Regeneration of full-thickness skin defects by differentiated adipose-derived stem cells into fibroblast-like cells by fibroblast-conditioned medium
Woojune Hur, Hoon Young Lee, Hye Sook Min, Maierdanjiang Wufuer, Chang-Won Lee, Ji An Hur, Sang Hyon Kim, Byeung Kyu Kim, Tae Hyun Choi, Woojune Hur, Hoon Young Lee, Hye Sook Min, Maierdanjiang Wufuer, Chang-Won Lee, Ji An Hur, Sang Hyon Kim, Byeung Kyu Kim, Tae Hyun Choi
Abstract
Background: Fibroblasts are ubiquitous cells in the human body and are absolutely necessary for wound healing such as for injured skin. This role of fibroblasts was the reason why we aimed to differentiate human adipose-derived stem cells (hADSCs) into fibroblasts and to test their wound healing potency. Recent reports on hADSC-derived conditioned medium have indicated stimulation of collagen synthesis as well as migration of dermal fibroblasts in wound sites with these cells. Similarly, human fibroblast-derived conditioned medium (F-CM) was reported to contain a variety of factors known to be important for growth of skin. However, it remains unknown whether and how F-CM can stimulate hADSCs to secrete type I collagen.
Methods: In this study, we obtained F-CM from the culture of human skin fibroblast HS27 cells in DMEM media. For an in-vivo wound healing assay using cell transplantation, balb/c nude mice with full-thickness skin wound were used.
Results: Our data showed that levels of type I pro-collagen secreted by hADSCs cultured in F-CM increased significantly compared with hADSCs kept in normal medium for 72 h. In addition, from a Sircol collagen assay, the amount of collagen in F-CM-treated hADSC conditioned media (72 h) was markedly higher than both the normal medium-treated hADSC conditioned media (72 h) and the F-CM (24 h). We aimed to confirm that hADSCs in F-CM would differentiate into fibroblast cells in order to stimulate wound healing in a skin defect model. To investigate whether F-CM induced hADSCs into fibroblast-like cells, we performed FACS analysis and verified that both F-CM-treated hADSCs and HS27 cells contained similar expression patterns for CD13, CD54, and CD105, whereas normal medium-treated hADSCs were significantly different. mRNA level analysis for Nanog, Oct4A, and Sox2 as undifferentiation markers and vimentin, HSP47, and desmin as matured fibroblast markers supported the characterization that hADSCs in F-CM were highly differentiated into fibroblast-like cells. To discover the mechanism of type I pro-collagen expression in hADSCs in F-CM, we observed that phospho-smad 2/3 levels were increased in the TGF-β/Smad signaling pathway. For in-vivo analysis, we injected various cell types into balb/c nude mouse skin carrying a 10-mm punch wound, and observed a significantly positive wound healing effect in this full-thickness excision model with F-CM-treated hADSCs rather than with untreated hADSCs or the PBS injected group.
Conclusions: We differentiated F-CM-treated hADSCs into fibroblast-like cells and demonstrated their efficiency in wound healing in a skin wound model.
Keywords: Cell transplantation; Human adipose-derived stem cell-derived conditioned medium; Type I collagen; Wound healing.
Figures
References
- Machens HG, Berger AC, Mailaender P. Bioartificial skin. Cells Tissues Organs. 2000;167(2–3):88–94. doi: 10.1159/000016772.
- Moiemen NS, Yarrow J, Kamel D, Kearns D, Mendonca D. Topical negative pressure therapy: does it accelerate neovascularisation within the dermal regeneration template, integra? A prospective histological in vivo study. Burns. 2010;36(6):764–8. doi: 10.1016/j.burns.2010.04.011.
- Nolte SV, Xu W, Rennekampff HO, Rodemann HP. Diversity of fibroblasts—a review on implications for skin tissue engineering. Cells Tissues Organs. 2008;187(3):165–76. doi: 10.1159/000111805.
- van der Veen VC, van der Wal MBA, van Leeuwen MCE, van Leeuwen MC, Ulrich MM, Middelkoop E. Biological background of dermal substitutes. Burn. 2010;36(3):305–21. doi: 10.1016/j.burns.2009.07.012.
