Advertisement

Production of endothelial progenitor cells from skin fibroblasts by direct reprogramming for clinical usages

  • Phuc Van Pham
  • Ngoc Bich Vu
  • Thuy Thi-Thanh Dao
  • Ha Thi-Ngan Le
  • Lan Thi Phi
  • Ngoc Kim Phan
Article

Abstract

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14 d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12 ± 0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

Keywords

Endothelial progenitor cells Direct conversion ETV2 Hindlimb ischemia Ischemia Stem cell therapy Epigenetic reprogramming 

Notes

Acknowledgment

This research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106-YS.06-2013.37.

Competing interests

The authors declare they have no competing interests.

References

  1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–967CrossRefPubMedGoogle Scholar
  2. Astori G, Amati E, Bambi F, Bernardi M, Chieregato K, Schafer R, Sella S, Rodeghiero F (2016) Platelet lysate as a substitute for animal serum for the ex-vivo expansion of mesenchymal stem/stromal cells: present and future. Stem Cell Res Ther 7:93CrossRefPubMedPubMedCentralGoogle Scholar
  3. Batta K, Florkowska M, Kouskoff V, Lacaud G (2014) Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Rep 9:1871–1884CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bernardi M, Albiero E, Alghisi A, Chieregato K, Lievore C, Madeo D, Rodeghiero F, Astori G (2013) Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells. Cytotherapy 15:920–929CrossRefPubMedGoogle Scholar
  5. Bogoslovsky T, Maric D, Gong Y, Qu B, Yang K, Spatz M, Hallenbeck J, Diaz-Arrastia R (2015) Preservation and enumeration of endothelial progenitor and endothelial cells from peripheral blood for clinical trials. Biomark Med 9:625–637CrossRefPubMedGoogle Scholar
  6. Bogoslovsky T, Wang D, Maric D, Scattergood-Keepper L, Spatz M, Auh S, Hallenbeck J (2013) Cryopreservation and enumeration of human endothelial progenitor and endothelial cells for clinical trials. J Blood Disord Transfus 4Google Scholar
  7. Borch TH, Engell-Noerregaard L, Zeeberg Iversen T, Ellebaek E, Met Ö, Hansen M, Andersen MH, Thor Straten P, Svane IM (2016) mRNA transfected dendritic cell vaccine in combination with metronomic cyclophosphamide as treatment for patients with advanced malignant melanoma. Onco Immunol :00–00Google Scholar
  8. Chen R, Yu H, An YL, Chen HJ, Jia Z, Teng GJ (2016) Endothelial progenitor cells combined with cytosine deaminase-endostatin for suppression of liver carcinoma. J Biomed Nanotechnol 12:1174–1182CrossRefPubMedGoogle Scholar
  9. Coosemans A, Tuyaerts S, Morias K, Corthals J, Heirman C, Thielemans K, Van Gool SW, Vergote I, Amant F (2016) mRNA electroporation of dendritic cells with WT1, survivin, and TriMix (a mixture of caTLR4, CD40L, and CD70). Synthetic mRNA: production, introduction into cells, and physiological consequences, pp 277–283Google Scholar
  10. D'Avola D, Fernandez-Ruiz V, Carmona-Torre F, Mendez M, Perez-Calvo J, Prosper F, Andreu E, Herrero JI, Inarrairaegui M, Fuertes C, et al. (2016) Phase 1–2 pilot clinical trial in patients with decompensated liver cirrhosis treated with bone marrow-derived endothelial progenitor cells. Transl ResGoogle Scholar
  11. Donndorf P, Lube L, Lux C, Skorska A, Steinhoff G, Kraft K (2015) Mobilization of bone marrow-derived endothelial progenitor cells following Finnish sauna: a pilot study. Forsch Komplementmed 22:246–250CrossRefPubMedGoogle Scholar
  12. Eggermann J, Kliche S, Jarmy G, Hoffmann K, Mayr-Beyrle U, Debatin KM, Waltenberger J, Beltinger C (2003) Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood. Cardiovasc Res 58:478–486CrossRefPubMedGoogle Scholar
