Investigation on HaCaT cell condition medium combined with all-trans-retinoic acid initiating differentiation of human adipose-derived stem cells into epidermal cells_West China Medical Journal

Authors：

LIN Wentao ^1,2 ,  LIU Xiaoxue ¹ , LIU Yong ¹ , CHEN Junjie ¹ ,  CEN Ying ¹

1. Department of Burn and Plastic Surgery, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Department of Plastic Surgery, the First Affiliated Hospital, Chongqing Medical University, Chongqing 400042, P. R. China;

Corresponding author：

LIU Xiaoxue, Email: liuxiaoxue0930@126.com; CEN Ying, Email: cenying0141@163.com

Keywords：

Human adipose-derived stem cells; Epidermal cells; All-trans-retinoic acid; HaCaT cell

DOI：

10.7507/1002-0179.201611154

Video：

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Objective To investigate the possibility of enhancing the inducing rate of adipose-derived stem cells (ASCs) into epidermal cells in the medium containing all-trans retinoic acid (ATRA) by supplementing with HaCaT condition medium. Methods ASCs were isolated and identified by detecting the expression of CD34, CD45, CD73, CD90, and CD105 with flow cytometry and differentiating into adipose and osteoblast lineage in the induction medium. The air-liquid interface cell culture model was established with the Transwell Room. The induction medium A contained ATRA, epidermal growth factor (EGF), and keratinocyte growth factor (KGF), while the induction medium B contained ATRA, EGF, KGF, and HaCaT condition medium. Experiment was divided into three groups cultured for 12 days: induction medium A (group A), induction medium B (group B), basic medium (group C). The epidermal cell surface markers: cytokeratin (CK) 14, 15, 16, 19 (Pan-CK) were detected by flow cytometry and CK14 were identified by immunofluorescence stain. Results After induction for 12 days, flow cytometry showed that the positive rate of Pan-CK in group B [(22.0±3.5)%] was higher than that in group A [(11.9±2.7)%], which were both higher than that in group C [(1.1±0.3)%], and the differences were statistical significantly (P<0.01). Immunofluorescence stain showed that the positive rate of CK14 in group B was higher than that in group A [(19.5±7.0)%vs. (10.8±5.7)%, P<0.01], and the expression of CK14 was negative in group C. Conclusion HaCaT condition medium can enhance the ability of ASCs differentiation into epidermal cells in the culture medium containing ATRA.

Citation： LIN Wentao, LIU Xiaoxue, LIU Yong, CHEN Junjie, CEN Ying. Investigation on HaCaT cell condition medium combined with all-trans-retinoic acid initiating differentiation of human adipose-derived stem cells into epidermal cells. West China Medical Journal, 2018, 33(3): 325-331. doi: 10.7507/1002-0179.201611154 Copy

1.	Nyame TT, Chiang HA, Leavitt T, et al. Tissue-engineered skin substitutes. Plast Reconstr Surg, 2015, 136(6): 1379-1388.
2.	Skubis A, Gola J, Sikora B, et al. Impact of antibiotics on the proliferation and differentiation of human adipose-derived mesenchymal stem cells. Int J Mol Sci, 2017, 18(12): 2522.
3.	Naderi N, Combellack EJ, Griffin M, et al. The regenerative role of adipose-derived stem cells (ADSC) in plastic and reconstructive surgery. Int Wound J, 2017, 14(1): 112-124.
4.	Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med, 2005, 54(3): 132-141.
5.	Brzoska M, Geiger H, Gauer S, et al. Epithelial differentiation of human adipose tissue-derived adult stem cells. Biochem Biophys Res Commun, 2005, 330(1): 142-150.
6.	Long JL, Zuk P, Berke GS, et al. Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering. Laryngoscope, 2010, 120(1): 125-131.
7.	龙剑虹, 刘芳芬, 祁敏. 体外诱导骨髓间充质干细胞分化为表皮干细胞. 中南大学学报: 医学版, 2006, 31(6): 866-871.
8.	李薇, 李松泽. HaCaT细胞诱导兔骨髓间充质干细胞表达角蛋白的初步研究. 皮肤性病诊疗学杂志, 2014, 21(2): 104-107.
9.	赵周婷, 胡大海, 陶克, 等. 人脂肪间充质干细胞向表皮细胞表型转化的研究. 中国美容医学, 2014, 23(22): 1899-1903.
10.	Baer PC, Bereiter-Hahn J, Missler C, et al. Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells. Cell Prolif, 2009, 42(1): 29-37.
11.	Li H, Xu Y, Fu Q, et al. Effects of multiple agents on epithelial differentiation of rabbit adipose-derived stem cells in 3D culture. Tissue Eng Part A, 2012, 18(17/18): 1760-1770.
12.	da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci, 2006, 119(Pt 11): 2204-2213.
13.	Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
14.	Bajek A, Gurtowska N, Olkowska J, et al. Adipose-derived stem cells as a tool in cell-based. Arch Immunol Ther Exp (Warsz), 2016, 64(6):443-454.
15.	Mizuno, H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch, 2009, 76(2): 56-66.
16.	Sugii S, Kida Y, Kawamura T, et al. Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells. Proc Natl Acad Sci USA, 2010, 107(8): 3558-3563.
17.	Bourin P, Bunnell BA, Casteilla LA, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy, 2013, 15(6): 641-648.
18.	Baglioni S, Francalanci M, Squecco R, et al. Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J, 2009, 23(10): 3494-3505.
19.	Vishnubalaji R, Al-Nbaheen M, Kadalmani B, et al. Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis. Cell Tissue Res, 2012, 347(2): 419-427.
20.	Ross AC. Cellular metabolism and activation of retinoids: roles of cellular retinoid-binding proteins. FASEB J, 1993, 7(2): 317-327.
21.	Zhang S, Chen X, Hu Y, et al. All-trans retinoic acid modulates Wnt3A-induced osteogenic differentiation of mesenchymal stem cells via activating the PI3K/AKT/GSK3β signalling pathway. Mol Cell Endocrinol, 2016, 422: 243-253.
22.	Ma K, Laco F, Ramakrishna S, et al. Differentiation of bone marrow-derived mesenchymal stem cells into multi-layered epidermis-like cells in 3D organotypic coculture. Biomaterials, 2009, 30(19): 3251-3258.
23.	Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol, 2008, 129(6): 705-733.
24.	Kwon YW, Kwon KS, Moon HE, et al. Insulin-like growth factor-Ⅱ regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT. J Invest Dermatol, 2004, 123(1): 152-158.
25.	Pozzi G, Guidi M, Laudicina F, et al. IGF-I stimulates proliferation of spontaneously immortalized human keratinocytes (HACAT) by autocrine/paracrine mechanisms. J Endocrinol Invest, 2004, 27(2): 142-149.

