Most AB ECFCs were poorly

Most AB-ECFCs were poorly clonogenic, entered rapidly in senescence and had limited functional capacities compared to CB-ECFCs. Moreover, their transcriptional level for the stemness genes DNMT3b, GDF3 and SOX2, that we have previously described (Guillevic et al., 2016), were similar to those found in adult mature endothelial cells. Although the efficacy of AB-ECFC injection as an autologous cell therapy remains challenging, these cells could be useful to study vascular aging in vitro as they may be obtained using a minimally invasive method. According to our results, senescence in AB-ECFCs is mainly mediated by p16 and p15 CDK inhibitors and seems to be associated to oncogenic signals such as an increased expression of RAF1. Furthermore in AB-ECFCs, the transcript level of eNOS was significantly decreased and associated with a low NO production, reflecting an endothelial dysfunction which is known to characterize pathophysiological vascular aging like atherosclerosis (Minamino and Komuro, 2004). Besides, a clinical study based on a NOS overexpression strategy to enhance autologous angiogenic cell therapy in myocardial infarction is ongoing (ClinicalTrials.gov, Identifier: NCT00936819). Highly clonogenic CB-ECFCs overexpressed eNOS and constitutively released high levels of NO, so they appear particularly interesting for angiogenic cell therapy, especially as they can form numerous long calcitonin gene related peptide as observed in 3D tubulogenesis assay. On this point, although Matrigel™ assay is often used to quickly test angiogenic potential, we can note that 3D tubulogenesis assay allows revealing a more marked difference between the tested samples. Our study also provided new information about endothelial aging since a loss of immaturity in AB-ECFS compared to CB-ECFCs was evidenced by the downregulation of CD34 and the defined set of stemness genes. CD34 is described as a marker of quiescence. In vivo, both very immature stem cells and mature endothelial cells are CD34+ and quiescent, thus CD34 is not a genuine stemness marker. Nevertheless, according to a recent study, the most proliferative CB-ECFCs are derived from the CD34+ fraction (Patel et al., 2016). Accordingly, we observed that 40–50% of highly and intermediate clonogenic CB-ECFCs were CD34+ while 4 times less low clonogenic CB-ECFCs and AB-ECFCs were CD34+. It could be assumed that the initial number of quiescent cells could be higher in the most clonogenic samples, supplying the pool of proliferative cells with a progressive loss of quiescence throughout their expansion. DNMT3b could be an interesting target since this de novo DNA methyltransferase plays a role in epigenetic stemness and early regulation of mesodermal differentiation (Jackson et al., 2004). Moreover, its expression in adult peripheral MNC linearly decreases during aging (Ciccarone et al., 2016). GDF3 (Growth differentiation Factor 3), a member of the TGF-β superfamily, is involved in the maintenance of embryonic stem cells at an undifferentiated stage (Levine and Brivanlou, 2006). Its role in endothelial cells or aging is not yet described. According to our results, its transcriptional expression in ECFCs appeared more related to their clonogenicity than to their proliferation, since intermediate clonogenic samples, whose proliferative potential was similar to that of highly clonogenic ECFCs, expressed significantly less GDF3. Sox2 belongs to the master transcriptional factors of the feedback loops maintaining pluripotency and self-renewal in mouse and human embryonic stem cells (Boyer et al., 2010). Interestingly, according to a study published in 2010, human endothelial/mesenchymal-like progenitor cells transduced with SOX2 show an enhanced cardiovascular differentiation leading to better angiogenic capacities (Koyanagi et al., 2010). Moreover in a pathological context, VEGF may promote Sox2 expression in breast tumor cells leading to an enhanced self-renewal (Zhao et al., 2014). Its potential involvement in physiological progenitor cell-mediated angiogenesis remains to be elucidated.