Factors affecting optimal culture of haematopoietic stem cells
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Haematopoietic stem cells (HSC) are invaluable, due to their potential to treat malignant and non-malignant diseases. Modern medicine requires a reliable source of human HSCs (hHSCs) for efficient transplantations, which in many cases cannot be obtained from a single donor. Therefore, the ability to amplify donor hHSCs ex vivo would be an ideal alternative. Past attempts to expand hHSCs in vitro, demonstrated that the protocols developed so far have limited success. My research studied the factors which can affect the optimal culture of transplantable HSCs using a 3D culture system that had previously been used to culture HSCs derived from the aorta-gonad-mesonephros (AGM) region of the mouse embryo. This system involved cell culturing at the gas-liquid interface which is particularly sensitive to mechanical disturbances. To overcome this problem, floating Polypropylene support (rings) were designed and tested and I demonstrated that this was able to prolong aggregate culturing for up to 21 days. Further optimisation tests included altering factors such as oxygen levels, and the presence of antioxidants and apoptosis inhibitors in mouse HSCs culture. I have shown that moderate hypoxia (6% O2) did not affect HSCs in culture, while 2% of O2 led to a significant decrease of HSCs activity. Normoxia resulted in higher reactive oxygen species generation, which would likely be detrimental to cells. However, unexpectedly no improvement in repopulation efficiency of cultured HSCs was achieved by the addition of antioxidant. I also found that when the AGM region was dissociated and co-aggregated in the presence of Rho kinase inhibitor a higher level of repopulation was achieved. In addition, troloxpifitrin-a and p38 inhibitor blocked HSC development without affecting progenitor frequency or the total number of live cells. Subclones of mouse stromal cell line (OP9) were used to create a defined haematopoietic niche for hHSC. Functional screening of these lines in co-aggregate culture re- vealed that 3 of the 34 subclones tested were able to maintain hHSC in culture and repopulate immunodeficient mice at a comparable level to uncultured CD34+ cells. The repopulation in engrafted recipients persisted for over 6 months and showed both myeloid and lymphoid potential. These 3 subclones therefore appeared to create a functional niche for hHSCs and were subsequently used to study the impact of a number of factors including SCF, rock inhibitor, TGFb inhibitor, StemRegenin1 (SR), and prolonged culture technique on hHSC expansion. A significant level of fluctuation between experiments was observed and no definitive conclusions could be drawn. I also attempted to establish stromal cell lines from the human AGM region, more specifically from the ventral (AoV) and dorsal (AoD) regions of the dorsal aorta. Despite attempts to immortalise primary stromal cells, all lines went through a growth crisis. Nevertheless, 30 lines were screened for their ability to support haematopoietic cells in co-aggregate culture with results suggesting that lines derived from AoV expanded haematopoietic precursors more efficiently than AoD lines and OP9 control. Many of the tested lines were able to maintain long-term repopulating human HSCs but the level of repopulation was not as high as that achieved from uncultured CD34+ cells. Unfortunately, these human stromal cell lines have an unstable karyotype which may have an impact on their functional characteristics and they may not represent the nature of the primary cells.