BackgroundEndothelial progenitor cell (EPC) transplantation is a promising therapy for ischemic diseases such as ischemic myocardial infarction and hindlimb ischemia. However, limitation of EPC sources remains a major obstacle. Direct reprogramming has become a powerful tool to produce EPCs from fibroblasts. Some recent efforts successfully directly reprogrammed human fibroblasts into functional EPCs; however, the procedure efficacy was low. This study therefore aimed to improve the efficacy of direct reprogramming of human fibroblasts to functional EPCs.MethodsHuman fibroblasts isolated from foreskin were directly reprogrammed into EPCs by viral ETV2 transduction. Reprogramming efficacy was improved by culturing transduced fibroblasts in hypoxia conditions (5 % oxygen). Phenotype analyses confirmed that single-factor ETV2 transduction successfully reprogrammed dermal fibroblasts into functional EPCs.ResultsHypoxia treatment during the reprogramming procedure increased the efficacy of reprogramming from 1.21 ± 0.61 % in normoxia conditions to 7.52 ± 2.31 % in hypoxia conditions. Induced EPCs in hypoxia conditions exhibited functional EPC phenotypes similar to those in normoxia conditions, such as expression of CD31 and VEGFR2, and expressed endothelial gene profiles similar to human umbilical vascular endothelial cells. These cells also formed capillary-like networks in vitro.ConclusionOur study demonstrates a new simple method to increase the reprogramming efficacy of human fibroblasts to EPCs using ETV2 and hypoxia.

Ischemia are common conditions related to lack of blood supply to tissues. Depending on the ischemic sites, ischemia can cause different diseases, such as hindlimb ischemia, heart infarction and stroke. This study aims to evaluate how extracellular vesicles (EVs) derived from ETV2 transfected fibroblasts affect endothelial cell proliferation and neovascularization in a murine model of hindlimb ischemia. Human fibroblasts were isolated and cultured under standard conditions and expanded to the 3th passage before use in experiments. Human fibroblasts were transduced with a viral vector containing the ETV2 gene. Transduced cells were selected by puromycin treatment. These cells were further cultured for collection of EVs, which were isolated from culture supernatant. Following co-culture with endothelial cells, EVs were evaluated for their effect on endothelial cell proliferation and were directly injected into ischemic tissues of a murine model of hindlimb ischemia. The results showed that EVs could induce endothelial cell proliferation in vitro and improved neovascularization in a murine model of hindlimb ischemia. Our results suggest that EVs derived from ETV2-transfected fibroblasts can be promising non-cellular products for the regeneration of blood vessels.

Introduction: Embryonic stem cells are pluripotent, thus capable of differentiating into all types of cells derived from the three germ layers. However, the application of embryonic stem cells (ESCs) for preclinical and clinical studies is difficult due to ethical concerns. Induced pluripotent stem cells (iPSCs) are derived from differentiation and have many ESC characteristics. The study herein examines the production of iPSCs from reprogramming of mouse embryonic fibroblasts (MEFs) via transduction with defined factors. Methods: MEFs were collected from mouse embryos via a previously published protocol. The cells were transduced with a single polycistronic viral vector encoding mouse cDNAs of Oct3/4, Sox2, Klf4 and c-Myc. Transduced cells were treated and subcultured with ESC medium. The cells were evaluated as iPSCs with specific morphology, and expression SSEA-1, Oct3/4, Sox2 and Nanog. In addition, they also were evaluated for pluripotency by assessing alkaline phosphatase (AP) activity and in vivo teratoma formation. Results: Under the reprogrammed conditions, the transduced cells displayed a change in morphology, forming ESC-like clusters. These cell clusters strongly expressed pluripotent markers as well as ESC-specific genes. Furthermore, the colonies exhibited higher AP activity and formed teratomas when injected into the murine testis.

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Abstract-Introduction:Human fibroblasts can be differentiated into endothelial progenitor cells by direct reprogramming via ETV-2 transfection. Previously, we have shown that the efficacy of direct reprogramming can be enhanced by hypoxia treatment. In this study, we aim to investigate whether the efficacy of direct reprogramming of fibroblasts into EPCs via Ets variant gene 2 (ETV2) transfection can be increased with hepatocyte growth factor (HGF) treatment. Methods: Foreskin-derived fibroblasts were cultured in standard medium (DMEM/F12 supplemented with fetal bovine serum). They were then transduced with a viral vector expressing ETV2 in culture medium supplemented with HGF. The transduced fibroblasts were cultured in endothelial cell medium supplemented with HGF for 28 days. The efficacy of direct reprogramming was evaluated based on expression of CD31 and VEGFR2 markers by transduced cells. Phenotypic and functional characterization of induced EPCs were also confirmed by expression of particular genes and in vitro angiogenesis assays. Results: Our results showed that HGF significantly increased the efficacy of direct reprogramming of fibroblasts towards EPCs via ETV2 transcription factors; efficiency increased from 5.41±1.51% for ETV2 transduction alone to 12.31±2.15% for ETV2 transduction combined with HGF treatment. Conclusion: These findings suggest the rationale for combined use of ETV2 and HGF in direct in vitro reprogramming of fibroblasts into EPCs.