Self-Renewal and High Proliferative Colony Forming Capacity of Late-Outgrowth Endothelial Progenitors Is Regulated by Cyclin-Dependent Kinase Inhibitors Driven by Notch Signaling.

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 + ECFC had the capacity to reproduce high proliferative potential (HPP) colonies on replating, whereas CD34- ECFCs formed only small clusters. CD34 + ECFCs were the only ones to self-renew in stringent single-cell cultures and gave rise to both CD34 + and CD34- cells. Upon replating, CD34 + ECFCs were always found at the centre of HPP colonies and were more likely in G0/1 phase of cell cycling. Functionally, CD34 + 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 + ECFC. Blocking the Notch pathway using a γ-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.

Subtype-Specific Analyses Reveal Infiltrative Basal Cell Carcinomas Are Highly Interactive with their Environment.

Little is known regarding the molecular differences between basal cell carcinoma (BCC) subtypes, despite clearly distinct phenotypes and clinical outcomes. In particular, infiltrative BCCs have poorer clinical outcomes in terms of response to therapy and propensity for dissemination. In this project, we aimed to use exome sequencing and RNA sequencing to identify somatic mutations and molecular pathways leading to infiltrative BCCs. Using whole-exome sequencing of 36 BCC samples (eight infiltrative) combined with previously reported exome data (58 samples), we determine that infiltrative BCCs do not contain a distinct somatic variant profile and carry classical UV-induced mutational signatures. RNA sequencing on both datasets revealed key differentially expressed genes, such as POSTN and WISP1, suggesting increased integrin and Wnt signaling. Immunostaining for periostin and WISP1 clearly distinguished infiltrative BCCs, and nuclear ß-catenin staining patterns further validated the resulting increase in Wnt signaling in infiltrative BCCs. Of significant interest, in BCCs with mixed morphology, infiltrative areas expressed WISP1, whereas nodular areas did not, supporting a continuum between subtypes. In conclusion, infiltrative BCCs do not differ in their genomic alteration in terms of initiating mutations. They display a specific type of interaction with the extracellular matrix environment regulating Wnt signaling.

Immunosuppression Agent Cyclosporine Reduces Self-Renewal and Vessel Regeneration Potentiation of Human Endothelial Colony Forming Cells.

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.