  13. Flex A, Biscetti F, Iachininoto MG, Nuzzolo ER, Orlando N, Capodimonti S, Angelini F, Valentini CG, Bianchi M, Larocca LM (2016) Human cord blood endothelial progenitors promote post-ischemic angiogenesis in immunocompetent mouse model. Thromb Res 141:106–111CrossRefPubMedGoogle Scholar
  14. Franca CN, Amaral JB, Tuleta ID, Siviero F, Ferreira CE, Izar MC, Fonseca FA (2016) Challenges facing the use of endothelial progenitor cells in stem cell therapies. Crit Rev Eukaryot Gene Expr 26Google Scholar
  15. Fu Y, Huang C, Xu X, Gu H, Ye Y, Jiang C, Qiu Z, Xie X (2015) Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails. Cell Res 25:1013–1024CrossRefPubMedPubMedCentralGoogle Scholar
  16. Goto T, Fukuyama N, Aki A, Kanabuchi K, Kimura K, Taira H, Tanaka E, Wakana N, Mori H, Inoue H (2006) Search for appropriate experimental methods to create stable hind-limb ischemia in mouse. The Tokai journal of experimental and clinical medicine 31:128–132PubMedGoogle Scholar
  17. Han C, Sun X, Liu L, Jiang H, Shen Y, Xu X, Li J, Zhang G, Huang J, Lin Z et al (2016a) Exosomes and their therapeutic potentials of stem cells. Stem Cells Int 2016:7653489PubMedGoogle Scholar
  18. Han JK, Chang SH, Cho HJ, Choi SB, Ahn HS, Lee J, Jeong H, Youn SW, Lee HJ, Kwon YW et al (2014) Direct conversion of adult skin fibroblasts to endothelial cells by defined factors. Circulation 130:1168–1178CrossRefPubMedGoogle Scholar
  19. Han YC, Lim Y, Duffieldl MD, Li H, Liu J, Abdul Manaph NP, Yang M, Keating DJ, Zhou XF (2016b) Direct reprogramming of mouse fibroblasts to neural stem cells by small molecules. Stem Cells Int 2016:4304916PubMedGoogle Scholar
  20. Iacovides D, Rizki G, Lapathitis G, Strati K (2016) Direct conversion of mouse embryonic fibroblasts into functional keratinocytes through transient expression of pluripotency-related genes. Stem Cell Res Ther 7:98CrossRefPubMedPubMedCentralGoogle Scholar
  21. Jamiolkowski RM, Kang SD, Rodriguez AK, Haseltine JM, Galinat LJ, Jantzen AE, Carlon TA, Darrabie MD, Arciniegas AJ, Mantilla JG et al (2015) Increased yield of endothelial cells from peripheral blood for cell therapies and tissue engineering. Regen Med 10:447–460CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jin Y, Seo J, Lee JS, Shin S, Park HJ, Min S, Cheong E, Lee T, Cho SW (2016) Triboelectric nanogenerator accelerates highly efficient nonviral direct conversion and in vivo reprogramming of fibroblasts to functional neuronal cells. Adv MaterGoogle Scholar
  23. Kim KL, Han DK, Park K, Song SH, Kim JY, Kim JM, Ki HY, Yie SW, Roh CR, Jeon ES et al (2009) Enhanced dermal wound neovascularization by targeted delivery of endothelial progenitor cells using an RGD-g-PLLA scaffold. Biomaterials 30:3742–3748CrossRefPubMedGoogle Scholar
  24. Kim SM, Kim JW, Kwak TH, Park SW, Kim KP, Park H, Lim KT, Kang K, Kim J, Yang JH et al (2016) Generation of integration-free induced neural stem cells from mouse fibroblasts. J Biol Chem 291:14199–14212CrossRefPubMedGoogle Scholar
  25. Kurian L, Sancho-Martinez I, Nivet E, Aguirre A, Moon K, Pendaries C, Volle-Challier C, Bono F, Herbert JM, Pulecio J et al (2013) Conversion of human fibroblasts to angioblast-like progenitor cells. Nat Methods 10:77–83CrossRefPubMedGoogle Scholar
  26. Le PM, Tran TT-B, Vu BT, Van Pham P (2016) A preliminary comparison of dendritic cell maturation by total cellular RNA to total cellular lysate derived from breast cancer stem cells. Biomedical Research and Therapy 3:679–686Google Scholar
  27. Li J, Huang NF, Zou J, Laurent TJ, Lee JC, Okogbaa J, Cooke JP, Ding S (2013) Conversion of human fibroblasts to functional endothelial cells by defined factors. Arterioscler Thromb Vasc Biol 33:1366–1375CrossRefPubMedPubMedCentralGoogle Scholar
