Jung-Kyu Han1, Sung-Hwan Chang1, Hyun-Ju Cho1, Saet-Byeol Choi1, Hyo-Suk Ahn1, Jaewon Lee1, Heewon Jeong1, Seock-Won Youn1, Ho-Jae Lee1, Yoo-Wook Kwon1, Hyun-Jai Cho1, Byung-Hee Oh1, Peter Oettgen1, Young-Bae Park1, Hyo-Soo Kim2. 1. From National Research Laboratory for Cardiovascular Stem Cell, Seoul National University College of Medicine, Seoul, Republic of Korea (J-K.H., S-H.C, Hyun-Ju C., S-B.C., H-S.A., J.L., H.J., S-W.Y., H-J.L., Y-W.K., Hyun-Jai C., B-H.O., Y-B.P., H-S.K.); Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea (J-K.H., Hyun-Jai C., B-H.O., Y-B.P., H-S.K.); Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (P.O.); and Molecular Medicine & Biopharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea (H-S.K.). 2. From National Research Laboratory for Cardiovascular Stem Cell, Seoul National University College of Medicine, Seoul, Republic of Korea (J-K.H., S-H.C, Hyun-Ju C., S-B.C., H-S.A., J.L., H.J., S-W.Y., H-J.L., Y-W.K., Hyun-Jai C., B-H.O., Y-B.P., H-S.K.); Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea (J-K.H., Hyun-Jai C., B-H.O., Y-B.P., H-S.K.); Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA (P.O.); and Molecular Medicine & Biopharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea (H-S.K.). hyosoo@snu.ac.kr.
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 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.
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.