Re-Evaluation of Age-Related Dysregulation of Hematopoiesis Based on Oxygen Induced Changes Associated with Cell Collection/Processing in Ambient Air
Authors: Broxmeyer Hal E. , Capitano Maegan L. , O'leary Heather A. , Cooper Scott , Chua Hui Lin , Plett Artur , Orschell Christie M. 
Published: October 2019

Abstract
Hematopoietic stem cell (HSC) numbers and function are reported to be dysregulated/compromised in old mice (e.g. ≥20 months of age: Cho, et. al., Blood 2008; Dykstra, et. al., J. Exp. Med. 2011; Sun, et. al., Cell Stem Cell 2014, amongst other papers). HSC numbers apparently increase with age, with decreased lymphoid production and compromised engrafting capability. However, what we know about aged HSCs is based on evaluation of these cells after they have first been collected and processed in ambient air. This is an hyperoxic environment, much increased in oxygen compared to the in vivo bone marrow (BM) hypoxic (~ 1-5 %) environment that the HSCs reside in. Moreover, there is little information on hematopoietic progenitor cells (HPCs) in terms of numbers and functional capacity in aged animals. Because of the need to better understand HSCs and HPCs in aging, where hematopoietic cell function appears to be compromised, and in context of blood disorders associated with aging, it is important to assess HSCs and HPCs removed from the body for analysis in situations as close as possible to that in which these cells find themselves in the body. It has recently become apparent that collection of BM cells from relatively young mice (e.g. 6-12 weeks of age), even for short periods (≤20 minutes) in ambient air, exposes these cells to the phenomenon of extra physiologic oxygen shock/stress (EPHOSS). EPHOSS triggers the opening of the mitochondrial permeability transition pore, resulting in enhanced production of mitochondrial reactive oxygen species that results in rapid cell differentiation, with decreased numbers of phenotypically- and functionally-defined long term (LT) repopulating, self-renewing HSCs and concomitant increases in rapidly cycling HPCs (Mantel, et. al. Cell 161:1553, 2015). By rigorous attention to detail in collection and processing mouse BM cells from young mice under constant hypoxic conditions of 3 % oxygen, it became clear that, in fact, there were on average 3-5 fold greater numbers of phenotyped- and functional LT HSCs. But, there were many fewer HPCs, and these HPCs were in a slow or non-cycling state when compared to cells collected and processed in ambient air, or when collected in 3% oxygen and then exposed to ambient air. We thus hypothesized that BM HSCs and HPCs from aged mice collected/processed in ambient air may not reflect their true numbers and functional characteristics in vivo. This led us to re-evalaute hematopoiesis in aged mice, compared to that in young mice, but in which BM cells were collected and processed for numbers and functions of HSCs and HPCs in a more physiological oxygen tension of 3 %, as reported (Mantel, et. al. Cell, 2015). We evaluated BM from CB6, Balb/c, and C57Bl/6 mice at 20-25 months vs. 6-16 weeks of age, collected/processed in ambient air or 3% oxygen. Collection in air demonstrated that older mice had 2.6-2.8 and 1.5-1.9 fold more LT- and short term (ST)-HSCs, with 1.8-2.2 fold fewer phenotypically-defined common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMPs) and common lymphoid progenitors (CLPs), and 2-3 fold fewer functional myeloid HPCs (CFU-GM, BFU-E, CFU-GEMM) than younger mice. Moreover, HPCs of older mice, as assessed by colony assay, were in a slow cycling state. In contrast, BM of young and old mice collected/processed in hypoxia, demonstrated similar numbers of LT-HSC, ST-HSCs, CMPs, GMPs, and CLPs. Moreover, while CFU-GM, BFU-E, and CFU-GEMM in young mice were decreased after hypoxic collection/processing and were in slow cycle, those of older mice were greatly increased in numbers, and were in rapid cycle. Engrafting and other cell and intracellular studies are ongoing, but it is clear that hematopoietic cell studies previously reported in aged mice will have to be re-evaluated for better mechanistic understanding of their actual numbers and cell and intracellular characteristics as reflected in an in vivo hypoxic environment.

Disclosures
Broxmeyer: CordUse: Other: SAB Member .