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UBC Theses and Dissertations

Analysis of hematopoiesis from human pluripotent stem cells Kardel, Melanie Dawn


Human embryonic stem (ES) or induced pluripotent stem (iPS) cells have the potential ability to generate all of the cell types in the body. If their differentiation into relevant cell types of interest can be effectively controlled, they are attractive for developmental studies, disease modelling, drug testing, and advancing regenerative medicine. The generation of hematopoietic cells from human ES/iPS cells has been reported, but is highly variable and often inefficient. My specific objective in this thesis was to more fully characterize the process whereby hematopoietic cells are generated from primitive pluripotent precursors, to understand current limitations, and to design improvements that would increase the yield and reproducibility of hematopoietic cell generation. I first examined the effect of the conditions used to maintain the undifferentiated starting population of ES/iPS cells on their hematopoietic cell differentiation ability. The results showed that the initial maintenance conditions used do have a significant influence on the subsequent number and consistency of hematopoietic cells generated. In addition, I found that this process is separately influenced by optimization of sequentially manipulated (early and late) differentiation steps. Analysis of individual EBs revealed a previously unappreciated heterogeneity of hematopoietic output from single EBs in vitro. Even under the most optimal conditions studied, it was found that the majority of EBs did not generate any hematopoietic colony-forming cells (CFCs). This suggested that only a limited number of the initial ES/iPS cells were contributing to the hematopoietic progenitor cell output under these conditions. To investigate this latter phenomenon further, I developed a lentiviral system to track the subsequent hematopoietic progeny of marked undifferentiated or early differentiating ES/iPS cells. The use of this approach confirmed that few of the starting ES/iPS cells contribute to the hematopoietic output of individual EBs. Together these studies suggest that the genesis of hematopoietic progenitors from pluripotent precursors remains limited by multiple factors. Further studies to characterize cell types intermediate between fully pluripotent cells and those with hematopoietic activity are needed to define more rigorously and optimize the use of this strategy for various medical applications.

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