Abstract 18359: Maturation of Reprogrammed Endothelial Cells Using a Single Transcription Factor in a Prolonged Cultivation
Introduction: Endothelial cells (ECs) are an essential component of blood vessels and are crucial for repairing injured and ischemic tissues. Previously, we showed that a single EC transcription factor, ETV2, is able to directly reprogram human postnatal cells into functional ECs. This novel approach can avoid tumorigenic potential of stem cells and enable autologous cell therapy and personalized vascular disease investigation. However, the reprogrammed ECs (rECs) were still in an immature stage. Here, we aimed to investigate whether immature early rECs can be further reprogrammed into a mature EC.
Methods and Results: We transduced human dermal fibroblasts (HDFs) with doxycycline (DOX)-inducible lentiviral-ETV2. Then these transduced cells were sorted for KDR at day 7 (D7). The D7-sorted KDR+ cells showed EC characteristics and termed as early rECs. After culturing 20 days, these cells usually lose EC characteristics. We varied coating substrates (i.e. collagen, gelatin, Matrigel, OP9 cells), serum percentages, culture media, culture duration, and small molecular epigenetic modifiers to induce long-term culture of rECs to generate mature rECs. We found that subsequent reactivation of ETV2 with DOX for 6 days and administration of valproic acid, the levels of ECs genes were maintained or increased over the culture period of ~3 months with CDH5 and PECAM1 expression reaching the level of HUVECs at D93. Flow cytometry analyses confirmed gene expression data, showing 83% CDH5+ cells and 60% PECAM1+ cells at D93. We referred to these cells as late rECs. Late rECs formed tubular structures in a Matrigel assay with LDL uptake and binding of UEA1 lectin. We confirmed their vessel-forming capability in vivo using mouse skin wound and hindlimb ischemia models. We then compared transcriptome profiles of early and late rECs with HDFs and HUVECs. Heat map and PCA analyses demonstrated that both early- and late rECs showed significantly enriched EC gene expression, closer to the pattern of HUVECs.
Conclusions: Transient reinduction of ETV2 together with an epigenetic modifier can mature immature rECs into more mature phenotype. This induction system and various stages rECs can be beneficial for investigating reprogramming mechanisms as well as therapeutic purposes.
Author Disclosures: S. Lee: None. C. Park: None. J. Han: None. J. Kim: None. K. Cho: None. S. Kim: None. S. Lee: None. S. Sharma: None. Y. Yoon: None.
- © 2016 by American Heart Association, Inc.