The role of Schwann cells-like cells derived from human amniotic membrane mesenchymal stem cells transplantation in flap nerves regeneration_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GONG Feiyu ,  WEI Zairong , JIN Wenhu , LI Hai , DENG Chengliang , WU Bihua , NIE Kaiyu

Department of Burn and Plastic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi Guizhou, 563000, P.R.China;

Corresponding author：

WEI Zairong, Email: zairongwei@sina.com

Keywords：

Human amniotic membrane mesenchymal stem cells; Schwann cells-like cells; induced differentiation; nerve regeneration; flap

DOI：

10.7507/1002-1892.201708007

Video：

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Objective Inducing human amniotic membrane mesenchymal stem cells (hAMSCs) to Schwann cells-like cells (SCs-like cells) in vitro, and to evaluate the efficacy of transplantation of hAMSCs and SCs-like cells on nerves regeneration of the rat flaps. Methods hAMSCs were isolated from placenta via two-step digestion and cultured by using trypsin and collagenase, then identified them by flow cytometry assay and immunofluorescence staining. The 3rd generation of hAMSCs cultured for 6 days were induced to SCs-like cells in vitro; at 19 days after induction, the levels of S-100, p75, and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence staining, Western blot, and real-time fluorescence quantitative PCR (qPCR). The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were measured by ELISA in the supernatant of the 3rd generation of hAMSCs cultured for 6 days and the hAMSCs induced within 19 days. In addition, 75 female Sprague Dawley rats were taken to establish the rat denervated perforator flap model of the abdominal wall, and were divided into 3 groups (n=25). The 3rd generation of hAMSCs (1×10⁶ cells) in the proliferation period of culturing for 6 days, the SCs-like cells (1×10⁶ cells), and equal volume PBS were injected subcutaneously in the skin flap of the rat in groups A, B, and C, respectively. At 2, 5, 7, 9, and 14 days after transplantation, 5 rats in each group were killed to harvest the flap frozen sections and observe the positive expression of neurofilament heavy polypeptide antibody (NF-01) by immunofluorescence staining. Results The cells were identified as hAMSCs by flow cytometry assay and immunofluorescence staining. The results of immunofluorescence staining, Western blot, qPCR showed that the percentage of positive cells, protein expression, and gene relative expression of S-100, p75, and GFAP in SCs-like cells group were significantly higher than those in hAMSCs group (P<0.05). The results of ELISA demonstrated that the expression of BDNF and NGF was significantly decreased after added induced liquid 1, and the level of BDNF and NGF increased gradually with the induction of liquids 2 and 3, and the concentration of BDNF and NGF was significantly higher than that of hAMSCs group (P<0.05). Immunofluorescence staining showed that the number of regenerated nerve fibers in group B was higher than that in groups A and C after 5-14 days of transplantation. Conclusion The hAMSCs can be induced into SCs-like cells with the proper chemical factor regulation in vitro, and a large number of promoting nerve growth factor were released during the process of differentiation, and nerve regeneration in flaps being transplanted the SCs-like cells was better than that in flaps being transplanted the hAMSCs, which through a large number of BDNF and NGF were released.

Citation： GONG Feiyu, WEI Zairong, JIN Wenhu, LI Hai, DENG Chengliang, WU Bihua, NIE Kaiyu. The role of Schwann cells-like cells derived from human amniotic membrane mesenchymal stem cells transplantation in flap nerves regeneration. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(1): 80-90. doi: 10.7507/1002-1892.201708007 Copy

