Identification of Signaling and Functional Adaptations of Hematopoietic Stem/Progenitor Cells via Generation of a Low Oxygen Landscape.

Hematopoietic stem and progenitor cells (HSC/HPC) reside in low O2 (1-4%) bone marrow (BM) niches which provide critical signals for stem cell maintenance and function. Since the technology to directly compare broad signaling/functional differences of live HSC/HPC maintained in their native low O2 environment did not previously exist, historic studies have been performed in ambient air (~21% O2), utilizing whole (W) BM or fixed cells leading to a lack of mechanistic insight into endogenous signaling pathways/functional outcomes dependent on relevant microenvironmental O2. Using our novel technology to study live HSC/HPC in continuous low O2, we generated the first reference low O2 landscape of differential phenotypic/signaling/functional regulation in live HSC/HPC. Murine BM was harvested from long bones, enriched for SCA1+ cells, sorted for HSC/HPC (LSKCD150/LSK) fractions at continuous 3% O2 then split to remain in low O2 or be exposed to room air (60 minutes) with paired comparisons performed on the divided samples. Statistical analyses (t-test or ANOVA) are based on multiple experiments (n>3/experiment). All subsequent comparisons are between 3% O2 samples compared to room air exposed samples. Live cell analysis demonstrated enhanced phenotypic marker expression and frequency in low O2 of HSC (2-fold), HPC (2-fold), B-cell- (B220) and erythroid-lineage (Ter119) cells under low O2 (p<0.05) relative to both historic data (utilizing WBM, fixed cells) and our room air data and identified masking of phenotypic difference in low O2 upon cell fixation. Transcriptional analyses of HPC detected 23,611 total genes; 1,239 were identified as differentially expressed (DEGs). HSC analyses detected 13,719 total genes with 135 identified DEGs (FDR<0.05) in low O2. DAVID analysis of bulk mRNA sequencing identified differential pathway regulation of HPC (324 increased GO pathways, 230 decreased) and HSC (73 increased, 30 decreased) with significant enhancements of genes responsible for calcium regulation, transmembrane transport, and cell differentiation observed in low O2. Proteomic analysis of HPC detected 2,352 expressed proteins; 99 identified as differentially expressed (FDR<0.05) and DAVID analysis highlighting enhancements in metabolism, cell signaling, and membrane regulation (p<0.05) in low O2. Single cell sequencing of sorted HPC identified over 18,000 total genes in both air and low O2 with GSEA of significantly detected genes (p<0.05) demonstrating differential regulation of cell cycle, metabolism, protein stabilization, enzyme binding/kinetics and calcium pathways in low O2. Functional analyses of HPC maintained at native low O2 revealed enhanced maintenance of HSC/HPC over 3-4 re-platings and altered fate decisions resulting in enhanced numbers of B-cell-, myeloid- (CD11b) and erythroid-lineage (p<0.05). These data provide novel insight into HSC/HPC endogenous signaling and function via generation of the first low O2 landscape and identified unique populations and differentially regulated pathways/gene signatures in low O2 vs. room air. Thus, highlighting the critical importance of native experimental conditions on data generation and interpretation in studies of HSC/HPC with potential broad implications for other stem cell populations.