Mitigating Detrimental Influences of Ambient Air Collection of Bone Marrow By Hypoxic Collection Results in Detection of Functionally Enhanced Aged Mouse Hematopoiesis: Reflecting Stem Cell Expression Levels of CXCR4, CCR5 and ROS

Bone marrow (BM) of aged mice have significantly increased phenotypically-defined hematopoietic stem cells (HSCs), but greatly decreased competitive engrafting capability with a shift to decreased lymphoid capability when compared to young mice. HSCs and HPCs reside in vivo in a grossly hypoxic (e.g. ~3% O₂) environment compared to that of ambient air (~21% O₂). Yet, until recently all knowledge of HSCs and HPCs was based on cells initially collected in ambient air, which allows for the gross underestimation of the numbers of phenotypic and functional HSCs while overestimating HPCs. Within minutes of exposure to ambient air, levels of reactive oxygen species (ROS) are increased, inducing rapid differentiation, not death, of HSCs (Mantel, et. al., Cell 16:1553,2015). We hypothesized that BM HSCs and HPCs from aged mice collected/processed in ambient air does not reflect their true numbers and functional capabilities. We thus re-evaluated the aged hematopoietic system of mouse BM collected/processed in more physiologically relevant conditions of 3% O₂ in comparison to the artificially induced extra physiological oxygen shock stress (EPHOSS) situation associated with collecting/processing cells in ambient air in 6-10 week old and 20-28 month old C57Bl/6 mice (N = 8-15 mice/group in a total of 3-4 experiments). BM cells from old, compared to young, mice collected in air had significant increases in LT-HSC, ST-HSC, MPP, CMP, GMP, MEP, but decreases in CLP numbers per femur. When BM was collected under hypoxic conditions, LT-HSC numbers were increased in both young and old mice, with enhanced numbers of CLP in the older mice. When collected in air, old BM cells had ~50% decreased chimerism in a competitive repopulating assay over six months of primary engraftment compared to young BM cells with respective young vs. old competitive repopulating units (CRUs) of 19.4 vs. 4.5 per 10⁶ cells (p<0.02) based on limiting dilution analysis, and lymphoid/myeloid ratios of 3.54 vs. 0.95 (p<0.002). Secondary transplants over 4 months showed 75% reduced chimerism of old vs. young BM. However, when compared to BM collected in air, hypoxia collected cells from young mice demonstrated over a 50% increase in chimerism in 1° transplants (up to 6 months), with old and young hypoxia collected BM having much less drastic changes in primary chimerism and lymphoid:myeloid ratio (2.2±0.6 vs. 3.3.±0.47; p>0.05). CRUs of hypoxia collected old cells (14.2) and air collected young cells (19.4) were similar (p>0.05). There were 2-3 fold fewer CFU-GM, BFU-E, and CFU-GEMM from air collected old BM when compared to young BM. Old BM HPCs were in a slow/non-cycling state compared to the rapid cycling of the young BM HPCs. In contrast, when collected in hypoxia, there were fewer HPCs in the BM of young mice and they were in a slow cycling state when compared to young BM cells collected in air. In old BM collected under hypoxic conditions, there were more HPCs then when collected in air and these cells were in rapid cycle. Results for the numbers of phenotypic HSC and HPC and functional characteristics of HPC have been duplicated in CB6 and BALB/c mice. Hence, there are more phenotypic and functional HSCs and HPCs when old BM cells are collected/processed in hypoxia compared to that of ambient air, numbers equal or close to those when young BM is processed in ambient air. Mechanistically, the enhanced numbers and function of old HSCs/HPCs when BM is collected/processed in hypoxia is associated with changes in expression of CXCR4, CCR5, and ROS. CXCR4 signaling mediates HSC homing. CCR5 is a receptor for RANTES, a chemokine implicated in increasing lymphoid-biased HSCs. ROS is implicated in EPHOSS-mediated differentiation of LT-HSC. We found that expression of CXCR4 and CCR5 was lower in old vs. young LT-HSCs collected in air but was enhanced in hypoxia-collected old LT-HSCs to the level of air collected young cells. Total and mitochondrial ROS were higher in old vs. young LT-HSC from air collected cells, but were decreased in old cells collected in hypoxia to the level of young air collected cells. Therefore, although cells from older BM are more sensitive than younger BM cells to the stress of EPHOSS, enhanced numbers of functional HSCs and HPCs can be obtained from old BM by hypoxic collection and processing.