To determine a profile of gene

To determine a profile of gene activation within the hearts of AMI rats following EXnaEFC administration, we performed a comprehensive PCR analysis of cardiac tissue seven days post-infarct and treatment. We reveal that the rats receiving EXnaEFCs exhibited rescued gene expression of Tnf, Sdf1 and Cd14. TNFα has been implicated in stimulating fibroblast proliferation and assisting in heart repair post-AMI (Jacobs et al., 1999). SDF1 supports further EPC mobilisation from the bone marrow (Jin et al., 2006) and CD14 expressing macrophages may aid in the clearance of necrotic tissue (Ertl and Frantz, 2005). Rats administered expanded naEFCs also exhibited levels of Cx3cl1 closer to those found in control rats. Herein Ccl2 gene expression was highest in rats receiving EXnaEFCs. Notably, the CCL2 receptor, CCR2, is expressed by EPCs and vascular smooth muscle purchase AL 8697 and is associated with angiogenesis (Schober, 2008). Mmp9, which was significantly increased in rats administered EXnaEFCs, has been shown to liberate EPCs from the bone marrow (Heissig et al., 2002; Liu and Velazquez, 2008). Together this data suggest that EXnaEFCs trigger the production of a suite of rat-derived pro-angiogenic factors, many of which also mediate the clearance of damaged and necrotic tissue. While immunocompromised animals, (such as the T lymphocyte deficient CBH/Rnu rats used here), are commonly employed to mimic human AMI (Dick et al., 1997; Masuda et al., 2012; Shultz et al., 1995), studies indicate that immunosuppression prevents leukocyte recruitment into cardiac tissue post-AMI, reduces the clearance of dead cells and significantly delays recovery time (Kloner et al., 1978). Therefore the suitability of immunocompromised animals in AMI experiments generally is questionable and conclusions must be made prudently. Finally, if we are to consider allogeneic cells for therapeutic application understanding their immunogenicity is important. We show that EXnaEFCs express slightly higher levels of MHC class I and class II when compared to donor-matched ECs. However, when stimulated with the pro-inflammatory cytokines TNF or IFNγ, two cytokines routinely present at a transplant site (Ono et al., 1998), the increase in MHC I expression on EXnaEFCs was modest compared to the ECs. While more reactive than the MSCs, the EXnaEFCs were significantly lower in MHC expression when compared to the mDCs. Herein we have described the isolation, serum-free expansion and characterisation of a non-adherent population of human endothelial progenitor cells (EXnaEFCs). These cells express progenitor cell and EC markers and display pro-angiogenic properties that can assist EC tube formation in vitro and in vivo. Interleukin-3 significantly enhanced EXnaEFC expansion and these cells demonstrated a clinically relevant improvement of heart function seven days post-AMI with reduced serum creatinine levels and an increase in pro-angiogenic genes in the rat heart. The mechanisms underpinning the effect of IL3 on EXnaEFCs are yet to be fully elucidated, however data to support its use come from it enhancing CD34+ and proangiogenic cell survival (D\'Atri et al., 2011; Zeoli et al., 2008) as well as EC migration and tube formation in vitro (Dentelli et al., 1999). Our work contributes to this by demonstrating an increase in ERK activation in response to IL3. With studies showing that high-dose intervention of stem cells within two weeks is most effective in AMI (Dimmeler et al., 2008; Richardson et al., 2013), our rapid expansion of EXnaEFCs to clinically relevant numbers within a therapeutically advantageous window supports the further investigation of IL3 in cell therapy. Importantly, in our study, the EXnaEFCs did not form capillary-like tubes on their own in vivo and therefore do not merit the ‘endothelial’ status (Hirschi et al., 2008; Yoder, 2012). A broader pro-angiogenic phenotype is noted instead. Taken together, these expanded human cells differ significantly from those we originally characterised and phenotyped as naEFCs with CD133+ isolation from cord blood and enrichment for four days in defined media (Appleby et al., 2012). These results show IL3 as a potent growth factor for human CD133+ cell expansion with clear pro-angiogenic properties, in vitro and in vivo.