The unique pluripotent character of human embryonic stem cells (hESCs) places them in the forefront of scientific research, especially as they hold great promise for application in regenerative medicine, as well as drug discovery and toxicity analyses. Conventionally hESCs are cultured on mitotically inactivated mouse embryonic fibroblasts (MEFs) that are derived from E13.5 mouse embryos. One of the biggest challenges in the hESC field is the development of a reproducible and defined hESC culture system that would eliminate batch-to-batch variability of the MEFs as well as exposure to feeder cells that makes hESCs less applicable for clinical use. Previous studies have shown that maintenance of pluripotency can be achieved using Matrigel, a mixture of ECM components, or ECM derived from MEFs or human fibroblasts (Xu, et al., 2001, Klimanskaya, et al., 2005). Other groups have succeeded in culturing feeder-free hESCs by using extracellular matrix (ECM) proteins, such as fibronectin, vitronectin or laminin, as substrates for hESC culture in the absence of feeders, confirming that ECM plays a key role in maintaining hESC growth (Amit, et al., 2004, Braam, et al., 2008, Baxter, et al., 2009, Rodin, et al., 2010).The aim of this work was to investigate the ECM deposited by MEF feeder cells and to isolate and identify proteins in the ECM that support undifferentiated growth of hESCs in the absence of feeders. We have investigated whether matrices derived from different passage feeders differ in their ability to support pluripotency. I also assessed the integrin receptor profile of hESCs in order to define the mechanisms of ECM engagement. ECM was extracted from two strains of feeder cells, CD1 x CD1 and MF1 x CD1, at passages 4 (early passage), 9 and 14 (late passage), and assessed for its ability to support hESC self-renewal over at least 3 passages. Tandem mass spectrometry was used to analyse the ECM composition of each MEF line, thereby allowing a comparison between different passages and different cell lines. More than 100 proteins were identified for each sample, the majority of which were ECM proteins and shared between different passage feeders. As predicted, fibronectin, which is known to support hESC self-renewal was the most prevalent species in all MEF-derived matrices. Furthermore a proteomic analysis of matrix derived from hESCs cultured in feeder-free conditions on fibronectin coating substratum revealed a number of proteins shared between supportive MEF populations and hESC, suggesting other potential candidates that may either assist or interfere with the maintenance of pluripotent hESCs. Of the proteins identified fibrillin-1, perlecan, fibulin-2 were tested as substrates for culturing hESCs in the absence of feeders, with the prospect of developing an optimised feeder-free culturing system that uses a combination defined animal-free substrates. Finally this study sought to dissect the interaction between ECM and growth factors and how these extrinsic factors may affect self-renewal and maintenance of pluripotency-associated gene expression. Interruption of hESC attachment, as well as removal of growth factors appeared to affect transcript levels of pluripotency genes, OCT4 and NANOG, suggesting that the microenvironment can influence hESC fate.

The limited treatment options available for heart disease patients has lead to increased interest in the development of embryonic stem cell (ESC) therapies to replace heart muscle. The challenges of developing usable ESC therapeutic strategies are associated with the limited ability to obtain a pure, defined population of differentiated cardiomyocytes, and the design of in vivo cell delivery platforms to minimize cardiomyocyte loss. These challenges were addressed in Chapter 2 by designing a cardiomyocyte selectable progenitor cell line that permitted evaluation of a collagen-based scaffold for its ability to sustain stem cell-derived cardiomyocyte function (“A P19 Cardiac Cell Line as a Model for Evaluating Cardiac Tissue Engineering Biomaterials”). P19 cells enriched for cardiomyocytes were viable on a transglutaminase cross-linked collagen scaffold, and maintained their cardiomyocyte contractile phenotype in vitro while growing on the scaffold. The potential for a novel cell-surface marker to purify cardiomyocytes within ESC cultures was evaluated in Chapter 3, “Dihydropyridine Receptor (DHP-R) Surface Marker Enrichment of ES-derived Cardiomyocytes”. DHP-R is demonstrated to be upregulated at the protein and RNA transcript level during cardiomyogenesis. DHP-R positive mouse ES cells were fluorescent activated cell sorted, and the DHP-R positive cultured cells were enriched for cardiomyocytes compared to the DHP-R negative population. Finally, in Chapter 4, mouse ESCs were characterized while growing on a clinically approved collagen I/III-based scaffold modified with the RGD integrin-binding motif, (“Collagen (+RGD and –RGD) scaffolds support cardiomyogenesis after aggregation of mouse embryonic stem cells”). The collagen I/III RGD+ and RGD- scaffolds sustained ESC-derived cardiomyocyte growth and function. Notably, no significant differences in cell survival, cardiac phenotype, and cardiomyocyte function were detected with the addition of the RGD domain to the collagen scaffold. Thus, in summary, these three studies have resulted in the identification of a potential cell surface marker for ESC-derived cardiomyocyte purification, and prove that collagen-based scaffolds can sustain ES-cardiomyocyte growth and function. This has set the framework for further studies that will move the field closer to obtaining a safe and effective delivery strategy for transplanting ESCs onto human hearts.