1.	IJpma FF, Nicolai JP, Meek MF, et al. Sural nerve donor-site morbidity thirty-four years of follow-up. Ann Plast Surg, 2006, 57(4): 391-395.
2.	He Y, Zhang PZ, Sun D, et al. Wnt1 from cochlear Schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model. Cell Transplant, 2014, 23(6): 747-760.
3.	Tomita K, Nishibayashi A, Yano K, et al. Differentiated Adipose-derived Stem Cells Promote Reinnervation of Rat Skin Flaps. Plast Reconstr Surg Glob Open, 2013, 1(3): e22.
4.	Waris T, Rechardt L, Kyösola K. Reinnervation of human skingrafts: a histochemical study. Plast Reconstr Surg, 1983, 72(4): 439-447.
5.	Ward RS, Tuckett RP, English KB, et al. Substance P axons and sensory threshold increase in burn-graft human skin. J Surg Res, 2004, 118(2): 154-160.
6.	Nedelec B, Hou Q, Sohbi I, et al. Sensory perception and neuroanatomical structures in normal and grafted skin of burn survivors. Burn, 2005, 31(7): 817-830.
7.	Gu JX, Pan JB, Liu HJ, et al. Aesthetic and sensory reconstruction of finger pulp defects using free toe flaps. Aesthetic Plast Surg, 2014, 38(1): 156-163.
8.	Dobreva MP, Pereira PN, Deprest J, et al. On the origin of amniotic stem cells: of mice and men. Int J Dev Biol, 2010, 54(5): 761-777.
9.	甘文婷, 孙新, 陆琰. 人羊膜上皮细胞与人羊膜间充质干细胞生物学特性对比. 中国实验血液学杂志, 2015, 23(4): 1120-1124.
10.	Saito S, Lin YC, Murayama Y, et al. Human amnion-derived cells as a reliable source of stem cells. Curr Mol Med, 2012, 12(10): 1340-1349.
11.	庞希宁, 王竟, 施萍, 等. 人羊膜间充质干细胞的超微结构观察. 基础医学与临床, 2013, 33(11): 1371-1376.
12.	Sakuragawa N, Kakinuma K, Kikuchi A, et al. Human amniotic mesenchymal cells express phenoptypes of neuroglial progenitor cells. Neurosci Res, 2004, 78(2): 208-214.
13.	焦红亮, 王晓宁, 孙剑瑞, 等. 人羊膜间充质干细胞混合培养及向神经样细胞分化. 中华实验外科杂志, 2013, 30(9): 1864-1866.
14.	Nagami M, Tsuno H, Koike C, et al. Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation. Transplantation, 2012, 93(12): 1221-1228.
15.	Kim SW, Zhang HZ, Kim CE, et al. Amniotic mesenchymal stem cells have robust angiogenic properties and are effective in treating hindlimb ischaemia. Cardiovasc Res, 2012, 93(3): 525-534.
16.	Li Y, Guo L, Ahn HS et al. Amniotic mesenchymal stem cells display neurovascular tropism and aid in the recovery of injured peripheral nerves. J Cell Mol Med, 2014, 18(6): 1028-1034.
17.	Tomita K, Madura T, Sakai Y, et al. Glial differentiation of human adipose-derived stem cells: implications for cell-based transplantation therapy. Neuroscience, 2013, 236: 55-65.
18.	Banerjee A, Nurnberger S, Hennerbichler S, et al. In toto differentiation of human amniotic membrane towards the Schwann cell lineage. Cell Tissue Bank, 2014, 15(2): 227-239.
19.	肖露, 周晓华, 王克, 等. 人羊膜上皮细胞的临床应用前景. 现代妇产科进展, 2015, 24(6): 478-480.
20.	Miki T, Grubbs B. Therapeutic potential of placenta-derived stem cells for liver diseases: current status and perspectives. J Obstet Gynaecol Res, 2014, 40(2): 360-368.
21.	Peng L, Wang J, Lu G. Involvement of Gene Methylation Changes in the Differentiation of Human Amniotic Epithelial Cells into Islet-Like Cell Clusters. DNA Cell Biol, 2014, 33(9): 591-598.
22.	Gu XS, Ding F, Yang YM, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
