Generation of a Low Oxygen Hematopoietic Landscape Identifies Transcriptional and Functional Alterations in Calcium Regulation

• Published in: Blood 2020-11, Vol.136 (Supplement 1), p.29-30
• Main Authors: Dausinas, Paige, Slack, Jacob, Basile, Chris, Karlapudi, Anish, O'Leary, Heather A.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2020-142991

Introduction: The bone marrow (BM) microenvironment consists of low oxygen niches (~1-4%, low O2) that provide critical signals for hematopoietic stem and progenitor cell (HSC/HSPC) maintenance, self-renewal, and differentiation. However, the majority of HSC/HSPC studies are performed in room air (~21% O2), often using whole BM or fixed cells, and potentially leaving endogenous signaling mechanisms of HSC/HSPC unidentified. Our previous data, analyzing whole BM/fixed cells, detected increased HSC numbers and engrafting capability. No study thus far has directly compared, and identified, broad signaling and functional differences of HSC/HSPC populations in their native low O2. Using our novel technology to isolate/sort/analyze HSC/HSPC in continuous low O2, we generated the first reference landscape of phenotype/signaling/functional alterations in real time/live cells and identified critical pathways utilized in low O2. Methods: Murine BM was harvested from long bones under low O2 (3%). Samples were enriched and sorted for populations of interest HSC/HSPC (LSKCD150/LSK) in low O2, split into groups either exposed to room air or left in low O2 for ~60 minutes, then analyzed for direct comparison. All samples remain in assigned experimental conditions unless otherwise indicated and statistical analysis (t-test or ANOVA) is based on multiple experiments (N > 3 animals/experiment). Results: Low O2 analyses demonstrated enhanced phenotypic (CD150;ALCAM;EPCR) marker expression and frequency of HSC/HSPC relative to historic data (whole BM, fixed cells), including a ~3-fold increase in LSK (p=0.04) and LSKCD150 (p=0.03). The utilization of live cells provided additional phenotypic insight including masking of some differences in low O2 upon cell fixation. mRNA sequencing identified differential pathway regulation of LSK (324 increased, 230 decreased; 6,331 significant genes) and LSKCD150+ (73 increased, 30 decreased; 447 significant genes) cells in low O2 vs. air. Additionally, 82 significant genes overlapped between LSK/LSKCD150 (13,524 overlapping genes total). Genes/pathways (in air) were comparable to published data sets and suggest differences obtained in low O2 are tightly regulated by environmental alterations. Pathway analysis identified numerous significant signaling changes including calcium (Ca2+) ion binding (2-fold), voltage-gated ion (3-fold) and Ca2+ channels (3-fold) and altered activity of the Na+/H+ exchanger (NHE-1) in both LSK & LSKCD150+ in low