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UBC Theses and Dissertations

Regulation of human hematopoietic cells with short-term in vivo repopulating activity Miller, Paul H.


Transplantation of hematopoietic cells is a critical component of treatments used to cure a range of malignancies and congenital disorders. Cells with rapid but short-term repopulating ability (STRCs) are the main source of neutrophils and platelets early post-transplant, but methods to increase their availability are lacking. In this study the use of NOD/SCID-IL-2Receptor-γchain-null mice engineered to produce human interleukin-3, granulocyte-macrophage colony-stimulating factor and Steel factor as hosts of CD34⁺ cells was shown to improve the quantification of human STRCs. This mouse was found to support a 5-fold higher human myeloid cell output at early time-points post-transplant, sufficient to assess total engraftment from an analysis of circulating human cells. This strategy was then used to determine the optimal protocol for mobilizing STRCs in normal adult human donors, comparing administration of granulocyte colony stimulating factor (G-CSF) plus plerixafor (P) to administration of P alone. Blood and marrow samples were obtained from 10 normal adult donors before, during, and after treatment and then evaluated for their content of CD34⁺ cells, colony-forming cells (CFCs), long-term culture-initiating cell activity, and cells with in vivo STRC activity. The results show all activities were maximally increased in the blood 4 hours after administration of P, with or without pre-treatment with G-CSF. In vivo assessment showed that administration of P led to a 30-fold increase in STRC activity over baseline levels, with further enhancement (90-fold over baseline) by prior G-CSF treatment. The ability of currently available growth-factor (GF)-containing HSC expansion protocols to support the expansion of STRCs was then assessed. The results revealed a significant (10-fold) loss of STRC activity after being cultured for 7 to 10 days, in spite of an extensive increase in CD34⁺ cells and CFCs. This GF-induced loss of in vivo STRC activity occurred within 24 hours, and was paralleled in time and amount by a loss of CFC homing to the bone marrow. Together these findings provide a further understanding of STRC biology and provide a foundation for developing improved yields of manipulated STRCs for clinical use.

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