts dh 2 O for minutes. At the end of that time the dye was replaced with 70% EtOH followed immediately by 2-3 washes in dh 2 O. The cells were counterstained with hematoxylin for one minute and then washed twice with dh 2 O. At the completion of the staining

42 37 procedure, cells were observed under the light microscope for presence/absence of stain. 15. Von Kossa Staining for Osteoblasts Staining calcium deposits in MSC/P cultures, indicative of osteoblast differentiation was carried out using the von Kossa method as follows. The media was removed from MSC/P cultures and the cells were fixed in 4% formaldehyde for one hour (directly in the plates) after which time the fixative was replaced with 5% silver nitrate (Sigma) and left for one hour under a 60 watt lamp to activate the reaction or until the calcium turned black. At the end of the hour, the silver nitrate was washed off three times with dh2o and the cells were saturated with 5% sodium thiosulfate (Sigma) for five minutes. The cells were then washed once with tap water and once with dh2o. Then the cells were counter-stained with Nuclear-fast Red (Sigma) for five minutes and washed with dh2o. At the completion of the staining procedure, cells were observed under the light microscope for the presence/absence of brown or black staining, indicative of calcium deposits. 16. Alcian Blue Staining for Chondrocytes Staining for glycosylaminoglycan deposits in MSC/P cultures as produced by chondrocytes was carried out as follows. The media was removed from the confluent MSC/P cultures and the cells were fixed directly in 4% paraformaldehyde for minutes. The cells were washed 2-3 times with PBS

43 38 after fixing. Then the cells were treated with 1% Alcian Blue (Fisher Scientific) for 30 minutes. At the end of 30 minutes, excess stain was removed and the cells were washed with 0.1 M HCl (Fisher Scientific) for 3-10 minutes, followed by washing with PBS. At the completion of the staining procedure, cells were observed under the light microscope for presence/absence of light blue staining in the matrix, indicative of glycosylaminoglycan. 17. Alkaline Phosphatase Immunostaining for Osteoblasts Osteoblast differentiation is indicated by expression of alkaline phosphatase in MSC/P cultures. Detection of alkaline phosphatase in MSC/P cultures using alkaline phosphatase histochemical staining kit (Sigma) was carried out as follows; The media was removed from the confluent MSC/P cultures and the cells were fixed directly in 4% formaldehyde for minutes. The cells were washed 2-3 times with PBS after fixing. Then the cells were immersed with the alkaline dye mixture (diazonium salt and naphthol alkaline solution, provided in the kit) and incubated at RT for 15 minutes. After the incubation, the cells were washed with dh 2 O. Immediately afterwards, the cells were counterstained with hematoxylin (provided in the Kit) for 2 minutes at RT, and washed a few times with tap water. At the completion of the staining procedure, cells were observed under the light microscope for presence/absence of staining.

44 Labeling and Separation of MSC/P for Lin and c-kit Total BM cells immediately after BMH were suspended in PBS with 5% FCS and 2% mouse serum, then stained for 20 minutes at RT, using a cocktail of fluorescence (FITC)-conjugated monoclonal anti-mouse antibodies specific for the mature cell lineage antigens (Lin) including CD45R (B220, Clone RA3-6B2), Gr-1 (Ly6G, Clone RB6-8C5), CD4 (L3T4, Clone GK1.5), CD8a (Ly-2, Clone ), Mac-1 (CD11b, CloneM1/70), CD3e (Clone 145-2C11), and a biotinylated monoclonal antibody for TER119 (Ly-76) (all from BD, Pharmingen, and at a concentration of 1 µg/100 µl PBS). After a wash with copious amounts of PBS, cells were stained for 20 minutes at RT with Streptavidin-FITC (BD, Pharmingen), APC-conjugated anti-mouse CD117 (c-kit, clone 2B8) and in some experiments with PE-conjugated anti-mouse Sca-1 (Ly6A/E, clone D7). Finally, 1 µl of 7-AAD was added. Lin-/c-Kit+ and Lin-/c-Kit- cells were separated by fluorescence activated cell sorting in a FACS Vantage DiVa (Becton Dickinson, San Jose, CA). 19. Fibronectin Treatment of Tissue Culture Plates The tissue culture plates for use in culturing whole BM or MSC/P were treated with 4µg of bovine fibronectin per cm 2 by placing the fibronctin solution for two hours onto the surface of the plates, just prior to plating the cells. At the end of two hours the fibronectin was removed from the plates. The plates were then ready to be used for culturing purposes.

