Abstract 142: New Approach for Directly Reprogrammed Endothelial Cells

Abstract

Rationale: For therapeutic neovascularization through cell therapy to treat cardiovascular diseases, recently, we successfully generated reprogrammed ECs, directly from human dermal fibroblasts (HDF) with the ETV2 transcription factor. Although functional ECs were generated, ETV2 was delivered lentiviral vector, which is clinically unsuitable. Objective: In this study, we aimed to generate clinically compatible rECs, by employing an adenoviral vector to minimize genetic integration. Furthermore, to prolong the cell retention and improve cell survival, we applied synthetic biomaterial, polypeptide amphiphile (PA) nanomatrix gel and tested for experimental cell therapy in animal models. Methods and Results: After we transduced HDFs with Ad- ETV2 (Ad- ETV2 -HDF), EC gene expression was measured by qRT-PCR and flow cytometry. We found that Ad- ETV2- HDFs displayed a cobblestone-like morphology and showed notable induction of EC genes (CDH5, KDR, PECAM1). Flow cytometry analysis showed peak expression of CDH5 and KDR at D6 and continuous increase of PECAM1 over 9 days. Also, Ad- ETV2 -HDFs took up acetylated LDL and bound lectin, suggesting that these cells possess functional EC characteristics. To enrich cells showing EC characters, Ad- ETV 2-HDFs were sorted for KDR by FACS at day 6. qRT-PCR showed substantial enrichment of EC-specific gene expression in KDR + cells. Only KDR + cells (reprogrammed ECs, rECs) formed tube-like structures on Matrigel. In addition, immunostaining confirmed high expression of EC marker proteins in rECs. Next, we tested the functionality of rECs in hindlimb ischemia mouse model. At 4 weeks after injection of 4 x 10 5 rECs into the ischemic hindlimb, we observed rECs incorporated into host blood vessels or resided perivascular regions. Furthermore, we found more rECs in the tissues and were directly incorporated into the vessels when they were encapsulated in PA nanomatrix gel compared to bare rEC-injected mice. Conclusions: Together, these data strongly support the generation of Ad- ETV2 -induced rECs (Ad-rECs) and enhanced retention of rECs by PA nanomatrix gel in tissue. With this study, we can develop advanced concept and techniques for clinical application of rECs and establish a new platform for future cell therapy.