23.	Kim HA, Mindos T, Parkinson DB. Plastic fantastic: Schwann cells and repair of the peripheral nervous system. Stem Cells Transl Med, 2013, 2(8): 553-557.
24.	Arthur-Farraj PJ, Latouche M, Wilton DK, et al. c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration. Neuron, 2012, 75(4): 633-647.
25.	Court FA, Midha R, Cisterna BA, et al. Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons. Glia, 2011, 59(10): 1529-1539.
26.	Madduri S, Gander B. Schwann cell delivery of neurotrophic factors of peripheral nerve regeneration. J Peripher Nerv Syst, 2010, 15(2): 93-103.
27.	Shen M, Ji Y, Zhang S, et al. A proteome map of primary cultured rat Schwann cells. Proteome Sci, 2012, 10(1): 20.
28.	Fong CY, Gauthaman K, Cheyyatraivendran S, et al. Human umbilicalcord Wharton’s jelly stem cells and its conditioned medium support hematopoietic stem cells expansion ex vivo. J Cell Biochem, 2012, 113(2): 658-668.
29.	Orbay H, Uysal AC, Hyakusoku H, et al. Differentiated and undifferentiated adipose-derived stem cells improve function in rats with peripheral nerve gaps. J Plast Reconstr Aes, 2012, 65(5): 657-664.
30.	施剑明, 牛力, 杜桂花, 等. 骨髓间充质干细胞治疗周围神经损伤的研究进展. 中国矫形外科杂志, 2014, 22(12): 1081-1085.
31.	Lavasani M, Thompson SD, Pollett JB, et al. Human muscle-derived stem/progenitor cells promote functional murine peripheral nerve regeneration. J Clin Invest, 2014, 124(4): 1745-1756.
32.	Martens W, Sanen K, Georgiou M, et al. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro. Faseb J, 2014, 28(4): 1634-1643.
33.	朱玉梅, 冯世庆. 人脐带间充质干细胞和大鼠激活态雪旺氏细胞联合培养的实验研究. 天津医药, 2012, 40(3): 251-253.
34.	Yang L, Fang J, Liao D, et al. Schwann cells differentiated from adipose-derived stem cells for the treatment of brain contusion. Mol Med Rep, 2014, 9(2): 567-573.
35.	Gao S, Zheng Y, Cai Q, et al. Different methods for inducting adipose-derived stem cells to differentiate into Schwann-like cells. Arch Med Sci, 2015, 11(4): 886-892.
36.	Quan H, Wu X, Tian Y, et al. Overexpression of CDK5 in neural stem cells facilitates maturation of embryonic neurocytes derived from rats in vitro. Cell Biochem Biophys, 2014, 69(3): 445-453.
37.	付秀美, 杨振江, 王荣良, 等. 大鼠脂肪源性干细胞来源的施万样细胞的增殖能力. 吉林大学学报 (医学版), 2017, 43(1): 36-41.
38.	买霞, 陈小义, 杨丽颖, 等. bFGF 通过上调 ERK 表达诱导 BMSCs 向神经胶质样细胞分化. 解剖科学进展, 2012, 18(6): 541-544, 548.
39.	Zhao Y, Jiang H, Liu XW, et al. Erratum to: Neurogenic differentiation from adipose-derived stem cells and application for autologous transplantation in spinal cord injury. Cell Tissue Bank, 2015, 16(4): 649.
40.	Zhu H, Yang A, Du J, et al. Basic fibroblast growth factor is a key factor that induces bone marrow mesenchymal stem cells towards cells with Schwann cell phenotype. Neurosci Lett, 2014, 559: 82-87.
41.	Nawa H, Sotoyman H, Iwakura Y, et al. Neuropathologic implication of peripheral neuregulin-1 and EGF signals in dopaminergic dysfunction and behavioral deficits relevant to schizophrenia: their target cells and time window. Biomed Res Int, 2014: 697935.
42.	周青, 王冬芽, 刘星. Heregulin 和 VEGF 在结肠癌组织中的表达及其临床意义. 中国老年学杂志, 2014, 22(11): 6289-6291.
43.	Barrenschee M, Lange C, Cossais F, et al. Expression and function of Neuregulin 1 and its signaling system ERBB2/3 in the enteric nervous system. Front Cell Neurosci, 2015, 9: 360.