45 40 FACILITIES AND RESOURCES This study was conducted in its entirety at research facilities of Cincinnati Children s Hospital Medical Center, Division of Experimental Hematology under the direct supervision of Jose Cancelas, MD, Ph.D. Director of the Research Division at Hoxworth Blood Center, University of Cincinnati. Thomas Leemhuis, Ph.D. Director of the Cellular Therapies Division at Hoxworth Blood Center also advised on this project. Available resources at the Division of Experimental Hematology at Cincinnati Children s Hospital Medical Center were used in conducting this research.

46 41 RESULTS Isolation and Ex vivo Expansion of MSC/P Two methods of isolation of putative MSC/P were compared and their ex vivo expansion was analyzed. At least 9 experiments were conducted for each method of isolation. Efficiency of immunodepletion: After immunodepletion of hematopoietic cells for isolation Method A, and hematopoietic and endothelial cells for Method B, the efficiency of depletion was assessed by comparison of flow cytometer results from pre- and post-depletion samples for each method of isolation (Table 1 & 2). Immunodepletion of hematopoietic cells by Method A was 43.0 ± 20%, indicating that 53.3 ± 22.9 % of the cells had hematopoietic lineage origin. The target population after immunodepletion of hematopoietic/endothelial cells for method B was enriched to a 60.1 ± 20%, suggesting that 39.8 ± 20.1 % of the cells had hematopoeitic/endothelial lineage origin. Ex vivo Expansion: The number of stromal cell showed a greater than 5.7 x fold increase after 19 passages when MSC/P were cultured by Method A. In addition, a 471 fold increase in CFU-F frequency was observed (CFU-F frequency at BMH was.0017 ± % (or 1.7 ± 0.4 CFU-F per 10 5 cells), ± % at passage 2 (or CFU-F per 10 5 cells) and 0.97 ± 0.00% at passage 18 (or 970 CFU-F per 10 5 cells)). Method B showed a limited capacity for ex vivo expansion, with a 1.3 fold increase in absolute number of cells observed after 2 passages, and a 127 fold increase in CFU-F frequency observed at passage 1. The

47 42 cells isolated by Method B did not grow beyond passage 3. Refer to figures 1, 2, 3, 4 and 5 for depiction of the above results. Phenotype analysis of a putative MSC/P cell population: The phenotype of endothelial and hematopoietic depleted cell population (via Method B) was analyzed by flow cytometry, immediately after isolation (Table 3). The results indicate that 8.1 ± 6.0% of the cells were CD105+/CD106-, 10.2 ± 8.8% were CD105-/CD106+, 64.8 ± 26.8% were positive for both markers and 16.8 ± 18.8% were expressing neither marker on their surface. The cells that comprise the phenotype of the putative MSC/P had a limited capacity for expansion ex vivo under the conditions of this experiment. Because of these results the subsequent experiments involved in this project were carried out exclusively on cells isolated via Method A. Transduction Three transduction experiments with a marker gene were performed on isolated MSC/P (Method A) obtained from C57BL/6J mice. Experiment one: Transduction with MIEG3 viral supernatant was carried out after depletion of hematopoietic cells for four rounds (on four separate days) starting on day 2, post depletion. The efficiency of transduction was 10%. Experiment two: Transduction was carried out using a different batch of MIEG3 viral supernatant. Total bone marrow cells were treated for four rounds of transduction before depletion of hematopoietic cells starting on day -4 prior to depletion, then after hematopoietic depletion, four additional rounds of

