An investigation of the origins of normal and leukemic hematopoiesis by genetic modification of primary human hematopoietic cells.

摘要

Extremely rare stem cells, found at the apex of the human hematopoietic hierarchy, are ultimately responsible for maintaining hematopoiesis. Using the SCID mouse Repopulating Cell (SRC) xenotransplantation assay, the function of these stem cells can be assayed. However, early attempts to investigate SRC activity by retroviral labeling or modification were undermined by inadequate understanding of appropriate vectors and the ex vivo culture requirements of stem cells. In the first part of this thesis, my efforts to improve SRC gene transfer are described. Application of a refined retroviral vector, and an optimized culture environment permitted efficient SRC transduction. Interestingly, cells with a very primitive CD34+CD38− phenotype expanded 166-fold, but there was no increase in SRC number. Thus, CD38 expression is not a reliable indicator of SRC content in cultured cells. Subsequently, these tools and methods were used to look for functional subcategories of human repopulating cells. Clonal analysis of bone marrow from mice engrafted with retrovirally marked cells revealed oligoclonal repopulation, with extensive variability in the lifespan and proliferative capacities of individual SRC. Thus, the human hematopoietic stem cell compartment is heterogeneous. Finally, many of these methods were applied to study the initiation of leukemia in naïve primitive blood cells. Expression of HOX11 enhanced proliferation and self-renewal, and shifted differentiation towards the eosinophilic and promonocytic/monoblastic lineages. This oncogene may therefore predispose cells to leukemic progression by shifting and partially blocking normal differentiation. The influence of activated H-Ras on hematopoietic cell fate was determined by its level of activity. A high intensity of Ras signaling was anti-proliferative and promoted monocytic differentiation, but cells with a moderate level of Ras activity showed enhanced proliferation and caused an elevated frequency of primitive blast-like cells and self-renewing progenitors. Thus, modest Ras activation may be an initiating leukemogenic event.