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Hematopoietic stem cells (HSCs) sustain continuous turnover and maintenance of all blood lineages through organismal lifespan. The extensive regenerative potential of HSCs has been harnessed in transplantation medicine to enable curative therapies for numerous life-threatening conditions that require hematological reconstitution. However, the rarity of HSCs combined with the limited availability of immunologically matched donors have constrained the utility of HSC transplantation whose success and safety depend critically on the quantity donor HSCs; therefore, ex vivo expansion of HSCs has been a highly sought after goal in HSC research. In this thesis, I present a hypothesis driven approach toward identifying cocktails of small molecules that enable ex vivo maintenance and propagation of mouse and human HSCs. Specifically, using HSCs isolated from Fgd5ZsGreen HSC-specific fluorescent reporter mice along with previously identified immunophenotypic markers, I conducted a small scale combinatorial chemical screen of developmental signaling modulators to determine a condition that would preserve immunophenotypic HSCs ex vivo. The screen led to the discovery that murine HSCs can be maintained for at least 14 days ex vivo when the basal media was supplemented with cytokines and the minimal combination of a small molecule inhibitor of TGF-β signaling and two epigenetic inhibitors, namely LSD1 inhibitor and HDAC inhibitor, which were selected based on reports that they may derepress Notch target gene expression. Additionally, metabolic optimizations led to the identification of putrescine as a critical culture supplement for promoting HSC propagation. The three chemicals identified in the murine screen were conserved in their ability to promote the ex vivo preservation of primary human HSCs. However, as presence of the two epigenetic inhibitors caused substantial growth suppression, alternative, more specific means to activate the Notch pathway were sought. To this end, I hypothesized that inhibition of IKAROS transcription factor, a repressor of Notch target gene expression, would be able to derepress Notch pathway activity and hence replace the growth suppressive epigenetic inhibitors. Indeed, substitution of the epigenetic inhibitors with an IKAROS inhibitor rescued immunophenotypic HSC propagation, and additional supplementation with UM171, a recently identified small molecule enhancer of human HSC expansion, further improved HSC yield as well as the durability of immunophenotypic preservation over prolonged culture; in sum, three compounds, namely a TGF-β inhibitor, pomalidomide, and UM171, were found to be necessary for robust ex vivo maintenance and propagation of human HSCs. At the time of writing, xenotransplantation is underway to assess the in vivo function of ex vivo cultured human HSCs. Collectively, this body of work contributed to identifying chemically defined ex vivo culture conditions supportive of murine and human HSCs while underscoring the importance of combinatorial pathway modulation for maintaining HSC immunophenotype and function. Development of effective HSC culture conditions should not only benefit clinical medicine but also facilitate interrogation and manipulation of HSCs in vitro., Medical Sciences

Hematopoietic Stem Cells (HSCs) are characterized by their ability to both self-renew and give rise to all lineages of the blood system. A recent chemical genetic screen identified 17β-estradiol (estrogen) as a novel modifier of the expression of the conserved HSC markers runx1 and cmyb in the Aorta-Gonad-Mesonephros of developing zebrafish. Exposure to exogenous estrogen during the development of the hematopoietic niche impeded specification of hemogenic endothelium and the subsequent emergence of HSCs via antagonism of somitic-derived VEGF signaling. Conversely, inhibition of endogenous estrogen activity increased the number of functional HSCs present in the embryo and resulted in higher expression of VEGF target genes, suggesting that endogenous estrogen acts to define the ventral limit of VEGF activity and hemogenic endothelial specification. In contrast, when embryos were exposed to estrogen after niche specification, markers of HSCs were increased, indicating that estrogen has a biphasic effect on HSC formation; this effect appears to be at least partially mediated by enhanced cell cycling of the HSC population. Estrogen exposure during primitive erythropoiesis likewise increased the number of erythroid progenitors in the embryo, but their maturation into functional erythrocytes was impaired. Inhibition of erythrocyte maturation is also conserved in a mammalian model of in utero excess estrogen, causing propensity for embryonic lethality. Treatment of adult zebrafish with exogenous estrogen after ablation of the hematopoietic system by irradiation revealed that elevated estrogen levels improved hematopoietic regeneration. Consistent with a role for hormonal regulation of HSC homeostasis, accelerated recovery of hematopoietic stem and progenitor numbers was observed in female fish compared to males, suggesting an endogenous difference in regenerative capacity between the sexes. Together, these data identify multiple distinct roles for estrogen in HSC biology and indicate it is a physiologically relevant regulator of HSC development and homeostasis.