48 43 transduction were carried out starting on day 2 post depletion. Transduction efficiency was 71.32% (Figure 6). Experiment three: This transduction experiment was carried out on MSC/P that were previously transduced (10% efficiency) before depletion and then frozen at passage 1 immediately after hemidepletion. After thawing, four additional rounds of transduction were carried out on these putative MSC/P using a different batch of viral supernatant. The efficiency of transduction was 49% in this case. Transplantation a. Total bone marrow from transgenic FVB/N mice To analyze the engraftment of SMAA8-EYFP expressing marrow stromal cells that are derived from MSC/P, total bone marrow cells from transgenic mice were transplanted into recipient wild type mice. Two experiments were conducted for this objective. Experiment one: Two sets of mice were analyzed. One set of three FVB/N wild type mice, 8-12 weeks old, were transplanted with SMAA8-EYFP FVB/N total bone marrow cells. The other set of three recipient mice also 8-12 weeks old were transplanted with SMGA13.7-EGFP FVB/N total bone marrow cells. After 5 weeks the mice were sacrificed, fixed, and the organs harvested for analysis. Presence of florescence in two regions of the femur specifically areas surrounding some intermediate vessels (pre/post sinusoidal regions), and, the endosteal and periosteal areas were observed in two out of three mice that had received the

49 44 SMAA8-EYFP total bone marrow cells. The femur sections were counterstained with Texas Red conjugated anti smooth muscle alpha actin antibody. Red/orange fluorescence was observed surrounding the pre- or post-sinusoidal vessels of the femur indicating presence of smooth muscle "-actin at the corresponding regions of the femur that had previously shown yellow fluorescence (Figures 7 & 8). No fluorescence in femur sections was observed for any of the mice that were transplanted with SMGA13.7-EGFP transgenic cells, confirming absence of differentiation into (-actin-expressing cells in the bone marrow. Experiment two: In this experiment total bone marrow from transgenic mice (SMAA8-EYFP only) was transplanted into eight, 3-5 week old wild type FVB/N recipient mice. Two mice died within days of transplantation. After 5 weeks, 3 out of the six surviving mice were sacrificed and their bone marrow harvested for assessment of engraftment by analysis of chimeric content of CFU-F assay and by ex vivo expansion assay. No fluorescence was observed in any of the ex vivo expansion assays of the recipient mice. CFU-F assay also showed no fluorescent chimeric colonies. The remaining 50% of transplanted mice were fixed and organs harvested for analysis of engraftment. Of the recipient mice whose tissues were fixed for analysis, no fluorescence was observed in any of the femur sections. b. Transgenic Lin and c-kit Phenotyped BM Cells To determine whether the cells expressing SMAA8-EYFP were derived from hematopoietic or non hematopoietic lineages (Lin-/c-Kit+ phenotypic cells

50 45 or Lin-/c-Kit- phenotypic cells), 5 FVB/N non transgenic, sublethally irradiated, 3 week old mice were transplanted with SMAA8-EYFP FVB/N BM cells that had been separated via the flow cytometer into the phenotypes described above. Experiment One: Three recipient mice were transplanted with 23,000 transgenic BM cells with the Lin-, c-kit+ phenotype (CD45R-/Gr-1-/CD4-/CD8a-/CD11b- /CD3e-/TER119-/CD117+/ or Sca-1+). Five weeks later the mice were sacrificed. BM was harvested from one of the mice for assessment of engraftment by analysis of chimeric content of CFU-F assay and by ex vivo expansion culture. The other two mice were fixed for analysis of engraftment in tissue sections. There were no chimeric CFU-F colonies found in the CFU-F assay or in the ex vivo expansion assay. Furthermore, there was no fluorescence observed in the tissue sections which would indicate non engraftment of the donor cells. Experiment Two: Two recipient mice were transplanted with 18,500 transgenic BM cells with the Lin-, c-kit- phenotype (CD45R-/Gr-1-/CD4-/CD8a-/CD11b- /CD3e-/TER119-/CD117-/ or Sca-1-). Five weeks later the mice were sacrificed. BM was harvested from one of the mice for analysis of chimeric content via CFU-F assay and ex vivo culture. The other mice were fixed for analysis of engraftment in tissue sections. There were no chimeric colonies present in the CFU-F assay or the ex vivo expansion assay. Furthermore, there was no fluorescence observed in the tissue sections which would indicate non engraftment of the donor cells.