Objectives: The effects of estrogens on immune system formation and function are well documented. Diethylstilbestrol (DES), a synthetic estrogen, has been linked to neoplasia and immune cell dysfunction in humans and animals exposed in-utero. In-vitro effects of DES exposure of murine embryonic stem (ES) cells on the early embryonic immune system development and the expression of cellular surface markers associated with common hemangioblastic and hematopoietic precursors of the endothelial, lymphoid & myeloid lineages were investigated. Hypothesis: Early ES cell expression of CD45 a marker common to lymphoid lineage hematopoietic stem cells and differentiation of lymphoid lineage precursors are affected by in-vitro exposure to DES. Methods: Murine ES cells were cultured using a variety of techniques: an OP9 co-culture system, and formation of embryoid bodies (EBs) in a liquid medium and hanging drop system. The OP9 co-culture system did not appear to give rise to well differentiated lymphoid lineage cells during 12 days of differentiation. The hanging drop EB culture system, previously shown to promote differentiation of endothelial and lymphoid precursor cells, was chosen for further studies of ES cell differentiation. ES cells were harvested at five time points: undifferentiated (day 0), and differentiated (days 3, 8, 12 and 16). Differentiating ES cells were treated with DES beginning on day 3. The synthetic estrogen, DES, was chosen as a treatment because of its similar potency to 17β estradiol and documented association with neoplasia in women exposed in-utero. Surface marker expression, measured by real-time RT-PCR amplification, was recorded using fluorogenic TaqMan(R) probes designed specifically for the surface proteins of interest: oct4, c-Kit, Flk1, ERα, ERβ, CD45, Flt1, & VE-cadherin. Analysis & Results: Changes in surface marker gene expression between day 0 and day 16 of differentiation were analyzed using the RT-PCR threshold counts (CT) and the comparative threshhold cycle method. The expression of each target mRNA was normalized internally to a housekeeping gene (18s rRNA) and calculated relative to day 0. ANOVA (Type 3 fixed-effects analysis, SAS) was performed using the unexponentiated ΔΔCT values. The effects of DES, time, and the interaction between DES and time were evaluated for days 8, 12 and 16. Additionally, the effects of DES on the expression of each marker were evaluated for day 16. Expression of estrogen receptor receptor α & β (ERα & β) in the EBs was established, and did not appear to be affected at any time by treatment with DES. ERα was expressed in significant levels on day 16, while ERβ was expressed in low levels throughout the period of differentiation. The expression of the cell surface marker, c-Kit was significantly (P<0.0001) altered by the presence of DES between the three time points sampled. The expression of the VEGF receptor, Flt1, and the adhesion molecule, VE-cadherin, markers of endothelial cells, were also significantly (P<0.026) altered by treatment with DES on day 16 of differentiation. Treatment with DES appeared to have no effect on the expression of CD45, a marker common to lymphoid precursor cells. Conclusions: These results indicate the presence of estrogen receptors in differentiating ES cells as early as day three in-vitro (ERβ) until day 16 (ERα). DES alters expression of common hemangioblastic and hematopoietic precursor, as well as endothelial lineage markers, but has no effect on expression of the marker of lymphoid lineage development before day 16. These effects coincided with the expression of ERα. The enduring effects of DES exposure in-utero may not be manifest in this ES model, or may occur at later stages of differentiation or in selected subpopulations of CD45+ cells. / Master of Science

Embryonic stem (ES) cells, which possess proliferating and differentiating abilities, are a potential source of cells for regenerative medicine. Nowadays, the challenge in using ES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential towards the derivation of a specific cell fate. This study is aimed to establish a simple and efficient method to derive ES cells into neural lineage cells and examine the safety and efficacy of derived cells in a mouse ischemic stroke model. To explore the underlying mechanisms responsible for lineage commitment of stem cells, Notch signaling and serotonin responses are also studied. / In a non-contact coculture system, mouse ES cells (D3 and E14TG2a) were cocultured with the stromal cells MS5 for eight days. On the other hand, human ES cells (H9 and H14) were directly cocultured with MS5 in a contact manner for two weeks. Derived cells were further propagated in a serum-free medium and selected subsequently in a differentiating medium. The cell viability, numbers, phenotypes and lineage-specific gene expression profile were evaluated at stages of induction, propagation and selection. / In vivo, behavioral assessments of ischemic mice after transplantation of mouse ES cell derivatives revealed a significant improvement in spatial learning and memory ability as compared to ischemic mice without cell therapy. Histology of brain sections of transplanted mice demonstrated the migration of BrdU+ cells to the CA1 region of