Direct conversion of adult skin fibroblasts to endothelial cells by defined factors

Background - Cell-based therapies to augment endothelial cells (ECs) hold great therapeutic promise. Here, we report a novel approach to generate functional ECs directly from adult fibroblasts. Methods and Results - Eleven candidate genes that are key regulators of endothelial development were selected. Green fluorescent protein (GFP)-negative skin fibroblasts were prepared from Tie2-GFP mice and infected with lentiviruses allowing simultaneous overexpression of all 11 factors. Tie2-GFP⁺ cells (0.9%), representing Tie2 gene activation, were detected by flow cytometry. Serial stepwise screening revealed 5 key factors (Foxo1, Er71, Klf2, Tal1, and Lmo2) that were required for efficient reprogramming of skin fibroblasts into Tie2-GFP⁺ cells (4%). This reprogramming strategy did not involve pluripotency induction because neither Oct4 nor Nanog was expressed after 5 key factor transduction. Tie2-GFP⁺ cells were isolated using fluorescence-activated cell sorting and designated as induced ECs (iECs). iECs exhibited endothelium-like cobblestone morphology and expressed EC molecular markers. iECs possessed endothelial functions such as Bandeiraea simplicifolia-1 lectin binding, acetylated low-density lipoprotein uptake, capillary formation on Matrigel, and nitric oxide production. The epigenetic profile of iECs was similar to that of authentic ECs because the promoters of VE-cadherin and Tie2 genes were demethylated. mRNA profiling showed clustering of iECs with authentic ECs and highly enriched endothelial genes in iECs. In a murine model of hind-limb ischemia, iEC implantation increased capillary density and enhanced limb perfusion, demonstrating the in vivo viability and functionality of iECs. Conclusions - We demonstrated the first direct conversion of adult fibroblasts to functional ECs. These results suggest a novel therapeutic modality for cell therapy in ischemic vascular disease.