- Bahar MA, Nabai L, Ghahary A. Immunoprotective role of indoleamine 2,3-dioxygenase in engraftment of allogenic skin substitute in wound healing. J Burn Care Res. 2012;33(3):364–70. doi: 10.1097/BCR.0b013e318235836e.
- Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. 1999;341(10):738–46. doi: 10.1056/NEJM199909023411006.
- Park BS, Jang KA, Sung JH, Park JS, Kwon YH, Kim KJ, Kim WS. Adipose-derived stem cells and their secretory factors as a promising therapy for skin aging. Dermatol Surg. 2008;34(10):1323–6.
- Schachinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Hölschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Süselbeck T, Assmus B, Tonn T, Dimmeler S, Zeiher AM. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med. 2006;355(12):1210–21. doi: 10.1056/NEJMoa060186.
- Schachinger V, Erbs S, Elsässer A, Haberbosch W, Hambrecht R, Hölschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Süselbeck T, Werner N, Haase J, Neuzner J, Germing A, Mark B, Assmus B, Tonn T, Dimmeler S, Zeiher AM. Improved clinical outcome after intracoronary administration of bone-marrow-derived progenitor cells in acute myocardial infarction: final 1-year results of the REPAIR-AMI trial. Eur Heart J. 2006;27(23):2775–83. doi: 10.1093/eurheartj/ehl388.
- Shahrokhi S, Arno A, Jeschke MG. The use of dermal substitutes in burn surgery: acute phase. Wound Repair Regen. 2014;22(1):14–22. doi: 10.1111/wrr.12119.
- Nassar D, Letavernier E, Baud L, Aractingi S, Khosrotehrani K. Calpain activity is essential in skin wound healing and contributes to scar formation. PLoS One. 2012;7(5) doi: 10.1371/journal.pone.0037084.
- Kopecki Z, Luchetti MM, Adams DH, Strudwick X, Mantamadiotis T, Stoppacciaro A, Gabrielli A, Ramsay RG, Cowin AJ. Collagen loss and impaired wound healing is associated with c-Myb deficiency. J Pathol. 2007;211(3):351–61. doi: 10.1002/path.2113.
- Urich K, King PJ. Extracellular structural and secretory proteins. Comp Animal Biochem. 1994;9(6):378–83.
- Von der Mark K, Seibel MJ, Robins SP. Structure, biosynthesis and gene regulation of collagens in cartilage and bone. Dynamics of bone and cartilage metabolism. Princip Clin Appli. eBook ISBN: 9780080456263 2nd Edition. 2006;3-40(chapter 1).
- Baraniak PR, McDevitt TC. Stem cell paracrine actions and tissue regeneration. Regen Med. 2010;5(1):121–43. doi: 10.2217/rme.09.74.
- Casteilla L, Planat-Benard V, Laharrague P, Cousin B. Adipose-derived stromal cells: their identity and uses in clinical trials, an update. World J Stem Cells. 2011;3(4):25–33. doi: 10.4252/wjsc.v3.i4.25.
- Kim WS, Park BS, Park SH, Kim HK, Sung JH. Antiwrinkle effect of adipose-derived stem cell: activation of dermal fibroblast by secretory factors. J Dermatol Sci. 2009;53(2):96–102. doi: 10.1016/j.jdermsci.2008.08.007.
- Kim WS, Park SH, Ahn SJ, Kim HK, Park JS, Lee GY, Kim KJ, Whang KK, Kang SH, Park BS, Sung JH. Whitening effect of adipose-derived stem cells: a critical role of TGF-β1. Biol Pharm Bull. 2008;31(4):606–10. doi: 10.1248/bpb.31.606.
- Kim WS, Park BS, Sung JH, Yang JM, Park SB, Kwak SJ, Park JS. Wound healing effect of adipose derived stem cells: a critical role of secretory factors on human dermal fibroblasts. J Dermatol Sci. 2007;48(1):15–24. doi: 10.1016/j.jdermsci.2007.05.018.
- Kopanska KS, Powell JJ, Jugdaohsingh R, Bruggraber SF. Filtration of dermal fibroblast-conditioned culture media is required for the reliable quantitation of cleaved carboxy-terminal peptide of collagen type I (CICP) by ELISA. Arch Dermatol Res. 2013;305(8):741–5. doi: 10.1007/s00403-013-1370-5.
