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, which are key regulators of endothelial development, were selected. GFP- skin fibroblasts (SFBs) were prepared from Tie2-GFP mice, and infected with lentiviruses allowing for 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 (5F: Foxo1, Er71, Klf2, Tal1, and Lmo2) which were required for efficient reprogramming of SFBs into Tie2-GFP+ cells (4%). This reprogramming strategy did not involve pluripotency induction, as neither Oct4 nor Nanog was expressed after 5F transduction. Tie2-GFP+ cells were isolated using fluorescence-activated cell sorting, and designated as induced endothelial cells (iECs). iECs exhibited endothelial-like cobblestone morphology and expressed EC molecular markers. iECs possessed endothelial functions such as BS1 lectin binding, acLDL uptake, capillary formation on Matrigel and nitric oxide production. The epigenetic profile of iECs was similar to authentic ECs as 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 hindlimb 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.
- Received November 25, 2013.
- Revision received July 12, 2014.
- Accepted July 24, 2014.