Obtaining, Identifying and Marking of the Human Umbilical Cord-derived Mesenchymal Stem Cells <i>in Vitro</i>_West China Medical Journal

Authors：

 ZHANGDe-shuang ^1,2 ,  CHENJuan ¹

1. Department of Neonatology, West China Second Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Department of Neonatology, the Affiliated Hospital of Luzhou Medical College, Luzhou, Sichuan 646000, P. R. China;

Corresponding author：

CHENJuan, Email: chenjuan2000@163.com

Keywords：

Mesenchymal stem cells; umbilical cord; human; Isolation; Culture; Differentiation

DOI：

10.7507/1002-0179.20150586

Video：

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Objective To obtain the mesenchymal stem cells (MSCs) from human umbilical cord and mark in vitro, for further transplantation therapy. Methods The MSC were isolated from human umbilical cord by tissue explants culture method. After subculture in vitro, the morphology of hUC-MSC was observed; the surface antigens of hUC-MSC were detected by flow cytometry; adipogenic and osteogenic differentiation were determined by specific staining; hUC-MSC labelled with Brd U were identified by immunofluorescence. Results MSC could be isolated successfully by tissue explants culture method. When cultured about one week, the cells climbed out from the tissue block edge, proliferated and formed colonies; the hUC-MSCs of passage 5 were detected by flow cytometry, and they highly expressed CD73, CD90 and CD105, didn't express or lowly expressed CD14, CD34, CD45, CD79a and human leukocyte antigen-DR. After two weeks of adipogenic induction, they were positive in oil red O staining, and after three weeks of osteogenic induction, red precipitate could be seen by alizarin red staining, and the red fluorescence of the hUC-MSC labelled with Brd U could be detected by immunofluorescence detection. Conclusion The cells can be isolated from human umbilical cord by tissue explants culture method, with the characteristics of hUC-MSCs and can be labeled successfully in vitro, so it can be used for the research in the field of cell transplantation.

Citation： ZHANGDe-shuang, CHENJuan. Obtaining, Identifying and Marking of the Human Umbilical Cord-derived Mesenchymal Stem Cells in Vitro. West China Medical Journal, 2015, 30(11): 2067-2070. doi: 10.7507/1002-0179.20150586 Copy

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9.	Zhao Q, Hu J, Xiang J, Gu Y, et al. Intranasal administration of human umbilical cord mesenchymal stem cells-conditioned medium enhances vascular remodeling after stroke[J]. Brain Res, 2015, 1624:489-496.
10.	Li T, Zhang C, Ding Y, et al. Umbilical cord-derived mesenchymal stem cells promote proliferation and migration in MCF-7 and MDA-MB-231 breast cancer cells through activation of the ERK pathway[J]. Oncol Rep, 2015, 34(3):1469-1477.
11.	Feng T, Zhang J, Zeng G, et al. Therapeutic potential of umbilical cord mesenchymal stem cells in mice with acute hepatic failure[J]. Int J Artif Organs, 2015, 38(5):271-276.
12.	Sarugaser R, Ennis J, Stanford WL, et al. Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs) [J]. Methods Mol Biol, 2009, 482:269-279.
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14.	Seshareddy K, Troyer D, Weiss ML. Method to isolate mesenchymal-like cells from Wharton’s Jelly of umbilical cord[J]. Methods Cell Biol, 2008, 86:101-119.
15.	Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord[J]. Stem Cells, 2004, 22(7):1330-1337.
16.	何绍清, 罗振宇, 刘秋英, 等. 人脐带间充质干细胞分离培养及向脂肪与成骨细胞的分化[J]. 中国组织工程研究与临床康复, 2010, 14(14):2492-2496.
17.	Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells:paradoxes of passaging[J]. Exp Hematol, 2004, 32(5):414-425.
18.	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[J]. Cytotherapy, 2006, 8(4):315-317.

1. 张晓敏, 李筱荣. 间充质干细胞在眼部疾病中的应用研究进展[J]. 中华眼科杂志, 2012, 48(10):956-960.
2. Joe AW, Gregory-Evans K. Mesenchymal stem cells and potential applications in treating ocular disease[J]. Curr Eye Res, 2010, 35(11):941-952.
3. Fan CG, Zhang QJ, Zhou JR. Therapeutic potentials of mesenchymal stem cells derived from human umbilical cord[J]. Stem Cell Rev, 2011, 7(1):195-207.
4. Moretti P, Hatlapatka T, Marten D, et al. Mesenchymal stromal cells derived from human umbilical cord tissues:primitive cells with potential for clinical and tissue engineering applications[J]. Adv Biochem Eng Biotechnol, 2010, 123:29-54.
5. Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton’s jelly form neurons and glia[J]. Stem Cells, 2003, 21(1):50-60.
6. Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells[J]. Nature, 2001, 414(6859):92-97.
7. 张德双, 王华, 白小红, 等. 脐带间充质干细胞移植治疗新生儿缺氧缺血性脑损伤的最新研究进展[J]. 中华实用儿科临床杂志, 2013, 28(14):1112-1114.
8. Vangsness CT Jr, Sternberg H, Harris L. Umbilical cord tissue offers the greatest number of harvestable mesenchymal stem cells for research and clinical application:a literature review of different harvest sites[J]. Arthroscopy, 2015, 31(9):1836-1843.
9. Zhao Q, Hu J, Xiang J, Gu Y, et al. Intranasal administration of human umbilical cord mesenchymal stem cells-conditioned medium enhances vascular remodeling after stroke[J]. Brain Res, 2015, 1624:489-496.
10. Li T, Zhang C, Ding Y, et al. Umbilical cord-derived mesenchymal stem cells promote proliferation and migration in MCF-7 and MDA-MB-231 breast cancer cells through activation of the ERK pathway[J]. Oncol Rep, 2015, 34(3):1469-1477.
11. Feng T, Zhang J, Zeng G, et al. Therapeutic potential of umbilical cord mesenchymal stem cells in mice with acute hepatic failure[J]. Int J Artif Organs, 2015, 38(5):271-276.
12. Sarugaser R, Ennis J, Stanford WL, et al. Isolation, propagation, and characterization of human umbilical cord perivascular cells (HUCPVCs) [J]. Methods Mol Biol, 2009, 482:269-279.
13. Romanov YA, Svintsitskaya VA, Smirnov VN. Searching for alternative sources of postnatal human mesenchymal stem cells:candidate MSC-like cells from umbilical cord[J]. Stem Cells, 2003, 21(1):105-110.
14. Seshareddy K, Troyer D, Weiss ML. Method to isolate mesenchymal-like cells from Wharton’s Jelly of umbilical cord[J]. Methods Cell Biol, 2008, 86:101-119.
15. Wang HS, Hung SC, Peng ST, et al. Mesenchymal stem cells in the Wharton’s jelly of the human umbilical cord[J]. Stem Cells, 2004, 22(7):1330-1337.
16. 何绍清, 罗振宇, 刘秋英, 等. 人脐带间充质干细胞分离培养及向脂肪与成骨细胞的分化[J]. 中国组织工程研究与临床康复, 2010, 14(14):2492-2496.
17. Javazon EH, Beggs KJ, Flake AW. Mesenchymal stem cells:paradoxes of passaging[J]. Exp Hematol, 2004, 32(5):414-425.
18. 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[J]. Cytotherapy, 2006, 8(4):315-317.

West China Medical Journal

Obtaining, Identifying and Marking of the Human Umbilical Cord-derived Mesenchymal Stem Cells in Vitro

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