51 46 c. Transduced MSC/P (C57BL/6J derived) To assess the engraftment of ex vivo manipulated and EGFP transduced MSC/P, transduced MSC/P were intravenously transplanted into syngenic, sublethally irradiated, 3-5 week-old, recipient mice. The experiment was performed twice. Experiment one: Six recipient mice were transplanted with 2.5 x 10 5 passage one, 71.3% transduced MSC/P. After five weeks, three out of six mice were sacrificed and their BM was harvested for assessment of engraftment by analysis of chimeric content in CFU-F assay and by ex vivo expansion assay. The remaining recipient mice were fixed and their organs harvested for analysis of engraftment in tissue sections. Questionable fluorescence may have been observed in the femur sections of one of the recipient mice, but due to high background fluorescence and also because there were no other apparent patches of fluorescence, this observation was called negative. The femur sections of other mice showed no fluorescence. In addition, upon examination of the ex vivo cultures, no fluorescent cells or CFU-F were observed in any of the ex vivo expansion assays of the recipient mice. Experiment two: Five recipient mice were transplanted with 2.0 x 10 5 passage one, transduced cells that showed 49.5% transduction efficiency. Of the four mice that survived, two were fixed for analysis of engraftment by examination of the tissue sections. The BM from the other two mice were harvested for assessment of engraftment by analysis of chimeric content of CFU-F assay and by ex vivo expansion culture. No fluorescent colonies could be seen in either the CFU-F

52 47 assay or ex vivo expansion assay of any of the recipient mice. Tissue sections were negative for fluorescent chimeric content. d. Transgenic MSC/P To assess the engraftment of ex vivo manipulated and expanded MSC/P with a strong expression of fluorescent protein in the bone marrow stromal cells, SMAA8-EYFP FVB/N MSC/P were isolated and subsequently transplanted into sublethally irradiated recipient wild type FVB/N mice. Eight recipient mice were transplanted intravenously with 2.0 x 10 5 passage two transgenic MSC/P. Of the six mice that were kept alive until week five, three were sacrificed for BMH to assess engraftment by analysis of chimeric content in CFU-F assay and ex vivo expansion culture. The remaining three mice were fixed and organs harvested for engraftment analysis by tissue sections. No florescence was seen in either the tissue sections or in the ex vivo cultures that were assayed for CFU-F chimeric content and ex vivo expansion. Upon close examination of the tissue sections, it was apparent that, the quality of the tissue sections was very poor. This may have affected the outcome that was observed. Ex vivo Differentiation of MSC/P In order to assess whether the isolated cells were the putative MSC/P, differentiation into the three mesenchymal lineages was induced. At least two separate experiments using MSC/P frozen at different passages were set up for each lineage to be differentiated.

53 48 a. Ex vivo differentiation of MSC/P into adipocytes After four weeks, the cultures of the isolated MSC/P set up for adipocyte differentiation were analyzed. When observed by light microscopy, the cultures appeared to contain fat granules (Figure 9). The presence of neutral fatty acid vacuoles was confirmed by staining with Oil Red O. The cultures were counterstained with hematoxylin (Figure 10). Of the two cultures set up for differentiation of adipocytes, cells at passage 5 and passage 11, respectively, were used for differentiation. In both cultures, over 70% of the cells (as seen under the microscope) had fat granules in their cytoplasm, indicative of the MSC/P differentiation into adipocytes. The remaining 30% of the cells had the same phenotype as the cells prior to the initiation of differentiation, and showed a fibroblast like morphology without the presence of gat granules. b. Ex vivo differentiation of MSC/P into osteoblasts/osteocytes Experiment one: Of the two cultures set up for osteoblast differentiation, one was at passage 5 and the other at passage 11. After six weeks under specific differentiation conditions, the cultures of the isolated MSC/P for osteoblast differentiation were analyzed. The cultures were stained with silver nitrate (von Kossa s method) for the presence of calcium and alkaline phosphatase. Results of the silver nitrate staining were inconclusive. Alkaline phosphatase staining was positive, indicating differentiation of the MSC/P into osteoblasts in about 30% of the cultured cells (Figure 12).

