Direct Conversion of Adult Skin Fibroblasts to Endothelial Cells by Defined Factors

Abstract

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

Direct Cardiac Reprogramming

“…Recent studies demonstrate that direct reprogramming has the potential to yield a diverse range of cell types from fibroblasts, including neurons, cardiomyocytes, endothelial cells, hematopoietic stem/progenitor cells, and hepatocytes ( Figure 1). [16][17][18][19][20][21][22] These findings indicate that cell fate plasticity is much wider than previously anticipated, and that direct reprogramming may offer a new system to study the mechanisms underlying cell fate decisions during development, which is currently a major focus of investigation in basic biology.…”

Preexisting endothelial cells mediate cardiac neovascularization after injury

“…Fibroblasts are regarded as terminally differentiated cells, and excessive new fibroblasts are accumulated after tissue injury, such as myocardial infarction (5,6). With the advent of reprogramming technology that introduces exogenous transcription factors, these differentiated fibroblasts could be reprogrammed into functional endothelial cells in vitro, which are capable of mediating neovascularization in tissue-engineered vessels and ischemic tissues (7,8). Whether endogenous fibroblasts or mesenchymal stromal cells (MSCs) contribute to endothelial cells after tissue injury remains enigmatic.…”

Bioinformatic and Genomic Analyses of Cellular Reprogramming and Direct Lineage Conversion

“…With these datasets, investigators are able to generate candidate genes which may be capable of direct conversion as well as use defined lineage specific networks as a benchmark for proper lineage reprogramming. Many of these rely on a study of the gene expression networks in the target cells to confirm proper lineage conversion including hematopoietic stem and progenitor cells (iHSC, iHPC, [4,73,85]), embryonic sertoli-like cells (ieSCs, [13]), endothelial cells (iECs, [33]), and melanocytes (iMels, [101]). …”