Transdifferentiation of Human Adult Fibroblasts to Endothelial Cells by Defined Factors

Abstract

Background− Mouse fibroblasts could be directly converted into functional endothelial cells in previous study. Here, this study aimed to develop the skill of direct conversion of human differentiated somatic cell into endothelial cell, and improve the efficiency and quality of this technology for therapeutic applications.

Methods and Results− Based on previous results, we cloned a total of 11 genes that played an important role in endothelial development and function. We confirmed the combinations of 5 transcriptional factors, including Foxo1, Er71, Klf2, Tal1 and Lmo2, can directly convert mouse adult fibroblasts into endothelial cells. It is known that 5 transcriptional factors can transdifferentiate mouse skin fibroblast into endothelial cells on previous study, and we transduced those 5 transcriptional factors to human dermal fibroblasts (HDFs) via lentiviruses. After transduction, we confirmed the conversion of human endothelial cell using flow cytometry in every 7 days. Twenty-eight days later, human dermal fibroblast was transduced with 5 transcriptional factors. They exhibited endothelial-like cobblestone morphology and expressed specific markers of endothelial cells. Furthermore, we determined the combination of optimal factors for direct conversion to endothelial cell. By serial stepwise screening using 5 transcriptional factors and 8 other transcriptional factors. We found the most suitable 3 key factor combination (Er71, Klf2, Tal1). We confirmed that the expression rate of endothelial specific markers that were transduced by 3 factors was two-times more than that of 5 factors. After we transduced each 5 factors and 3 factors to HDFs, VE-cadherin+ and/or CD31+ cells were isolated using FACS sorting. We designated the isolated cells as human induced endothelial cells (hiECs). Both 5F-hiECs and 3F-hiECs showed endothelial-like cobblestone morphology but 3F-hiECs expressed more of VE-cadherin+ and CD31+ than 5F-hiECs. 3F-hiECs possessed characterizations of endothelial cells such as DiI-Ac-LDL uptake, UEA-1 lectin binding, tube formation on matrigel coated dish, NO synthesis and SEM image. This reprogramming strategy that using 3 factors did not involve pluripotency state, as Oct4/3, Nanog and Sox2 were not expressed after 3 factor transduction.

Conclusions− This study reports the novel strategy that human endothelial cells can be transdifferentiated from fibroblasts. We found that the combination of Er71, Klf2 and Tal1 is sufficient to induce endothelial conversion.