Thesis (M. Sc.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 111-119). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ59179.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 248-277). Also available in print.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 3 uppsatser.

Human pluripotent stem cells (hPSCs) hold the potential to revolutionize cardiac tissue engineering. Because of their ability to proliferate and differentiate into all cardiomyocyte subtypes they represent an opportunity to regenerate virtually any tissue lost from the over 1 million cardiac disease patients in the United States alone. Studies have shown, however, that hPSCs which are not terminally differentiated pose a variety of risks including teratoma formation and lack of appropriate cell engraftment. It is therefore important to ensure that only well characterized cardiac subtypes are implanted into patients or used for research purposes. Current differentiation protocols generate a mixture of cardiac subtypes, and research on cardiac subtype specification is hampered by the lack of a high throughput method to distinguish cardiac subtypes. This thesis establishes the ability to identify, enrich and characterize cardiac subtypes using MBs. This will provide a robust tool for clinical use of hPSCs in cardiac cell therapy and for analysis of differentiation protocol effects on cardiac subtype formation.

Due to the limited intrinsic healing ability of mature cartilage tissue, stem cell therapies offer the potential to restore cartilage lost due to trauma or arthritis. Mesenchymal stem cells (MSCs) are a promising cell source due to their ability to differentiate into various adult tissues under specific biochemical and physical cues. Current MSC chondrogenic differentiation strategies employ large pellets, however, we have previously developed a high-throughput technique to form small MSC aggregates (500-1,000 cells) that may reduce diffusion barriers while maintaining a multicellular structure that is analogous to cartilaginous condensations. The objective of this study was to examine the effects on chondrogenesis of incorporating chondroitin sulfate methacrylate (CSMA) microparticles (MPs) within these small MSC spheroids when cultured in the presence of transforming growth factor-β1 (TGF-β1) over 21 days. Spheroids +MP induced earlier increases in collagen II and aggrecan gene expression (chondrogenic markers) than spheroids -MP, although no large differences in immunostaining for these matrix molecules were observed by day 21. Collagen I and X was also detected in the ECM of all spheroids by immunostaining. Interestingly, histology revealed that CSMA MPs clustered together near the center of the MSC spheroids and induced circumferential alignment of cells and ECM around the material core. Because chondrogenesis was not hindered by the presence of CSMA MPs, this study demonstrates the utility of this culture system to further examine the effects of matrix molecules on MSC phenotype, as well as potentially direct differentiation in a more spatially controlled manner that better mimics the architecture of specific target tissues.

Embryonic stem (ES) cells provide a potentially unlimited cell source for cellular therapies; however, reliable methods must be developed to provide clinically-relevant numbers of homogeneous therapeutic cell populations. Dynamic cultures may encourage ES cell differentiation and amenable to large-scale cell production. Our goal was to optimize dynamic culture parameters (bioreactor type, speed, cell seeding density, conditioned medium, and hypoxia) to maximize the generation of hematopoietic stem and progenitor cells (HSPCs) from ES cells and also to investigate the ability of dynamic culture-derived HSPCs to generate terminally differentiated hematopoietic cells. Our results indicate that varying cell seeding density and speed in two different bioreactors significantly affects embryoid body formation and ES cell differentiation efficiency into progenitor cells. In general, increased cell seeding density generated higher percentages of HSPCs in both bioreactors. In addition, rotary (Synthecon) bioreactors produced more sca-1⁺ progenitors, and spinner flasks generated more c-kit⁺ progenitors, demonstrating their unique differentiation profiles. cDNA microarray analysis of genes involved in pluripotency, germ layer formation, and hematopoietic differentiation showed that unique gene expression profiles were observed in the two bioreactors with the expression of specific hematopoietic genes more up regulated in the Synthecon cultures compared to spinner flasks. Combining bioreactor cultures with directed differentiation strategies via conditioned medium and hypoxic culture may further encourage hematopoietic differentiation. Dynamically cultured ES cell-derived hematopoietic stem and progenitor cells were further differentiated into a phenotype typical of dendritic cells which had the ability to process antigen. Additionally, microarray analysis of isolated ES cell-derived HSPCs demonstrated differences in the gene expression from native HSCs isolated from the fetal liver or bone marrow of mice. Insight gained from this work should be continued by comparing the differentiation efficiency of HSPCs derived in dynamic and traditional static culture methods into functional, terminally differentiated hematopoietic cells to generate clinically-relevant numbers of transplantable, therapeutic cells. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy