EXPERIMENTAL STUDY OF DIFFERENTIATION OF UMBILICAL CORD MESENCHYMAL STEM CELLS INTO SMOOTH MUSCLE CELLS INDUCED BY BLADDER SMOOTH MUSCLE CELLS CONDITIONED MEDIUM_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YUAN Haichao , LIU Liangren , ZHENG Shuo , LIU Zhenhua , YANG Lu , PU Chunxiao , LI Jinhong , LONG Dan , WEI Qiang , HAN Ping.

Department of Urology, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P.R.China. Corresponding author: HAN Ping, E-mail: hanping@scu.edu.cn;

Corresponding author：

Keywords：

Umbilical cord mesenchymal stem cells; Bladder smooth muscle cells conditioned medium; Smooth muscle cells; Differentiation

DOI：

10.7507/1002-1892.20130330

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Objective To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system. Methods UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1 ∶ 1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of α-smooth muscle actin (α-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days. Results The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of α-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of α-SMA could be observed in groups A and B; at 14 days, the positive expression of α-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of α-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining. Conclusion UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.

Citation： YUAN Haichao,LIU Liangren,ZHENG Shuo,LIU Zhenhua,YANG Lu,PU Chunxiao,LI Jinhong,LONG Dan,WEI Qiang,HAN Ping.. EXPERIMENTAL STUDY OF DIFFERENTIATION OF UMBILICAL CORD MESENCHYMAL STEM CELLS INTO SMOOTH MUSCLE CELLS INDUCED BY BLADDER SMOOTH MUSCLE CELLS CONDITIONED MEDIUM. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(12): 1505-1511. doi: 10.7507/1002-1892.20130330 Copy

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29.	Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells, 2007, 25(2): 319-331.
30.	Schneider RK, Puellen A, Kramann R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds. Biomaterials, 2010, 31(3): 467-480.
31.	刘晓芳, 王延洲, 徐惠成, 等. 细胞间接触诱导骨髓间充质干细胞向平滑肌细胞分化的实验研究. 第三军医大学学报, 2010, 32(6): 516-519.
32.	Mohana Kumar B, Maeng GH, Lee YM, et al. 212 porcine bone marrow-derived mesenchymal stem cells differentiate in vitro into smooth muscle cells. Reprod Fertil Dev, 2011, 24(1): 218.
33.	Zhang R, Jack GS, Rao N, et al. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment. Stem Cells, 2012, 30(3): 481-490.