1. IJpma FF, Nicolai JP, Meek MF, et al. Sural nerve donor-site morbidity thirty-four years of follow-up. Ann Plast Surg, 2006, 57(4): 391-395.
2. He Y, Zhang PZ, Sun D, et al. Wnt1 from cochlear Schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model. Cell Transplant, 2014, 23(6): 747-760.
3. Tomita K, Nishibayashi A, Yano K, et al. Differentiated Adipose-derived Stem Cells Promote Reinnervation of Rat Skin Flaps. Plast Reconstr Surg Glob Open, 2013, 1(3): e22.
4. Waris T, Rechardt L, Kyösola K. Reinnervation of human skingrafts: a histochemical study. Plast Reconstr Surg, 1983, 72(4): 439-447.
5. Ward RS, Tuckett RP, English KB, et al. Substance P axons and sensory threshold increase in burn-graft human skin. J Surg Res, 2004, 118(2): 154-160.
6. Nedelec B, Hou Q, Sohbi I, et al. Sensory perception and neuroanatomical structures in normal and grafted skin of burn survivors. Burn, 2005, 31(7): 817-830.
7. Gu JX, Pan JB, Liu HJ, et al. Aesthetic and sensory reconstruction of finger pulp defects using free toe flaps. Aesthetic Plast Surg, 2014, 38(1): 156-163.
8. Dobreva MP, Pereira PN, Deprest J, et al. On the origin of amniotic stem cells: of mice and men. Int J Dev Biol, 2010, 54(5): 761-777.
9. 甘文婷, 孙新, 陆琰. 人羊膜上皮细胞与人羊膜间充质干细胞生物学特性对比. 中国实验血液学杂志, 2015, 23(4): 1120-1124.
10. Saito S, Lin YC, Murayama Y, et al. Human amnion-derived cells as a reliable source of stem cells. Curr Mol Med, 2012, 12(10): 1340-1349.
11. 庞希宁, 王竟, 施萍, 等. 人羊膜间充质干细胞的超微结构观察. 基础医学与临床, 2013, 33(11): 1371-1376.
12. Sakuragawa N, Kakinuma K, Kikuchi A, et al. Human amniotic mesenchymal cells express phenoptypes of neuroglial progenitor cells. Neurosci Res, 2004, 78(2): 208-214.
13. 焦红亮, 王晓宁, 孙剑瑞, 等. 人羊膜间充质干细胞混合培养及向神经样细胞分化. 中华实验外科杂志, 2013, 30(9): 1864-1866.
14. Nagami M, Tsuno H, Koike C, et al. Isolation and characterization of human amniotic mesenchymal stem cells and their chondrogenic differentiation. Transplantation, 2012, 93(12): 1221-1228.
15. Kim SW, Zhang HZ, Kim CE, et al. Amniotic mesenchymal stem cells have robust angiogenic properties and are effective in treating hindlimb ischaemia. Cardiovasc Res, 2012, 93(3): 525-534.
16. Li Y, Guo L, Ahn HS et al. Amniotic mesenchymal stem cells display neurovascular tropism and aid in the recovery of injured peripheral nerves. J Cell Mol Med, 2014, 18(6): 1028-1034.
17. Tomita K, Madura T, Sakai Y, et al. Glial differentiation of human adipose-derived stem cells: implications for cell-based transplantation therapy. Neuroscience, 2013, 236: 55-65.
18. Banerjee A, Nurnberger S, Hennerbichler S, et al. In toto differentiation of human amniotic membrane towards the Schwann cell lineage. Cell Tissue Bank, 2014, 15(2): 227-239.
19. 肖露, 周晓华, 王克, 等. 人羊膜上皮细胞的临床应用前景. 现代妇产科进展, 2015, 24(6): 478-480.
20. Miki T, Grubbs B. Therapeutic potential of placenta-derived stem cells for liver diseases: current status and perspectives. J Obstet Gynaecol Res, 2014, 40(2): 360-368.
21. Peng L, Wang J, Lu G. Involvement of Gene Methylation Changes in the Differentiation of Human Amniotic Epithelial Cells into Islet-Like Cell Clusters. DNA Cell Biol, 2014, 33(9): 591-598.
22. Gu XS, Ding F, Yang YM, et al. Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration. Prog Neurobiol, 2011, 93(2): 204-230.
23. Kim HA, Mindos T, Parkinson DB. Plastic fantastic: Schwann cells and repair of the peripheral nervous system. Stem Cells Transl Med, 2013, 2(8): 553-557.