the hippocampus, which was evident of both an increase of pyramidal neuron density and normalized morphology. Teratoma development was found in one out of 17 transplanted mice. / MS5 was noted to express genes encoding neurotrophins and neuroprotective factors. Functional tests showed that MS5 exerted neurotrophism on neuroblastoma cell lines (SK-N-AS, SH-SY5Y, and SK-N-MC) and ES cells. The numbers of viable cells and the proportion of neural subtypes derived from ES cells at three stages of the culture system were significantly higher than those of the control cultures without MS5 induction, respectively. MS5 cocultures generate a relatively higher yield of neural lineage cells but select against the mesodermal and endodermal lineage derivatives. Together with non-contact MS5 coculture, serotonin could further increase the proportion of neural precursors and accelerate maturation of neural progenitor cells in a synergistic manner. During the induction phase with non-contact MS5 coculture, the Notch inhibitor could significantly decrease the number of derived neural precursors and instigate non-neural differentiation. With the supplement of the Notch inhibitor, serotonin could neither promote the expression of neuroectodermal genes nor enhance the proportion of neural precursors in MS5-cocultured ES cells. Notably, in the propagation of undifferentiated human ES cells, Notch signaling was also found to play an active role in maintaining cell survival. / The Notch inhibitor (gamma-secretase inhibitor) and serotonin were supplemented into induction cultures to investigate the roles of Notch signaling and the neurotransmitter serotonin in neural differentiation. For in vivo study, mouse ES cell-derived cells were labeled with BrdU and implanted onto the caudate putamens of mice having undergone transient occlusion of bilateral common carotid arteries and reperfusion to induce cerebral ischemia. Spatial learning and memory ability of transplanted mice were assessed in a water maze system. Histological assessment was also conducted on brain sections of mice three weeks post transplant to examine the migration and homing of implanted cells. / This study describes a simple and efficient differentiation protocol to derive mouse ES cells and human ES cells into neural lineage cells. Derived cells appear to significantly improve cognitive functions in a mouse ischemic stroke model. Data of the study suggest that MS5 cells may exert a neurotrophic effect on ES cells. With MS5 coculture, serotonin synergistically promotes neural commitment and facilitates maturation of derived neural precursors in ES cell cultures. In contact coculture with MS5, Notch signaling is shown to play a role in the directed neural differentiation of human ES cells, whereas in maintenance culture, Notch signaling is also important to cell survival of human ES cells. Thus, Notch signaling through cell-cell interaction may explain, at least partially, the difference between mouse ES cells and human ES cells in cell growth ability when seeded at low cell densities. / Yang Tao. / Adviser: Ho Keung Ng. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: November 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 161-194). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Mouse embryonic stem (ES) cells have played a crucial role in studying developmental processes and gene function in vivo. They are extremely useful in the generation of transgenic animals as they can be genetically manipulated and subsequently microinjected into blastocyst stage embryos, where they combine with the inner cell mass and contribute to the developing embryo. Some of the resulting pups are chimaeric, consisting of a mixture of cells derived from the host blastocyst and the injected ES cells. We have identified several ES cell clones arising from gene targeting experiments with an impaired capacity to generate viable chimaeras. When injected into blastocysts, these clones cause embryonic death during mid to late gestation, suggesting that the cells are able to contribute to the embryo but interfere with normal embryonic development. The aim of this work was to identify the underlying changes in the transcriptome, epigenome or cell surface markers that have occurred in these compromised ES cells and to further define the developmental phenotype of the chimaeric embryos. Different stages during development were analysed and whereas there was little difference in embryonic death at gestational day e13.5, there was a significant decrease in embryos surviving to gestational day e17.5. Additionally, severe haemorrhaging was observed in all the dead embryos and small foci of haemorrhaging could also be seen in a number of embryos that were still alive. This was also observed at e13.5, albeit to a less severe extent. Using RNA sequencing to discover differences in the transcriptome between control ES cells and the compromised ES cells, five genes were identified that were downregulated in the compromised cells. Four of these, Gtl2, Rtl1as, Rian and Mirg are all located in the imprinted Dlk1-Dio3 region on chromosome 12 and are normally expressed from the maternal genome. This pattern was also validated in tissues from e17.5 chimaeric embryos. The expression of this locus