Since the discovery of endothelial colony forming cells (ECFC), there has been significant interest in their therapeutic potential to treat vascular injuries. ECFC cultures display significant heterogeneity and a hierarchy among cells able to give rise to high proliferative versus low proliferative colonies. Here we aimed to define molecularly this in vitro hierarchy. Based on flow cytometry, CD34 expression levels distinguished two populations. Only CD34 1 ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD342 ECFCs formed only small clusters. CD34 1 ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 1 and CD342 cells. Upon replating, CD34 1 ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 1 ECFC were superior at restoring perfusion and better engrafted when injected into ischemic hind limbs. Transcriptomic analysis identified cyclin-dependent kinase (CDK) cell cycle inhibiting genes (p16, p21, and p57), the Notch signaling pathway (dll1, dll4, hes1, and hey1), and the endothelial cytokine il33 as highly expressed in CD34 1 ECFC. Blocking the Notch pathway using a c-secretase inhibitor (DAPT) led to reduced expression of cell cycle inhibitors, increased cell proliferation followed by a loss of self-renewal, and HPP colony formation capacity reflecting progenitor exhaustion. Similarly shRNA knockdown of p57 strongly affected self-renewal of ECFC colonies. ECFC hierarchy is defined by Notch signalling driving cell cycle regulators, progenitor quiescence and self-renewal potential. STEM CELLS 2016;34:902-912 SIGNIFICANCE STATEMENTWe demonstrate here the mechanism by which endothelial colony forming cells (ECFC) are able to self-renew and maintain their survival for long-term culture. This is driven by strong Notch signaling within the progenitor population that allows for the self-renewing of the progenitor population and differentiation into a mature endothelial cell. This discovery has significant implications in the development of ECFC into a mainstream cellular therapy as it is now established how ECFC can be expanded and maintained, which will impact the use of ECFC in treating ischemic disease.

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Endothelial colony forming cells (ECFC) and mesenchymal stem cells (MSC) combined have great potential to be used for cell therapy of ischemic vascular diseases. However, to improve allogeneic stem cell engraftment the use of immunosuppression, such as cyclosporine has been suggested. Our aim was to assess the impact of cyclosporine on hind limb revascularisation upon MSC and ECFC combination therapy. Balb/c immunocompetent mice subjected to hind limb ischemia (right femoral artery ligation) were given both human ECFC and MSC (weekly intramuscular injections) with or without cyclosporine (daily injection). Surprisingly, mice receiving cyclosporine had a significant decrease in reperfusion based on laser Doppler imaging compared to vehicle controls and had poorer limb survival. In vitro, the downstream calcineurin target NFATC4 was highly expressed in the self‐renewing fraction of ECFCs. ECFCs cultured with cyclosporine had reduced colony formation capacity and tube formation in Matrigel. Lastly, ECFC displayed increased proliferation and loss of capacity for long term culture when in the presence of cyclosporine clearly showing a loss of quiescence and progenitor function. Our findings demonstrate the deleterious impact of cyclosporine on ECFC function, with significant impact on ECFC‐based allogeneic cellular therapy. stem cells translational medicine 2019;8:162&7

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Background: Although endothelial progenitors have long been described, there remains significant controversy around their identity in vivo . The endothelial colony forming (ECFC) assays suggested a hierarchy among endothelial cells in vivo . Our aim was to systematically test different endothelial cell populations sorted from the human placenta, a highly vascularised tissue, based on various cell surface markers for their ECFC potential. Methods and Results: Upon sorting based on key markers CD45, CD34 and CD34 it was easily established that most ECFC potential was concentrated in the CD45-CD34+ fraction. Among this population, single cell culture assays (>300 wells per cell type) were performed on sorted CD31neg, CD31int and CD31hi cells. Only the CD31int cells were able to grow high proliferative potential ECFC. CD31neg populations contained mesenchymal stem cells (MSC) whereas CD31hi cells only produced mature endothelial clusters. RNA sequencing of each fraction identified Notch signalling as a key driver of endothelial progenitors as opposed to MSC. When the CD31int population was further characterised it was found to be expressing VE-Cadherin as predicted by the RNAseq, however, was not in contact with the circulation as it did not stain for lectins injected intravenously. In accordance the self-renewing fraction of ECFC cultures in vitro was dependent on Notch signalling and controlled the expression of IL33 and CDKN1C (p57) to maintain progenitors in quiescence. This was validated in vitro and in vivo by performing shRNA and pharmacological inhibition of the different pathways. Conclusion: Our study uncovers a population of endothelial progenitors in vivo at the cellular and molecular level and identified a novel role for Notch signalling in maintaining progenitor self-renewal in vivo and in vitro .