Hypoxia's role in regulating stressed hematopoietic stem cells - (PLEASE KEEP IN WORD, DO NOT PDF) Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Hematopoietic stem cells (HSCs) reside in the bone marrow microenvironment (BM-ME) with distinctive low oxygen tension. In contrast to the physiological status of HSCs in the BM (~1-5% O2), almost all ex vivo HSC studies have been conducted by collecting and processing BM in ambient air with extra-physiologic oxygen tension (~21% O2). Our previous work evaluating normal mouse BM and human cord blood, revealed a significant loss in the number and function of HSCs upon exposure to extraphysiologic oxygen shock/stress (EPHOSS) in ambient air. HSCs collected and processed under physioxia demonstrate higher engraftment and self-repopulating capacity than HSCs collected and processed under ambient air. The exposure to ambient air (~21% O2) and sudden oxygenation induces rapid differentiation of HSCs to multipotent and committed HPCs. While these paradigm changing findings have been exciting to report; the molecular basis behind this distinction are poorly understood but have the potential to improve hematopoietic cell transplantation (HCT) for both malignant and non-malignant diseases as well as for gene therapy and/or correction of diseased HSCs. One example of how diseased HSCs result in bone marrow failure (BMF) involves Fanconi Anemia (FA), a rare genetic disease characterized by BMF and cancer. BM from FA patients and from fanca-/- and fancc-/- mice are defective in HSC function, which compromises their use in human and mouse HCT and gene therapy to correct genetic defects causing FA. We provide strong preliminary data to suggest that fanca-/- and fancc-/- HSCs when collected and processed in physioxia (i.e., hypoxia) show a profound increase in the recovery of long-term (LT)-HSCs compared to those harvested in normoxia (i.e., ambient air). Whether these HSCs are functionally more competent than those harvested in normoxia will be one of the focuses of this application. Additionally, we have identified novel antioxidant pathways in hypoxic HSCs, which we will try to manipulate in FA HSCs to improve HSC function. Lastly, we have established the Spacer-Nick-mediated gene correction approach, which barely induces double-stranded breaks and DNA damage response in cells. We hypothesize that implementing the Spacer-Nick-mediated gene correction approach under hypoxia and in fancc-/- HSCs lacking stress pathways will be more efficient and, importantly, will enhance the function(s) of gene corrected HSCs in vitro and in vivo.