  28. Margariti A, Winkler B, Karamariti E, Zampetaki A, Tsai TN, Baban D, Ragoussis J, Huang Y, Han JD, Zeng L et al (2012) Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels. Proc Natl Acad Sci U S A 109:13793–13798CrossRefPubMedPubMedCentralGoogle Scholar
  29. Morita R, Suzuki M, Kasahara H, Shimizu N, Shichita T, Sekiya T, Kimura A, Sasaki K, Yasukawa H, Yoshimura A (2015) ETS transcription factor ETV2 directly converts human fibroblasts into functional endothelial cells. Proc Natl Acad Sci U S A 112:160–165CrossRefPubMedGoogle Scholar
  30. Papadimitrious M, Fromm P, Losio NVK, Bryant C, Clark G, Anguille S, Berneman Z, Horvath L, Bradstock K, Hart D (2016) mRNA loaded CMRF-56 blood dendritic cells augment anti-cancer immune responses in conjunction with nivolumab. Cytotherapy 18:S100CrossRefGoogle Scholar
  31. Park SJ, Moon SH, Lee HJ, Lim JJ, Kim JM, Seo J, Yoo JW, Kim OJ, Kang SW, Chung HM (2013) A comparison of human cord blood- and embryonic stem cell-derived endothelial progenitor cells in the treatment of chronic wounds. Biomaterials 34:995–1003CrossRefPubMedGoogle Scholar
  32. Pham PV, Vu NB, Pham VM, Truong NH, Pham TL, Dang LT, Nguyen TT, Bui AN, Phan NK (2014a) Good manufacturing practice-compliant isolation and culture of human umbilical cord blood-derived mesenchymal stem cells. J Transl Med 12:56CrossRefPubMedPubMedCentralGoogle Scholar
  33. Pham PV, Vu NB, Phan NLC, Le DM, Truong NC, Truong NH, Bui KHT, Phan NK (2014b) Good manufacturing practice-compliant isolation and culture of human adipose-derived stem cells. 2014 1:9Google Scholar
  34. Preskey D, Allison TF, Jones M, Mamchaoui K, Unger C (2016) Synthetically modified mRNA for efficient and fast human iPS cell generation and direct transdifferentiation to myoblasts. Biochem Biophys Res Commun 473:743–751CrossRefPubMedGoogle Scholar
  35. Rohani L, Fabian C, Holland H, Naaldijk Y, Dressel R, Löffler-Wirth H, Binder H, Arnold A, Stolzing A (2016) Generation of human induced pluripotent stem cells using non-synthetic mRNA. Stem Cell Res 16:662–672CrossRefPubMedGoogle Scholar
  36. Rustemeyer P, Wittkowski W, Greve B, Stehling M (2007) Flow-cytometric identification, enumeration, purification, and expansion of CD133+ and VEGF-R2+ endothelial progenitor cells from peripheral blood. J Immunoassay Immunochem 28:13–23CrossRefPubMedGoogle Scholar
  37. Sabry D, Noh O, Samir M (2016) Comparative evaluation for potential differentiation of endothelial progenitor cells and mesenchymal stem cells into endothelial-like cells. Int J Stem Cells 9:44–52CrossRefPubMedPubMedCentralGoogle Scholar
  38. Suh W, Kim KL, Kim JM, Shin IS, Lee YS, Lee JY, Jang HS, Lee JS, Byun J, Choi JH et al (2005) Transplantation of endothelial progenitor cells accelerates dermal wound healing with increased recruitment of monocytes/macrophages and neovascularization. Stem Cells 23:1571–1578CrossRefPubMedGoogle Scholar
  39. Talkhabi M, Pahlavan S, Aghdami N, Baharvand H (2015) Ascorbic acid promotes the direct conversion of mouse fibroblasts into beating cardiomyocytes. Biochem Biophys Res Commun 463:699–705CrossRefPubMedGoogle Scholar
  40. Telpalo-Carpio SA, Díaz-Mitoma F, Moreno-Cuevas JE, Aguilar-Yanez JM (2016) Direct comparison of DNA versus mRNA on the efficiency of induced pluripotent stem cells generation from human primary fibroblasts. Cell Mol Med Open AccessGoogle Scholar
  41. Treutlein B, Lee QY, Camp JG, Mall M, Koh W, Shariati SA, Sim S, Neff NF, Skotheim JM, Wernig M et al (2016) Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534:391–395CrossRefPubMedPubMedCentralGoogle Scholar
