Abstract
Background: Hematopoietic stem and progenitor cells (HSC/HSPCs) inhabit niches within bone marrow that have significantly lower oxygen tension (1-4% O2, LowO2) than ambient air (21% O2) and provide signals regulating HSC signaling/function/self-renewal. Despite this knowledge, the majority of HSC/HSPC experiments are performed in ambient air, not replicating the endogenous environment. Our previous studies of HSC/HSPCs in LowO2 demonstrated enhanced phenotypic (CD150) marker expression, and frequency, of HSC/HSPC in live cells (~3fold LSK/LSKCD150 p=0.04) relative to historic data (fixed cells) as well as alterations in novel signaling/functional pathways. 
 
 Methods: Our novel technology facilitates the isolation/sorting, and analysis of rare HSC populations, in live cells, continuously at their native LowO2 environments (3% O2). All assays have been adapted and validated in LowO2. 
 
 Results: Time course (0 min, 15 min, 40 min, 60 min, 180 min) analysis comparing phenotypic markers of HSC/HSPC populations at LowO2 and ambient air, showed HSC/HSPC enhancement at baseline in LowO2 that were significantly blunted by exposure to air (p-value =.00007) and time beginning at 40 min (p-value =.00003). Phenotypic expression, as shown by the marker CD150, was also diminished by exposure to air (p-value =.0008) and via time (p-value =.05). Analysis via mRNA sequencing showed enhancement in 324 and 73 (LSK, LSKCD150) pathways including calcium channel complex and calcium ion binding when HSC/HSPC remained in LowO2 compared to air. Analysis of calcium regulation via FURA staining correlated with these data confirming increased calcium influx in LowO2 HSC/HSPC populations. 
 
 Potential Impact: Our findings demonstrate significant negative impacts of ambient air, fixation, and time from harvest on HSC/HSPC phenotype and function. These findings suggest the critical importance of studying HSC/HSPC, live, quickly, and in their native environment, highlight potential caveats of current approaches, and rationale for lack of comparable results for studies of HSC/HSPC in the literature.
Highlights
Indiana University School of Medicine1, Indiana University School of Medicine Department Anatomy, Cell Biology, and Physiology2, Indiana University School of Medicine Department of Medicine Division of Oncology/Hematology2,3,4
Hematopoietic stem and progenitor cells (HSC/HSPCs) inhabit niches within bone marrow that have significantly lower oxygen tension (1-4% O2, LowO2) than ambient air (21% O2) and provide signals regulating HSC signaling/function/self-renewal
Time course (0 min, 15 min, 40 min, 60 min, 180 min) analysis comparing phenotypic markers of HSC/HSPC populations at LowO2 and ambient air, showed HSC/HSPC enhancement at baseline in LowO2 that were significantly blunted by exposure to air (p-value =.00007) and time beginning at 40 min (p-value =.00003)
Summary
Indiana University School of Medicine1, Indiana University School of Medicine Department Anatomy, Cell Biology, and Physiology2, Indiana University School of Medicine Department of Medicine Division of Oncology/Hematology2,3,4 Background: Hematopoietic stem and progenitor cells (HSC/HSPCs) inhabit niches within bone marrow that have significantly lower oxygen tension (1-4% O2, LowO2) than ambient air (21% O2) and provide signals regulating HSC signaling/function/self-renewal.
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