is to a large extent regulated by a differentially methylated region located approximately 13kb upstream of the Gtl2 promoter, the IG-DMR. Whereas this is usually only methylated on the paternal copy, in the compromised ES cells both the paternal and the maternal copy were fully methylated, likely causing the silencing of Gtl2, Rtl1as, Rian and Mirg. Using the DNA methyltransferase inhibitor 5-azacytidine, expression of Gtl2 could be rescued. Injection of those 5-azacytidine treated cells into blastocysts did partially rescue the embryonic lethal phenotype. Additionally, cell surface markers were analysed in a phenotypic screen using phage display. NGS analysis of the phage outputs indicates that there may be additional differences in cell surface markers between the control and compromised ES cell clones, but their specific details remain to be identified. Overall, we have identified the maternally expressed genes of the Dlk1-Dio3 region as markers that can distinguish between ES cells with normal or compromised developmental potency and propose to include these genes in the pre-blastocyst injection screening routine for experiments involving the production of chimaeras or genetically modified mouse strains.

Leung, Sze Lee Cecilia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-153). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Adult stem cells --- p.2 / Chapter 1.1.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5 / Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6 / Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7 / Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7 / Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9 / Chapter 1.2.1 --- Causes and statistics of CVD --- p.9 / Chapter 1.2.2 --- Current treatment for CVD --- p.10 / Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11 / Chapter 1.3 --- Myosin light chain2v --- p.13 / Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14 / Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15 / Chapter 1.6 --- Aims of the Project --- p.19 / Chapter 1.7 --- Significance of the Project --- p.19 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Subcloning --- p.20 / Chapter 2.1.1 --- Amplification of MLC-2v --- p.20 / Chapter 2.1.2 --- Purification of DNA product --- p.21 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.21 / Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22 / Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22 / Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23 / Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23 / Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24 / Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25 / Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25 / Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26 / Chapter 2.2.1 --- Derivation of MEF --- p.26 / Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27 / Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28 / Chapter 2.3 --- HESC culture --- p.29 / Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29 / Chapter 2.3.2 --- Splitting hESCs --- p.30 / Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31 / Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies / Chapter b) --- "Remove differentiated cells by ""Picking to Remove""" / Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep""" / Chapter 2.3.4 --- Freezing hESCs --- p.31 / Chapter 2.3.5 --- Differentiation of hESCs --- p.32 / Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34 / Chapter a) --- Preparation of mTeSRTMl / Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots / Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix / Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl / Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36 / Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36 / Chapter 2.4.2 --- Immunofluorescence staining --- p.37 / Chapter 2.5 --- MESC culture --- p.38 / Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38 / Chapter 2.5.2 --- Splitting mESCs --- p.38 / Chapter 2.5.3 --- Differentiation of mESCs --- p.39 / Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40 / Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41 / Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41 / Chapter 2.6.2 --- LV titering --- p.42 / Chapter 2.7 --- MultipleTransduction --- p.43 / Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43 / Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43 / Chapter 2.8.2 --- Selection of stable clones --- p.44 / Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45 / Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45 / Chapter 2.9.2 --- FACS --- p.46 / Chapter 2.10 --- Gene expression study / Chapter 2.10.1 --- Primer design --- p.46 / Chapter 2.10.2 --- RNA extraction --- p.46 / Chapter 2.10.3 --- DNase Treatment --- p.47 / Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47 / Chapter 2.10.5 --- Quantitative real-time PCR --- p.48 / Chapter 2.10.6 --- Quantification of mRNA expression --- p.49 / Chapter 2.11 --- Protein Expression study --- p.49 / Chapter 2.11.1 --- Crude protein extraction --- p.49 / Chapter 2.11.2 --- Quantitation of protein samples --- p.50 / Chapter 2.11.3 --- SDS-PAGE --- p.50 / Chapter 2.11.4 --- Western Blot --- p.51 / Chapter 2.11.5 --- Western