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
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/ETD-UT-2010-12-2254 |
| Date | 14 February 2012 |
| Creators | Fridley, Krista Marie |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | thesis |
| Format | application/pdf |
Page generated in 0.0019 seconds