Functional analysis of the homeobox gene HOXB4 in primitive hematopoietic cells

Hematopoiesis involves the ordered production of mature blood cells from a rare population of undifferentiated, totipotent, stem cells in the bone marrow. A pool of stem cells is preserved through self-renewing divisions, to maintain lifelong hematopoiesis. Deregulation of stem cell self-renewal and differentiation can have severe clinical consequences, including leukemia. Hematopoietic stem cells are also highly valuable for their therapeutic uses, as they can help reconstitute the hematopoietic system when transplanted. Yet despite the central importance of stem cells, we know very little about the regulatory mechanisms controlling their behaviour. HOXB4, a member of the Hox family of transcriptional regulators, was previously shown to selectively enhance hematopoietic stem cell growth in vivo. Hox family members have gene-specific roles in hematopoiesis, controlling differentiation and proliferation along specific lineages or even causing leukemia. Retrovirus-mediated overexpression of HOXB4 in murine bone marrow cells resulted in enhanced stem cell growth in vivo, without altering lympho-myeloid differentiation or predisposing to leukemia. The major objective of this thesis was to further explore the effects oiHOXB4 overexpression on primitive hematopoietic cells, and to test the utility and limitations of this strategy for stem cell expansion. Cells overexpressing HOXB4 had a competitive growth advantage in vitro, and a competitive transplant advantage in vivo. HOXB4 overexpressing bone marrow cell cultures showed increased proliferation over control cell cultures. Mice transplanted with //(9A34-transduced and control-transduced cells were selectively repopulated by the HOXB4 overexpressing cells. Importantly, the growth enhancement induced by HOXB4 overexpression did not come at the expense of normal differentiation. Further analysis of the //C7A7J4-mediated enhancement to stem cell expansion in vivo revealed that the rate of expansion was elevated. Accelerated stem cell regeneration mediated by HOXB4 allowed stem cell levels to reach 100% of pre-transplant levels within the first three months post-transplant. Stem cells did not expand beyond the normal level in HOXB4 mice, suggesting retained responsiveness to negative feedback mechanisms. Thus, //CTAB^-transduced stem cells remained responsive to positive and negative feedback on expansion and to differentiation-promoting signals. This work also addressed the potential for HOXB4 to serve as a stem cell expanding factor ex vivo. Although significant biological and clinical advances await effective stem cell expansion regimes, previous efforts have been largely unsuccessful. HOXB4 overexpression mediated rapid, extensive, and highly polyclonal stem cell expansions, resulting in over 1000-fold higher stem cell levels relative to controls and a 40-fold net stem cell expansion. These results show that HOXB4 can be used to expand stem cells ex vivo. The results presented in this thesis add to the recognition of Hox genes as important hematopoietic regulators. HOXB4 in particular can play a key role in the regulation of the rate and/or probability of hematopoietic stem cell self-renewal. These studies further suggest the complex biomolecular pathways controlling stem cell fate, and point to new avenues to manipulate HSC expansion.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Kit regulates maintenance of quiescent hematopoietic stem cells.

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (LSK)CD34(-)Flt3(-) long-term HSCs by 12 wk of age, whereas LSKCD34(+)Flt3(-) short-term HSCs and LSKCD34(+)Flt3(+) multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/W41) HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit(W41/W41) HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis

Cytokines regulate postnatal hematopoietic stem cell expansion: opposing roles of thrombopoietin and LNK

The role of cytokines as regulators of hematopoietic stem cell (HSC) expansion remains elusive. Herein, we identify thrombopoietin (THPO) and the cytokine signaling inhibitor LNK, as opposing physiological regulators of HSC expansion. Lnk(−/−) HSCs continue to expand postnatally, up to 24-fold above normal by 6 mo of age. Within the stem cell compartment, this expansion is highly selective for self-renewing long-term HSCs (LT-HSCs), which show enhanced THPO responsiveness. Lnk(−/−) HSC expansion is dependent on THPO, and 12-wk-old Lnk(−/−)Thpo(−/−) mice have 65-fold fewer LT-HSCs than Lnk(−/−) mice. Expansions of multiple myeloid, but not lymphoid, progenitors in Lnk(−/−) mice also proved THPO-dependent