54 49 Experiment two: The experiment was repeated for osteoblast differentiation after changing the FCS from 20% to 2%, as a constituent of the differentiation media, while all other conditions remained constant. One week after initiation of differentiation, the cultures were stained with silver nitrate. A few regions of the wells (approximately 10%) were stained brown/black, indicating calcium deposition as a result of osteoblast differentiation (Figure 11). The result of alkaline phosphatase immunostaining of these cultures was complimentary to the calcium staining by silver nitrate, and confirmed initiation of MSC/P differentiation into calcium depositing osteocytes. c. Ex vivo differentiation of MSC/P into chondrocytes Experiment one: The three cultures were at passages 5, 10 and 11 when chondrocyte differentiation was initiated. After four weeks under specific differentiation conditions, the cultures were observed for the presence of chondrocytes by Alcian Blue staining. No glycosylaminoglycan indicative of differentiation of the MSC/P into chondrocyte was observed. Experiment two: Experiment 1 was repeated with two of the cultures set up at passages 10 and 11 for chondrocyte differentiation by changing the FCS from 20% to 2% as a constituent of differentiation media, while all other conditions were kept constant. One week after initiation of differentiation, over 60% of the cultured cells were stained blue by Alcian Blue staining, indicative of the presence of glycosilaminoglycan and differentiation of the MSC/P into chondrocytes (Figure 13).

55 50 A B B Figure 1. Marrow stromal cultures, A. Phase contrast; B. Epifluorescent SMAA8-EYFP FVB/N transgenic cells under UV light. O.M. 200x. A B Figure 2. A. CFU-F morphology (Diff-Quick staining); B. SMAA8-EYFP FVB/N transgenic CFU-F (epifluorescence). O.M. 200x.

56 51 A B Figure 3. Morphological differences between putative MSC/P isolated via two different methods. A: Marrow stromal cells isolated by Method A at passage 4. B: Marrow stromal cells isolated by Method B at passage 3. O.M. 200x.

57 52 Fold Expansion 1.00E E E E E E-01 Method A Method B Passage Number Fold Expansion 1.00E E E E E+03 Method A Method B 1.00E Average Numer of Days in Culture (at each passage) Figure 4. BM MSC/P fold-expansion over time in culture after selection by Methods A and B. (top: as depicted by passage numbers. bottom: as depicted by number of days in culture, starting at passage #2.)

58 Method A Clonogenic Efficiency (%) Method B 0.0 BMH Post depletion Passage Number Clonegenic Efficiency (%) Method A Method B Average Number of Days in Culture (at each passage) Figure 5. CFU-F frequency variation over time in culture after selection by Methods A and B. (Top: as depicted by passage number. Bottom: as depicted by number of days in culture.)

59 54 R3 R1 R % Figure 6. Flow cytometry analysis of MIEG3 transduced marrow stromal cells showing 71.32% transduction efficiency.

60 Figure 7. Top: Femur section of a wild type recipient mouse, showing donor transgenic cell engraftment (GFP filter) surrounding an intermediate size bone marrow vessel. Bottom: Same femur section counter-stained with Texas Red conjugated anti-smooth muscle "-actin antibody (rhodamine filter) suggestive of smooth muscle differentiation. O.M. 400x. 55

61 56 Cortical bone Periosteum Figure 8. Periosteal region of femur showing graft of EYFP+ donor cells in a chimeric mouse. O.M. 400x.

62 57 Figure 9. Ex vivo adipocytic differentiation of MSC/P (phase contrast). O.M. 200x. Figure 10. Fig. 9, after Oil Red O staining, demonstrating the accumulation of neutral fatty acids in cytoplasmic vacuoles. O.M. 200x.

63 58 Figure 11. Presence of calcium deposits (black arrows) as stained by silver nitrate indicative of calcium deposition by differentiated MSC/P into osteoblasts/osteocytes. O.M. 200x. Figure 12. Presence of alkaline phosphatase in differentiated MSC/P indicative of osteoblasts. O.M. 400x.

64 Figure 13. Presence of glycosylaminoglycans in differentiated MSC/P indicative of chondrocyte differentiation. Alcian Blue staining. O.M. 200x. 59