24. Arthur-Farraj PJ, Latouche M, Wilton DK, et al. c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration. Neuron, 2012, 75(4): 633-647.
25. Court FA, Midha R, Cisterna BA, et al. Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons. Glia, 2011, 59(10): 1529-1539.
26. Madduri S, Gander B. Schwann cell delivery of neurotrophic factors of peripheral nerve regeneration. J Peripher Nerv Syst, 2010, 15(2): 93-103.
27. Shen M, Ji Y, Zhang S, et al. A proteome map of primary cultured rat Schwann cells. Proteome Sci, 2012, 10(1): 20.
28. Fong CY, Gauthaman K, Cheyyatraivendran S, et al. Human umbilicalcord Wharton’s jelly stem cells and its conditioned medium support hematopoietic stem cells expansion ex vivo. J Cell Biochem, 2012, 113(2): 658-668.
29. Orbay H, Uysal AC, Hyakusoku H, et al. Differentiated and undifferentiated adipose-derived stem cells improve function in rats with peripheral nerve gaps. J Plast Reconstr Aes, 2012, 65(5): 657-664.
30. 施剑明, 牛力, 杜桂花, 等. 骨髓间充质干细胞治疗周围神经损伤的研究进展. 中国矫形外科杂志, 2014, 22(12): 1081-1085.
31. Lavasani M, Thompson SD, Pollett JB, et al. Human muscle-derived stem/progenitor cells promote functional murine peripheral nerve regeneration. J Clin Invest, 2014, 124(4): 1745-1756.
32. Martens W, Sanen K, Georgiou M, et al. Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro. Faseb J, 2014, 28(4): 1634-1643.
33. 朱玉梅, 冯世庆. 人脐带间充质干细胞和大鼠激活态雪旺氏细胞联合培养的实验研究. 天津医药, 2012, 40(3): 251-253.
34. Yang L, Fang J, Liao D, et al. Schwann cells differentiated from adipose-derived stem cells for the treatment of brain contusion. Mol Med Rep, 2014, 9(2): 567-573.
35. Gao S, Zheng Y, Cai Q, et al. Different methods for inducting adipose-derived stem cells to differentiate into Schwann-like cells. Arch Med Sci, 2015, 11(4): 886-892.
36. Quan H, Wu X, Tian Y, et al. Overexpression of CDK5 in neural stem cells facilitates maturation of embryonic neurocytes derived from rats in vitro. Cell Biochem Biophys, 2014, 69(3): 445-453.
37. 付秀美, 杨振江, 王荣良, 等. 大鼠脂肪源性干细胞来源的施万样细胞的增殖能力. 吉林大学学报 (医学版), 2017, 43(1): 36-41.
38. 买霞, 陈小义, 杨丽颖, 等. bFGF 通过上调 ERK 表达诱导 BMSCs 向神经胶质样细胞分化. 解剖科学进展, 2012, 18(6): 541-544, 548.
39. Zhao Y, Jiang H, Liu XW, et al. Erratum to: Neurogenic differentiation from adipose-derived stem cells and application for autologous transplantation in spinal cord injury. Cell Tissue Bank, 2015, 16(4): 649.
40. Zhu H, Yang A, Du J, et al. Basic fibroblast growth factor is a key factor that induces bone marrow mesenchymal stem cells towards cells with Schwann cell phenotype. Neurosci Lett, 2014, 559: 82-87.
41. Nawa H, Sotoyman H, Iwakura Y, et al. Neuropathologic implication of peripheral neuregulin-1 and EGF signals in dopaminergic dysfunction and behavioral deficits relevant to schizophrenia: their target cells and time window. Biomed Res Int, 2014: 697935.
42. 周青, 王冬芽, 刘星. Heregulin 和 VEGF 在结肠癌组织中的表达及其临床意义. 中国老年学杂志, 2014, 22(11): 6289-6291.
43. Barrenschee M, Lange C, Cossais F, et al. Expression and function of Neuregulin 1 and its signaling system ERBB2/3 in the enteric nervous system. Front Cell Neurosci, 2015, 9: 360.

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The role of Schwann cells-like cells derived from human amniotic membrane mesenchymal stem cells transplantation in flap nerves regeneration

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