br Materials and methods br Results

Materials and methods
Results
Discussion The ability to repair and generate blood vessels is critical to improve outcomes in a number of diseases including cardiovascular disease and diabetes. Endothelial progenitor Sulindac sulfide contribute to blood vessel formation and vascular repair in animal models of ischemia (reviewed in (Timmermans et al., 2009; Yoder, 2012)) but human trials have yielded moderate success (Lipinski et al., 2007; Martin-Rendon et al., 2008). Human EPCs have been isolated by a variety of means that essentially fall into two broad categories, 1) cell culture conditions to enrich for EPC colonies (Hill et al., 2003; Ito et al., 1999; Kalka et al., 2000) and 2) the isolation of cells based on surface marker expression e.g. CD34 or CD133 (Appleby et al., 2012; Bompais et al., 2004). These methods warrant refining as they (i) lack the specificity of EPC selection and (ii) are commercially prohibitive based on high cell culture costs and requiring ~5weeks of culture to reach an ECFC number sufficient for therapeutic application (Gulati et al., 2003; Hur et al., 2004). Herein we have isolated CD133+ naEFCs from cord blood and expanded the population ~350-fold in less than two weeks, using serum-free medium. Given that 1–3×106 CD133+ naEFCs can be isolated from ~60ml cord blood, this protocol can produce in excess of 5×108 cells within 14days. Serum-free expansion of CD133+ umbilical cord blood cells has previously been achieved using medium supplemented with SCF (100ng/ml), TPO (20ng/ml), Flt3L (100ng/ml), IL3 (20ng/ml), plus VEGF and IL6 (Masuda et al., 2012). Here, using significantly lower concentrations of SCF (40ng/ml), TPO (10ng/ml) and Flt3L (40ng/ml), together with IL3 (20ng/ml), we were able to achieve similar expansion in the same time period. Our investigation into cell viability also suggests that the IL3-induced expansion of the naEFCs was not merely via pro-survival effect as the rate of cell viability was similar across all media combinations assessed. We were also able to show that isolated CD133+ EXnaEFCs could be frozen, thawed and expanded equally well as the freshly isolated cells, an advantage for the use of these cells as a potential allogeneic cell therapy. The expression of CD133 by EXnaEFCs differentiates them significantly from the well characterised ECFCs which are CD133− and suggests that the EXnaEFCs are a more primitive circulating EPC rather than the EC-like ECFCs. EXnaEFCs also express CD117, CD31, and low levels of CD45, but do not express CD14, CD38, CD144 or CD146. The expression of CD45 is a contentious issue in this field but increasing evidence supports that cells expressing this marker are clinically relevant. For example, Asahara\'s recent development of a clonogenic assay for cells with endothelial potential showed that these cells clearly expressed CD45 (Masuda et al., 2011). A study by Estes et al. showed a CD34+CD133+CD45lowCD31+CD14− population capable of promoting tumour blood vasculature in mice (Estes et al., 2010). Furthermore, reduced frequency of circulating CD34+VEGFR2+CD45low and CD34+CD133+CD45low cells correlates with peripheral and coronary artery disease, while CD34+VEGFR2+CD45− shows no such correlation (Estes et al., 2010; Schmidt-Lucke et al., 2010). Our data demonstrate that co-culture of EXnaEFCs with HUVECs in Matrigel™ in vitro promoted tube-like structure formation. Fluorescence microscopy suggested that expanded naEFCs were in close proximity to and possibly incorporated into HUVEC structures. The infusion of a heterogeneous population of progenitor cells, either from bone marrow or peripheral blood into myocardial infarction patients has been shown to improve left ventricle ejection fraction, end-systolic and diastolic volumes and increase coronary blood flow of the affected vessel (Assmus et al., 2002; Jiang et al., 2010; Schachinger et al., 2004; Strauer et al., 2002). As delivery of human EPCs into the infarct site of rats with surgically induced AMI has shown increased cardiac function as measured by ejection fraction (Hong et al., 2013), fraction shortening (Schuh et al., 2008) and left ventricle end systolic and diastolic pressure (Hu et al., 2009) we executed similar experiments to examine the restorative effect of the human EXnaEFCs. Intracardial injection of 1×106 EXnaEFCs immediately following AMI demonstrated a decrease in serum creatinine levels when compared to AMI rats administered PBS alone. Notably, the creatinine levels measured in the untreated control CBH/Rnu rats were below published values for immunocompetent rats such as Sprague Dawley which we have previously used in a similar model of AMI (Palm and Lundblad, 2005; Richardson et al., 2013). As measurement of serum creatinine is an indirect determinant of cardiac damage that rises acutely post-myocardial infarction, measurements within the first two to three days would better represent cardiac damage. This will be examined in future studies. Furthermore, the CBH/Rnu rats in this study were routinely ~80–150g, half the weight of Sprague Dawley rats which weigh ≥250g each at the same age (Hong et al., 2013; Schuh et al., 2008). A higher level of AMI-induced mortality was observed in the CBH/Rnu rats and the ejection fraction in our rats was significantly lower than documented elsewhere, both prior to surgery (63% compared to 82%) and post-surgery (20% compared to 39%) (Hong et al., 2013). While we demonstrate a modest 5% increase in EF seven days following administration of EXnaEFCs versus PBS controls, it must be noted that a recent meta-analysis from 2625 patients showed that compared to the standard treatment, bone marrow cell transplantation delivered intracoronary exhibited a 3.96% improvement in EF and correlated with a significant decrease in all-cause mortality in these patients (Jeevanantham et al., 2012). Based on these observations, the contribution of EXnaEFCs to alleviate ischemic injury warrants further investigation. Improved cardiac performance in animal models has been shown four-weeks post-EPC transfer (Hong et al., 2013; Hu et al., 2009; Masuda et al., 2012; Schuh et al., 2008), suggesting a longer time period within our study may well have improved outcomes even further. In addition, our in vitro work suggested that the EXnaEFCs expressed less MHC classes I and II when compared to donor matched mature DCs, and were less receptive to TNF and IFNγ for increased surface expression of MHC class I. Our in vivo experiments utilised immunocompromised rodents and thus conclusive evidence of ‘low immunogenicity’ will require immunocompetent animals, longer in vivo studies and would also benefit from a comparison to MSCs. This is part of an ongoing study within our laboratory.