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Control of the lympho-myeloid potential of murine hematopoietic stem cells

University of British Columbia

Little is known about the mechanisms that regulate the maintenance and expression of the self-renewal and differentiation properties of hematopoietic stem cells (HSC) in vivo or in vitro. Murine HSC can be detected and quantitated by in vivo assays and there is evidence to suggest that many, if not all, HSC can be detected in vitro by their ability to sustain the longterm production of colony-forming cells in culture. However, at the time this thesis was initiated, reproducible and specific assays for the in vitro detection and manipulation of lympho-myeloid progenitors had not been developed. I have devised such an assay which relies on the joint expression of lymphoid and myeloid differentiative potentials in the same culture. My studies have shown that the cells detected by this assay, termed LTC-IC[sub ML], are closely related to totipotent cells with long-term competitive in vivo repopulating potential (CRU) by their similar resistance to killing by 5-fluorouracil and their >500-fold co-enrichment in a -0.03% subpopulation of adult marrow cells. The myeloid LTC-IC and LTC-IC[sub ML] assays were used to determine whether the maintenance of long-term in vitro lympho-myeloid differentiation potential correlates with the persistence of in vivo multilineage reconstitution ability. We found that LTC-IC and LTC-IC[sub ML] are both better maintained in culture than CRU, suggesting that the ability to generate clonogenic progenitors in vitro for ≥4 weeks and the ability to competitively reconstitute all lineages in myeloablated recipients can be differently regulated. As a first step towards analyzing the mechanism(s) utilized by the stromal feeder layer in stimulating LTC-IC[sub ML] in vitro, its replacement with two stromal cell-derived growth factors, interleukin (IL)-11 and the Flt3 ligand (FL) were investigated. A subpopulation of fetal liver cells highly enriched for CRU was used as a starting material since both IL-11 and FL had previously been reported to act on primitive fetal liver-derived hematopoietic cells. The results of these experiments show that both IL-11 and FL support the development and maturation of early B lineage cells but that their activities in this regard are not identical.

Thesis/Dissertation

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2009-02-20

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http://hdl.handle.net/2429/4823

Genetics

University of British Columbia

UBCV

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