한빛사 논문
Abstract
Jung-Kyu Han, MD1, 2*; Sung-Hwan Chang, BS1*; Hyun-Ju Cho, PhD1*; Saet-Byeol Choi, BS1*; Hyo-Suk Ahn, MS1; Jaewon Lee, BS1; Heewon Jeong, BS1; Seock-Won Youn, PhD1; Ho-Jae Lee, BS1; Yoo-Wook Kwon, PhD1; Hyun-Jai Cho, MD1,2; Byung-Hee Oh, MD1,2; Peter Oettgen, MD3; Young-Bae Park, MD1,2; Hyo-Soo Kim, MD1,2,4
1National Research Laboratory for Cardiovascular Stem Cell, Seoul National University College of Medicine, Seoul, Republic of Korea; 2Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea; 3Center for Vascular Biology Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; 4Molecular Medicine & Biopharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea
*contributed equally.
Address for Correspondence:Hyo-Soo Kim, MD
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, 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.
Key Words: induced endothelial cells, direct conversion, transdifferentiation
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