br Introduction Stem progenitor cells SPCs capable of

Introduction Stem/progenitor pppa cost (SPCs) capable of multipotent differentiation can be mobilized from bone marrow and adipose tissue, enter the blood stream and migrate to peripheral sites where they may facilitate recovery from injuries (To et al., 1997; Gil-Ortega et al., 2013; Asahara et al., 1997). SPCs mobilization occurs after wounding, physical exertion and in response to a variety of chemical agents (Fiorina et al., 2010; Fukaya et al., 2013; Albanese et al., 2009; Asahara et al., 1999; Rehman et al., 2004; Reyes et al., 2002; Takahashi et al., 1999). Exposure to hyperbaric oxygen (HBO2) appears to be a reliable way to mobilize SPCs in humans and also has been shown in rodents and horses (Thom et al., 2006, 2011; Ma et al., 2011; Milovanova et al., 2009; Dhar et al., 2012). Animal studies indicate that one mechanism is based on activation of nitric oxide synthase type 3 (NOS-3) in bone marrow stromal cells with subsequent liberation of stem cell factor (Thom et al., 2006; Goldstein et al., 2006). Separate from mobilization, HBO2 improves engraftment and differentiation of several progenitor cell types in organs such as the spleen, bone marrow, brain, peripheral nerve, pancreas, cartilage and heart (Aljitawi et al., 2014; Lee et al., 2013; Cherng et al., 2012; Khan et al., 2012; Zhang et al., 2010, 2011; Pan et al., 2009). One area of interest with circulating SPCs is the identification of the sub-set having propensity to form vascular endothelium, so-called endothelial progenitor cells (EPCs) (Hirschi et al., 2008). Quantification of mobilized EPCs is based on flow cytometric detection of cell surface proteins and phenotypic manifestations of laboratory-grown clones (Hirschi et al., 2008; Mund et al., 2012). Cells mobilized by HBO2 exhibit many of these surface markers and when cultured, some clones show lectin binding consistent with an endothelial phenotype (Thom et al., 2006, 2011). Animal studies have documented that HBO2-mobilized SPCs form blood vessels in vivo and hasten wound healing (Milovanova et al., 2009; Goldstein et al., 2006; Gallagher et al., 2007). HBO2-mobilized SPCs have greater content of hypoxia inducible factors (HIFs) and thioredoxin-1 (Trx), which in the murine model confers improved neovascularization (Thom et al., 2011; Milovanova et al., 2008, 2009). Subsequent to HBO2 treatments of refractory wounds and diabetic patients, the number of wound margin SPCs is increased and local HIFs and Trx appear to be within these localized SPCs (Thom et al., 2011; Ma et al., 2011). This suggests that SPCs play a role in supplying factors required for wound healing. Hence, evaluating intracellular proteins may have greater importance to assess SPCs function versus ex vivo manipulations. Assessment of intracellular regulatory proteins of cells selected based on surface markers precludes studying ex vivo cell growth because of need to permeabilize the cell membranes. HBO2 treatment involves breathing 100% O2 at 2 to 3 atmospheres absolute (ATA) pressure for 1.5 to 2h once or twice daily. HBO2 has been shown to improve refractory diabetic wounds and delayed radiation injuries in randomized trials and use is supported by independent evidence-based reviews (Bennett et al., 2008; Clarke et al., 2008; Kranke et al., 2012; Goldman, 2009; Fife et al., 2007; Duzgun et al., 2008; Londahl et al., 2010). Several studies have failed to identify clinical efficacy (Annane et al., 2004; Margolis et al., 2013). Notably, these studies involved exposures to 2.0 ATA or use of face masks with questionable seals thus reducing the fraction of inspired O2; whereas several prospective randomized trials documenting therapeutic benefit utilized pressures of 2.4 or 2.5 ATA in pure O2-filled chambers or using head-covering hoods (Londahl et al., 2010; Marx et al., 1985). Whether clinical results may differ because of treatment protocols is unclear. The goal of this investigation was to evaluate whether mobilization of cells with surface markers considered consistent with SPCs (CD34+ and CD45-dim) and content of intracellular regulatory proteins differed between two commonly used HBO2 protocols (Pober, 2012).