blot luminal detection --- p.52 / Chapter 2.11.6 --- Quantification of protein expression --- p.52 / Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs / Chapter 3.1 --- Subcloning --- p.57 / Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58 / Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59 / Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60 / Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61 / Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61 / Chapter 3.2 --- Lentivirus (LV) packaging --- p.62 / Chapter 3.2.1 --- Transfection --- p.63 / Chapter 3.2.2 --- LV titering --- p.64 / Chapter 3.3 --- HESC culture --- p.66 / Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67 / Chapter 3.5 --- Differentiation after transduction --- p.69 / Chapter 3.6 --- Antibiotic selection --- p.71 / Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72 / Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72 / Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73 / Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74 / Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75 / Chapter 3.7 --- Differentiation of hESCs after selection --- p.76 / Chapter 3.8 --- FACS --- p.77 / Chapter 3.9 --- QPCR of cells after FACS --- p.80 / Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81 / Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82 / Chapter 3.9.3 --- Gene expression of c-TnT --- p.83 / Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84 / Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION / Chapter 4.1 --- mESC culture --- p.85 / Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86 / Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88 / Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94 / Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95 / Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98 / Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101 / Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104 / Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107 / Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110 / Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113 / Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114 / Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116 / Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118 / Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120 / Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122 / Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124 / Chapter CHAPTER 5 --- DISCUSSION / Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127 / Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135 / Chapter 5.3 --- Conclusion --- p.142 / REFERENCES --- p.144

Thesis (doctoral)--Humboldt-Universiẗat, Berlin, 2005. / Includes bibliographical references (p. 231-260) and index.

Source: Masters Abstracts International, Volume: 47-02, page: 0981. Adviser: Luisa Franzini. Includes bibliographical references.

The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.

Embryonic stem cells (ESCs) are being considered as a cell source for cardiac regeneration because of their potency and availability. We studied the electrophysiological implications using co-cultures of ESCs and neonatal rat ventricular myocytes (NRVM) grown on a multi-electrode array (MEA). To mimic expected engraftment rates 5% mouse ESCs were co-cultured with NRVMs. Comparing cultures without and with 5% ESCs at 4 days, the mean bipolar field potential duration (FPD) of NRVMs increased from 26.3 ± 2.2 ms (n=10) to 44.3 ± 6.2 ms (n=9; p < 0.05), the interspike interval (ISI) increased from 358.3 ± 62.8 ms (n=10) to 947.8 ± 214.6 ms (n=7; p < 0.01), and conduction velocity (CV) decreased from 14.2 ± 1.3 cm/s (n=8) to 4.6 ± 1.2 cm/s (n=5; p < 0.01). To evaluate whether ESC were having direct or paracrine effects on NRVMs, media conditioned by 3x106 ESCs for 24 hr was diluted 1:1 with fresh media and then introduced to NRVM cultures on the day of plating. Conditioned media was changed daily and altered mean FPD, ISI, and CV to 46.1 ± 7.8 ms, ISI to 682.0 ± 128.5 ms, and 4.2 ± 0.4 cm/s (n=8; p < 0.01 for each measure), respectively at 4 days. However, changes were not seen in media that was incubated for 24hrs and diluted 1:1 with fresh media and introduced to NRVM cultures in a similar fashion (n=7; p > 0.05). Slowed CV is associated with increased arrhythmic risk and reports demonstrate an inverse relationship between CV and nonphosphorylated Cx43(NP-Cx43). Western blots for total Cx43 expression revealed a decrease in ratio of P-Cx43/NP-Cx43 in the 5% mouse ESCs and ESC conditioned media cultures as compared to controls (n=8; p < 0.01 for each). There was not significant increase in the total Cx43 expression (n=6; p > 0.05). Culturing ESCs with NRVMs resulted in a decreased ISI, prolonged FPD, and slowed CV of the co-cultures as compared to controls leading to pro-arrhythmic conditions. Similar effects on NRVMs were observed when applying media conditioned by ESCs, suggesting that the electrophysiological changes were mediated by soluble factors. The increase in NP-Cx43 leads to gap junction uncoupling being a potential mechanism for these arrhythmogenic substrates. Further research into preventing NP-Cx43 in cultures is currently underway.