1. Nyame TT, Chiang HA, Leavitt T, et al. Tissue-engineered skin substitutes. Plast Reconstr Surg, 2015, 136(6): 1379-1388.
2. Skubis A, Gola J, Sikora B, et al. Impact of antibiotics on the proliferation and differentiation of human adipose-derived mesenchymal stem cells. Int J Mol Sci, 2017, 18(12): 2522.
3. Naderi N, Combellack EJ, Griffin M, et al. The regenerative role of adipose-derived stem cells (ADSC) in plastic and reconstructive surgery. Int Wound J, 2017, 14(1): 112-124.
4. Strem BM, Hicok KC, Zhu M, et al. Multipotential differentiation of adipose tissue-derived stem cells. Keio J Med, 2005, 54(3): 132-141.
5. Brzoska M, Geiger H, Gauer S, et al. Epithelial differentiation of human adipose tissue-derived adult stem cells. Biochem Biophys Res Commun, 2005, 330(1): 142-150.
6. Long JL, Zuk P, Berke GS, et al. Epithelial differentiation of adipose-derived stem cells for laryngeal tissue engineering. Laryngoscope, 2010, 120(1): 125-131.
7. 龙剑虹, 刘芳芬, 祁敏. 体外诱导骨髓间充质干细胞分化为表皮干细胞. 中南大学学报: 医学版, 2006, 31(6): 866-871.
8. 李薇, 李松泽. HaCaT细胞诱导兔骨髓间充质干细胞表达角蛋白的初步研究. 皮肤性病诊疗学杂志, 2014, 21(2): 104-107.
9. 赵周婷, 胡大海, 陶克, 等. 人脂肪间充质干细胞向表皮细胞表型转化的研究. 中国美容医学, 2014, 23(22): 1899-1903.
10. Baer PC, Bereiter-Hahn J, Missler C, et al. Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells. Cell Prolif, 2009, 42(1): 29-37.
11. Li H, Xu Y, Fu Q, et al. Effects of multiple agents on epithelial differentiation of rabbit adipose-derived stem cells in 3D culture. Tissue Eng Part A, 2012, 18(17/18): 1760-1770.
12. da Silva Meirelles L, Chagastelles PC, Nardi NB. Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci, 2006, 119(Pt 11): 2204-2213.
13. Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4): 315-317.
14. Bajek A, Gurtowska N, Olkowska J, et al. Adipose-derived stem cells as a tool in cell-based. Arch Immunol Ther Exp (Warsz), 2016, 64(6):443-454.
15. Mizuno, H. Adipose-derived stem cells for tissue repair and regeneration: ten years of research and a literature review. J Nippon Med Sch, 2009, 76(2): 56-66.
16. Sugii S, Kida Y, Kawamura T, et al. Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells. Proc Natl Acad Sci USA, 2010, 107(8): 3558-3563.
17. Bourin P, Bunnell BA, Casteilla LA, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy, 2013, 15(6): 641-648.
18. Baglioni S, Francalanci M, Squecco R, et al. Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB J, 2009, 23(10): 3494-3505.
19. Vishnubalaji R, Al-Nbaheen M, Kadalmani B, et al. Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis. Cell Tissue Res, 2012, 347(2): 419-427.
20. Ross AC. Cellular metabolism and activation of retinoids: roles of cellular retinoid-binding proteins. FASEB J, 1993, 7(2): 317-327.
21. Zhang S, Chen X, Hu Y, et al. All-trans retinoic acid modulates Wnt3A-induced osteogenic differentiation of mesenchymal stem cells via activating the PI3K/AKT/GSK3β signalling pathway. Mol Cell Endocrinol, 2016, 422: 243-253.
22. Ma K, Laco F, Ramakrishna S, et al. Differentiation of bone marrow-derived mesenchymal stem cells into multi-layered epidermis-like cells in 3D organotypic coculture. Biomaterials, 2009, 30(19): 3251-3258.
23. Moll R, Divo M, Langbein L. The human keratins: biology and pathology. Histochem Cell Biol, 2008, 129(6): 705-733.
24. Kwon YW, Kwon KS, Moon HE, et al. Insulin-like growth factor-Ⅱ regulates the expression of vascular endothelial growth factor by the human keratinocyte cell line HaCaT. J Invest Dermatol, 2004, 123(1): 152-158.
25. Pozzi G, Guidi M, Laudicina F, et al. IGF-I stimulates proliferation of spontaneously immortalized human keratinocytes (HACAT) by autocrine/paracrine mechanisms. J Endocrinol Invest, 2004, 27(2): 142-149.

West China Medical Journal

Investigation on HaCaT cell condition medium combined with all-trans-retinoic acid initiating differentiation of human adipose-derived stem cells into epidermal cells

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