1. Gage FH. Cell therapy. Nature, 1998, 392(6679 Suppl): 18-24.
2. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science, 1997, 276(5309): 71-74.
3. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002, 13(12): 4279-4295.
4. Seale P, Asakura A, Rudnicki MA. The potential of muscle stem cells. Dev Cell, 2001, 1(3): 333-342.
5. Hutson EL, Boyer S, Genever PG. Rapid isolation, expansion, and differentiation of osteoprogenitors from full-term umbilical cord blood. Tissue Eng, 2005, 11(9-10): 1407-1420.
6. RusCiucǎ D, Soritǎu O, Suşman S, et al. Isolation and characterization of chorionic mesenchyal stem cells from the placenta. Rom J Morphol Embryol, 2011, 52(3): 803-808.
7. Rao MS, Mattson MP. Stem ceils and aging: expanding the possibilities. Mech Ageing Dev, 2001, 122(7): 713-734.
8. Kern S, Eichler H, Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells, 2006, 24(5): 1294-1301.
9. Sinha S, Hoofnagle MH, Kingston PA, et al. Transforming growth factor-beta1 signaling contributes to development of smooth muscle cells from embryonic stem cells. Am J Physiol Cell Physiol, 2004, 287(6): C1560-1568.
10. Tian H, Bharadwaj S, Liu Y, et al. Differentiation of human bone marrow mesenchymal stem cells into bladder cells: potential for urological tissue engineering. Tissue Eng Part A, 2010, 16(5): 1769-1779.
11. Gonzalez R, Griparic L, Umana M, et al. An efficient approach to isolation and characterization of pre- and postnatal umbilical cord lining stem cells for clinical applications. Cell Transplant, 2010, 19(11): 1439-1449.
12. Oh SH, Muzzonigro TM, Bae SH, et al. Adult Bone marrow derived cells trans-differentiating in to insulin-producing cells for the treatment of type I diabetes. Lab Invest, 2004, 84(5): 607-617.
13. Ende N, Chen R, Reddi AS. Effect of human umbilical cord blood cells on glycemia and insulitis in type 1 diabetic mice. Biochem Biophys Res Commun, 2004, 325(3): 665-669.
14. Lim JH, Byeon YE, Ryu HH, et al. Transplantation of canine umbilical cord blood-derived mesenchymal stem cells in experimentally induced spinal cord injured dogs. J Vet Sci, 2007, 8(3): 275-282.
15. Coutts M, Keirstead HS. Stem cells for the treatment of spinal cord injury. Exp Neurol, 2008, 209(2): 368-377.
16. Pereira WC, Khushnooma I, Madkaikar M, et al. Reproducible methodology for the isolation of mesenchymal stem cells from human umbilical cord and its potential for cardiomyocyte generation. J Tissue Eng Regen Med, 2008, 2(7): 394-399.
17. Ichim TE, Alexandrescu DT, Solano F, et al. Mesenchymal stem cells as anti-inflammatories: implications for treatment of Duch-ennemuscular dystrophy. Cell Immunol, 2010, 260(2): 75-82.
18. Corcos J, Loutochin O, Campeau L, et al. Bone marrow mesenchymal stromal cell therapy for external urethral sphincter restoration in a rat model of stress urinary incontinence. Neurourol Urody, 2011, 30(3): 447-455.
19. Zhao W, Zhang C, Jin C, et al. Periurethral injection of autologous adipose-derived stem cells with controlled-release nerve growth factor for the treatment of stress urinary incontinence in a rat model. Eur Urol, 2011, 59(1): 155-163.
20. Fu Q, Cao YL. Tissue engineering and stem cell application of urethroplasty: from bench to bedside. Urology, 2012, 79(2): 246-253.
21. Sharma AK, Bury MI, Marks AJ, et al. A nonhuman primate model for urinary bladder regeneration using autologous sources of bone marrow-derived mesenchymal stem cells. Stem Cells, 2011, 29(2): 241-250.
22. Mitchell KE, Weiss ML, Mitchell BM, et al. Matrix cells from Wharton’s jelly form neurons and glia. Stem cells, 2003, 21(1): 50-60.
23. Wang L, Tran I, Seshareddy K, et al. A comparison of human bone marrow-derived mesenchymal stem cells and human umbilical cord-derived mesenchymal stromal cells for cartilage tissue engineering. Tissue Eng Part A, 2009, 15(8): 2259-2266.
24. Wang M, Yang Y, Yang D, et al. The immunomodulatory activity of human umbilical cord blood-derived mesenchymal stem cells in vitro. Immunology, 2008, 126(2): 220-232.
25. Ma L, Feng XY, Cui BL, et al. Human umbilical cord Wharton’s Jelly-derived mesenchymal stem cells differentiation into nerve-like cells. Chin Med J (Engl), 2005, 118(23): 1987-1993.
26. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med, 2000, 343(4): 230-238.
27. Campard D, Lysy PA, Najimi M, et al. Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology, 2008, 134(3): 833-848.
28. Zhang YN, Lie PC, Wei X. Differentiation of mesenchymal stromal cells derived from umbilical cord Wharton’s jelly into hepatocyte-like cells. Cytotherapy, 11(5): 548-558.
29. Karahuseyinoglu S, Cinar O, Kilic E, et al. Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys. Stem Cells, 2007, 25(2): 319-331.
30. Schneider RK, Puellen A, Kramann R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds. Biomaterials, 2010, 31(3): 467-480.
31. 刘晓芳, 王延洲, 徐惠成, 等. 细胞间接触诱导骨髓间充质干细胞向平滑肌细胞分化的实验研究. 第三军医大学学报, 2010, 32(6): 516-519.
32. Mohana Kumar B, Maeng GH, Lee YM, et al. 212 porcine bone marrow-derived mesenchymal stem cells differentiate in vitro into smooth muscle cells. Reprod Fertil Dev, 2011, 24(1): 218.
33. Zhang R, Jack GS, Rao N, et al. Nuclear fusion-independent smooth muscle differentiation of human adipose-derived stem cells induced by a smooth muscle environment. Stem Cells, 2012, 30(3): 481-490.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY OF DIFFERENTIATION OF UMBILICAL CORD MESENCHYMAL STEM CELLS INTO SMOOTH MUSCLE CELLS INDUCED BY BLADDER SMOOTH MUSCLE CELLS CONDITIONED MEDIUM

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