Hematopoietic Stem and Progenitor Cell Expansion

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Objective 1: Notch-mediated ex vivo expansion of human HSPCs by culture under hypoxia To investigate whether hypoxia can facilitate superior ex vivo expansion of human HSPCs than normoxia in the presence of Delta1ext-IgG, a total of 1 x 105 human MPB CD34+ cells were cultured under normoxic or hypoxic conditions in vessels coated with fibronectin alone or combined with increasing concentrations of Delta1ext-IgG (2.5, 5, 10 and 20 g/mL). After 21 days in culture, cells were counted and characterized by flow cytometry and functional assays. We demonstrate that ex vivo culture of human adult HSPCs with Delta1ext-IgG under low oxygen tension (2% O2) limits ER stress in LTR-HSCs and, to a lesser extent, in lineage committed progenitors compared to normoxic (21% O2) cultures. A distinct HSC gene expression signature was upregulated in cells cultured with Delta1ext-IgG in hypoxia and, after 21 days of culture, the frequency of long-term repopulating (LTR) HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared to the normoxia group, as measured by limiting dilution analysis in NSG mice. Notch and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured CD34+ cells, and both hypoxia inducible factor-1 and the intracellular domain of Notch1 receptor were central in the convergence point between the two signaling pathways. Thus, our work underscores the importance of mitigating ER stress perturbations to preserve functional HSCs in extended cultures, and offers a clinically feasible platform for the expansion of human HSPCs. This work was published Stem Cell reports 2021. Objective 2: Notch-mediated ex vivo expansion of human HSPCs by culture with 17-AAG In a recent study, murine and human cord blood-derived HSPCs cultured over a 10-day period in standard medium/cytokines supplemented with 17-AAG were functionally similar to fresh HSCs. However, 17-AAG alone did not support significant HSC expansion. In FY22, we have initiated investigations to determine whether highly proliferative conditions, such as those promoted in culture with Delta1ext-IgG, could act synergistically with Hsf1 inhibition and promote HSC expansion ex vivo. We have optimized doses of 17-AAG for culture of human adult HSPCs and long-term xenogeneic transplants are ongoing to assess efficacy of this approach.