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

Jung Kyu Han, Sung Hwan Chang, Hyun Ju Cho, Saet Byeol Choi, Hyo Suk Ahn, Jaewon Lee, Heewon Jeong, Seock Won Youn, Ho Jae Lee, Yoo Wook Kwon, Hyun Jai Cho, Byung Hee Oh, Peter Oettgen, Young Bae Park, Hyo Soo Kim

Research output: Contribution to journalArticle

50 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1168-1178
Number of pages11
JournalCirculation
Volume130
Issue number14
DOIs
StatePublished - Jan 1 2014

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Endothelial Cells
Fibroblasts
Green Fluorescent Proteins
Skin
Cell- and Tissue-Based Therapy
Flow Cytometry
Extremities
Lentivirus
Regulator Genes
Vascular Diseases
Epigenomics
Transcriptional Activation
Genes
Endothelium
Cluster Analysis
Nitric Oxide
Ischemia
Perfusion
Messenger RNA
Therapeutics

Keywords

  • Cell transdifferentiation
  • Endothelial cells
  • Fibroblasts

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

Han, J. K., Chang, S. H., Cho, H. J., Choi, S. B., Ahn, H. S., Lee, J., ... Kim, H. S. (2014). Direct conversion of adult skin fibroblasts to endothelial cells by defined factors. Circulation, 130(14), 1168-1178. https://doi.org/10.1161/CIRCULATIONAHA.113.007727

Direct conversion of adult skin fibroblasts to endothelial cells by defined factors. / Han, Jung Kyu; Chang, Sung Hwan; Cho, Hyun Ju; Choi, Saet Byeol; Ahn, Hyo Suk; Lee, Jaewon; Jeong, Heewon; Youn, Seock Won; Lee, Ho Jae; Kwon, Yoo Wook; Cho, Hyun Jai; Oh, Byung Hee; Oettgen, Peter; Park, Young Bae; Kim, Hyo Soo.

In: Circulation, Vol. 130, No. 14, 01.01.2014, p. 1168-1178.

Research output: Contribution to journalArticle

Han, JK, Chang, SH, Cho, HJ, Choi, SB, Ahn, HS, Lee, J, Jeong, H, Youn, SW, Lee, HJ, Kwon, YW, Cho, HJ, Oh, BH, Oettgen, P, Park, YB & Kim, HS 2014, 'Direct conversion of adult skin fibroblasts to endothelial cells by defined factors', Circulation, vol. 130, no. 14, pp. 1168-1178. https://doi.org/10.1161/CIRCULATIONAHA.113.007727
Han, Jung Kyu ; Chang, Sung Hwan ; Cho, Hyun Ju ; Choi, Saet Byeol ; Ahn, Hyo Suk ; Lee, Jaewon ; Jeong, Heewon ; Youn, Seock Won ; Lee, Ho Jae ; Kwon, Yoo Wook ; Cho, Hyun Jai ; Oh, Byung Hee ; Oettgen, Peter ; Park, Young Bae ; Kim, Hyo Soo. / Direct conversion of adult skin fibroblasts to endothelial cells by defined factors. In: Circulation. 2014 ; Vol. 130, No. 14. pp. 1168-1178.
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AU - Chang, Sung Hwan

AU - Cho, Hyun Ju

AU - Choi, Saet Byeol

AU - Ahn, Hyo Suk

AU - Lee, Jaewon

AU - Jeong, Heewon

AU - Youn, Seock Won

AU - Lee, Ho Jae

AU - Kwon, Yoo Wook

AU - Cho, Hyun Jai

AU - Oh, Byung Hee

AU - Oettgen, Peter

AU - Park, Young Bae

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N2 - 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.

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