- Chowdhury SR, Aminuddin BS, Ruszymah BHI. Effect of supplementation of dermal fibroblast conditioned medium on expansion of keratinocytes through enhancing attachment. Indian J Exp Biol. 2012;50(5):332–9.
- Millis AJ, Hoyle M, Field B. Human fibroblast conditioned media contains growth promoting activities for low density cells. J Cell Physiol. 1977;93(1):17–24. doi: 10.1002/jcp.1040930104.
- Lim JS, Yoo G. Effect of adipose-derived stromal cells and of their extract on wound healing in a mouse model. J Korean Med Sci. 2010;25(5):746–51. doi: 10.3346/jkms.2010.25.5.746.
- Schaffler A, Buchler C. Concise review: adipose tissue-derived stromal cells—basic and clinical implications for novel cell-based therapies. Stem Cells. 2007;25(4):818–27. doi: 10.1634/stemcells.2006-0589.
- Wagner W, Wein F, Seckinger A, Frankhauser M, Wirkner U, Krause U, Blake J, Schwager C, Eckstei B, Ansorge W, Ho AD. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp Hematol. 2005;33(11):1402–16. doi: 10.1016/j.exphem.2005.07.003.
- Lee RH, Kim B, Choi I, Kim H, Choi HS, Suh K, Bae YC, Jung JS. Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue. Cell Physiol Biochem. 2004;14(4-6):311–24. doi: 10.1159/000080341.
- Spiekstra SW, Breetveld M, Rustemeyer T, Scheper RJ, Gibbs S. Wound-healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes. Wound Repair Regen. 2007;15(5):708–17. doi: 10.1111/j.1524-475X.2007.00280.x.
- Hunt TK, Burke J, Barbul A, Gimbel ML. Wound healing. Science. 1999;284(5421):1775. doi: 10.1126/science.284.5421.1773d.
- Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells. 2007;25(10):2648–59. doi: 10.1634/stemcells.2007-0226.
- Badiavas EV, Abedi M, Butmarc J, Falanga V, Quesenberry P. Participation of bone marrow derived cells in cutaneous wound healing. J Cell Physiol. 2003;196(2):245–50. doi: 10.1002/jcp.10260.
- Nakagawa H, Akita S, Fukui M, Fujii T, Akino K. Human mesenchymal stem cells successfully improve skin-substitute wound healing. Br J Dermatol. 2005;153(1):29–36. doi: 10.1111/j.1365-2133.2005.06554.x.
- Liu P, Deng Z, Han S, Liu T, Wen N, Lu W, Geng X, Huang S, Jin Y. Tissue-engineered skin containing mesenchymal stem cells improves burn wounds. Artif Organs. 2008;32(12):925–31. doi: 10.1111/j.1525-1594.2008.00654.x.
- Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen. 2008;16(5):585–601. doi: 10.1111/j.1524-475X.2008.00410.x.
- Sievert KD, Tanagho EA. Organ-specific a cellular matrix for reconstruction of the urinary tract. World J Urol. 2000;18(1):19–25. doi: 10.1007/s003450050004.
- Xu L. Regulation of Smad activities. Biochem Biophys Acta. 2006;1759(11–12):503–13.
- Verrecchia F, Mauviel A. Transforming growth factor-beta and fibrosis. World J Gastroenterol. 2007;13(22):3056–62.
- Moustakas A, Souchelnytskyi S, Heldin CH. Smad regulation in TGF beta signal transduction. J Cell Sci. 2001;114(24):4359–69.
- Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997;390(6659):465–71. doi: 10.1038/37284.
- Schiller M, Javelaud D, Mauviel A. TGF-beta-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing. J Dermatol Sci. 2004;35(2):83–92. doi: 10.1016/j.jdermsci.2003.12.006.
- Cui W, Jin HB, Li ZW. Mechanism of the transforming growth factorbeta induction of fibronectin expression in hepatic stem-like cells. Braz J Med Biol Res. 2010;43(1):36–42. doi: 10.1590/S0100-879X2009007500017.
Source: PubMed