Session: 501. Hematopoietic Stem and Progenitor Biology: Poster II
Hematology Disease Topics & Pathways:
HSCs, cell regulation, Biological Processes, Technology and Procedures, epigenetics, Cell Lineage, hematopoiesis, flow cytometry, microenvironment, RNA sequencing, proteomics, signal transduction
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 O2. Proteomic assessment of air vs low O2 LSK confirmed enhancement of differential Ca2+ pathway regulation. Live cell analysis of functional 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. Conversely, Ca2+ flux decreased under low O2 in terminally differentiated effector B (B220; p=0.002), myeloid (CD11b; p=0.0009), erythroid (Ter119; p=0.003) and T-cells (CD3; p=0.02). To elucidate mechanisms of Ca2+ regulation in HSC/HPC, we inhibited NHE-1, a known modulator of Ca2+ flux (via cariporide) in air vs low O2 and observed a 1.5-fold blunting of the low O2 Ca2+ influx (p=0.02), with no effect on Ca2+ detected in air. Functional serial re-plating assays of LSK sorted cells revealed a 2.6 fold increase (p=0.02) in LSK and LSKCD150+ frequency, and proliferation, in low O2 vs. air (day 14 & up) that was blunted in the presence of cariporide.
Finally, to identify the potential functional implications of differential Ca2+ regulation in air vs low O2, LSKs were isolated/sorted in low O2 for subpopulations of Ca2+ FURA hi vs low cells, and serially re-plated in air or low O2. Functional re-plating assays showed increased proliferation (CFSE) and frequency of phenotypic LSKs in FURA High compared to FURA dim plated cells in low O2.
Conclusions: Taken together, these data provide insight into the low O2 landscape, highlighting that unique pathways and gene signatures are identified in low O2 compared to room air. Further, we show differential regulation of novel and known pathways (Ca2+), with phenotypic and functional consequences, in HSC/HSPC populations in low O2. This highlights the relevance of experimental context in data interpretation and the differential response to inhibitors, functional re-plating, signaling, and phenotypic analysis detected using live cells in native low O2 compared to room air.
Disclosures: No relevant conflicts of interest to declare.
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