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Although the bone marrow low oxygen niche (∼1-4% low O2) provides critical signals for hematopoietic stem and progenitor cell (HSC/HSPC) maintenance, self-renewal and differentiation, the majority of studies are performed in room air (∼21% O2), often in fixed cells. This leaves endogenous signaling mechanisms of HSC/HSPC unidentified. By isolating/sorting/analyzing HSC/HSPC in continuous low O2, we generated the first reference landscape of phenotype/signaling/functional alterations in real time/live cells. Low O2 analyses demonstrated enhanced phenotypic (CD150;ALCAM;EPCR) marker expression and frequency of HSC/HSPC relative to historic data (in fixed cells), including a ∼3-fold increase in LSK (p=0.04) and LSKCD150 (p=0.03). Serial re-plating assays of LSK sorted cells revealed a 2.6 fold increase (p=0.02) in LSKCD150+ frequency in low O2 vs. air. mRNA sequencing uncovered differential pathway regulation of LSK (324 increased, 230 decreased) and LSKCD150+ (73 increased, 30 decreased) in low O2 vs. air. Analysis identified enrichment of genes/pathways for Ca2+ ion binding (2-fold), voltage-gated ion channel (3-fold), voltage-gated Ca2+ channel (3-fold) and altered activity of the Na+/H+ exchanger (NHE-1) in both LSK & LSKCD150+ in low O2. Although roles for cytosolic Ca2+ have been identified in HSC/HSPC function the regulation/roles of Ca2+ in low O2 have not been investigated. Live cell analysis of cytosolic Ca2+ flux (FURA, MFI) showed a 2.6 fold increase in HSC (p=0.02) and a 1.4-fold increase in HSPC (p=0.003) in low O2 vs. air. NHE-1 inhibition (cariporide) resulted in a 1.5-fold decrease of the Ca2+ influx detected in low O2 (p=0.02), with no effect detected in air. LSKs were isolated/sorted in low O2 for subpopulations of Ca2+ FURA hi vs low, in low O2 or air, and serially re-plated. Functional re-plating assays showed increased proliferation and frequency of phenotypic LSKs in FURA High vs FURA dim cells in low O2, with cariporide blunting this effect. Taken together, pathways identified in the low O2 landscape showed differential regulation, phenotypic and functional consequences, of altered Ca2+ in low O2 is HSC/HSPC populations. |
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