  42. Tu S, Shao A, Ren L, Chen T, Yao D (2014) Angiogenesis effect of Astragalus polysaccharide combined with endothelial progenitor cells therapy in diabetic male rat following experimental hind limb ischemia. Chin Med J 127:2121–2128PubMedGoogle Scholar
  43. Van Pham P, Truong NC, Le PT, Tran TD, Vu NB, Bui KH, Phan NK (2016a) Isolation and proliferation of umbilical cord tissue derived mesenchymal stem cells for clinical applications. Cell Tissue Bank 17:289–302CrossRefPubMedGoogle Scholar
  44. Van Pham P, Vu NB, Nguyen HT, Huynh OT, Truong MT (2016b) Significant improvement of direct reprogramming efficacy of fibroblasts into progenitor endothelial cells by ETV2 and hypoxia. Stem Cell Res Ther 7:104CrossRefPubMedPubMedCentralGoogle Scholar
  45. Van Pham P, Vu NB, Nguyen HT, Phan NK (2016c) Isolation of endothelial progenitor cells from human adipose tissue. Biomedical Research and Therapy 3:645–652Google Scholar
  46. Vu NB, Bui ANT, Ngoc-Le Trinh V, Phi LT, Phan NK, Van Pham P (2014) A comparison of umbilical cord blood-derived endothelial progenitor and mononuclear cell transplantation for the treatment of acute hindlimb ischemiaGoogle Scholar
  47. Wang Y, Fan L, Meng X, Jiang F, Chen Q, Zhang Z, Yan H (2016) Transplantation of IL-10-transfected endothelial progenitor cells improves retinal vascular repair via suppressing inflammation in diabetic rats. Graefes Arch Clin Exp OphthalmolGoogle Scholar
  48. Wong WT, Cooke JP (2016) Therapeutic transdifferentiation of human fibroblasts into endothelial cells using forced expression of lineage-specific transcription factors. J Tissue Eng 7:2041731416628329CrossRefPubMedPubMedCentralGoogle Scholar
  49. Yamamoto K, Kishida T, Sato Y, Nishioka K, Ejima A, Fujiwara H, Kubo T, Yamamoto T, Kanamura N, Mazda O (2015) Direct conversion of human fibroblasts into functional osteoblasts by defined factors. Proc Natl Acad Sci U S A 112:6152–6157CrossRefPubMedPubMedCentralGoogle Scholar
  50. Yang R, Zheng Y, Li L, Liu S, Burrows M, Wei Z, Nace A, Herlyn M, Cui R, Guo W et al (2014) Direct conversion of mouse and human fibroblasts to functional melanocytes by defined factors. Nat Commun 5:5807CrossRefPubMedPubMedCentralGoogle Scholar
  51. Yu P, Li Q, Liu Y, Zhang J, Seldeen K, Pang M (2015) Pro-angiogenic efficacy of transplanting endothelial progenitor cells for treating hindlimb ischemia in hyperglycemic rabbits. J Diabetes Complicat 29:13–19CrossRefPubMedGoogle Scholar
  52. Zhang Y, Chen Z, Wang T, Yang J, Li R, Wang S, Liu J, Ye Z (2016) Treatment of diabetes mellitus-induced erectile dysfunction using endothelial progenitor cells genetically modified with human telomerase reverse transcriptase. OncotargetGoogle Scholar
  53. Zhao Z, Xu M, Wu M, Ma K, Sun M, Tian X, Zhang C, Fu X (2015) Direct reprogramming of human fibroblasts into sweat gland-like cells. Cell Cycle 14:3498–3505CrossRefPubMedPubMedCentralGoogle Scholar
  54. Zheng J, Choi KA, Kang PJ, Hyeon S, Kwon S, Moon JH, Hwang I, Kim YI, Kim YS, Yoon BS et al (2016) A combination of small molecules directly reprograms mouse fibroblasts into neural stem cells. Biochem Biophys Res Commun 476:42–48CrossRefPubMedGoogle Scholar
  55. Zhou L, Xia J, Qiu X, Wang P, Jia R, Chen Y, Yang B, Dai Y (2015) In vitro evaluation of endothelial progenitor cells from adipose tissue as potential angiogenic cell sources for bladder angiogenesis. PLoS One 10:e0117644CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2016

Authors and Affiliations

  • Phuc Van Pham
    • 1
  • Ngoc Bich Vu
    • 1
  • Thuy Thi-Thanh Dao
    • 1
  • Ha Thi-Ngan Le
    • 1
  • Lan Thi Phi
    • 1
  • Ngoc Kim Phan
    • 1
  1. 1.Laboratory of Stem Cell Research and Application, University of ScienceVietnam National UniversityHo Chi Minh CityVietnam

Personalised recommendations