ADIPOSE-DERIVED STEM CELLS DIFFERENTIATION INTO NEURON-LIKE CELLS INDUCED BY CO-CULTURE WITH SCHWANN CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGZhenhui ¹ , LIDong ¹ , SUNKai ¹ , LIRuixin ² , ZHUXiang ³ ,  CHENXuyi ³ ,  XUYunqiang ¹

1. Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, 300052, P. R. China;
2. Institute of Medical Equipment, Academy of Military and Medical Sciences;
3. Department of Brain, Affiliated Brain Hospital, Logistics University of People's Armed Police Force;

Corresponding author：

CHENXuyi, Email: chenxuyi1979@126.com; XUYunqiang, Email: docxu@sina.com

Keywords：

Nerve tissue engineering; Schwann cells; Adipose-derived stem cells; Co-culture; Induceddifferentiation; Rats

DOI：

10.7507/1002-1892.20150020

Video：

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Objective To investigate the differentiation of rat adipose-derived stem cells (ADSCs) into neuronlike cells by indirect co-culture with Schwann cells (SCs) in vitro so as to look for the ideal seed cells for tissue engineering. Methods SCs were isolated from sciatic nerves of 1-2 days old Sprague-Dawley rats with enzymatic digestion method. Immunofluorescence staining was used to identify SCs with the marker S-100. ADSCs were isolated from the epididymal fat pads of adult male Sprague-Dawley rats by means of differential attachment. And the cell phenotypes (CD29, CD34, CD45, CD73, CD90, and CD105) of ADSCs at passage 3 were determined by flow cytometry analysis. Primary SCs and ADSCs at passage 3 were co-cultured at a ratio of 2:1 in Transwell culture dishes (experimental group), and ADSCs cultured alone served as control group. Immunofluorescence and flow cytometry were adopted to investigate the neural differentiation of ADSCs at 14 days. The expression differences for neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), neuronal nuclei protein (NeuN), and glial fibrillary acidic protein (GFAP) were detected, and the percentage of positive cells was calculated. Results ADSCs were successfully extracted and can passage in a considerable large amount. Flow cytometry analysis showed that ADSCs at passage 3 were positive for CD29, CD90, CD73, and CD105 expression, but negative for CD34 and CD45 expression. The ADSCs of the experimental group showed contraction of nucleus, increasing of soma refraction, and several long and thick protrusions of cell body. The cell shape had no obvious change in the control group. Both immunofluorescence and flow cytometry analysis results showed the expressions of MAP2, NSE, NeuN, and GFAP at 14 days after co-cultured with SCs, and the positive cell ratios were significantly higher than those in the control group (P<0.01). Conclusion Co-culture with SCs not only can promote the survival regeneration of ADSCs, but also can induce the differentiation of ADSCs into neuron-like cells.

Citation： ZHANGZhenhui, LIDong, SUNKai, LIRuixin, ZHUXiang, CHENXuyi, XUYunqiang. ADIPOSE-DERIVED STEM CELLS DIFFERENTIATION INTO NEURON-LIKE CELLS INDUCED BY CO-CULTURE WITH SCHWANN CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(1): 97-102. doi: 10.7507/1002-1892.20150020 Copy

1.	Kaengkan P, Baek SE, Kim JY, et al. Administration of mesenchymal stem cells and ziprasidone enhanced amelioration of ischemic brain damage in rats. Mol Cells, 2013, 36(6):534-541.
2.	Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res, 2000, 61(4):364-370.
3.	Zurita M, Vaquero J, Oya S, et al. Schwann cells induce neuronal differentiation of bone marrow stromal cells. Neuroreport, 2005, 16(5):505-508.
4.	Ito R, Morimoto N, Liem PH, et al. Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor. J Tissue Eng Regen Med, 2014, 8(12):1000-1008.
5.	Fraser JK, Wulur I, Alfonso Z, et al. Fat tissue:an underappreciated source of stem cells for biotechnology. Trends Biotechnol, 2006, 24(4):150-154.
6.	Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
7.	Hiwatashi N, Hirano S, Mizuta M, et al. Adipose-derived stem cells versus bone marrow-derived stem cells for vocal fold regeneration. Laryngoscope, 2014, 124(12):E461-E469.
8.	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.
9.	Armati PJ, Mathey EK. Clinical implications of Schwann cell biology. J Peripher Nerv Syst, 2014, 19(1):14-23.
10.	Xiaojie L, Dapeng L, Ping G, et al. Neural differentiation of adiposederived stem cells by indirect co-culture with Schwann cells. Arch Biol Sci, 2009, 61(4):703-711.
11.	Kingham PJ, Kalbermatten DF, Mahay D, et al. Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neurol, 2007, 207(2):267-274.
12.	Ni WF, Wu AM, Li QL, et al. Induced human bone marrow stromal cells differentiate into neural cells by bFGF and cocultured with olfactory ensheathing cells. Curr Stem Cell Res Ther, 2014, 9(4):291-296.
13.	Jang S, Cho HH, Cho YB, et al. Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biol, 2010, 11:25.
14.	Qian DX, Zhang HT, Ma X, et al. Comparison of the efficiencies of three neural induction protocols in human adipose stromal cells. Neurochem Res, 2010, 35(4):572-579.
15.	Wrage PC, Tran T, To K, et al. The neuro-glial properties of adiposederived adult stromal (ADAS) cells are not regulated by Notch 1 and are not derived from neural crest lineage. PLoS One, 2008, 3(1):e1453.
16.	Lu P, Blesch A, Tuszynski MH. Induction of bone marrow stromal cells to neurons:differentiation, transdifferentiation, or artifact? J Neurosci Res, 2004, 77(2):174-191.
17.	Duan P, Xu H, Zeng Y, et al. Human bone marrow stromal cells can differentiate to a retinal pigment epithelial phenotype when co-cultured with pig retinal pigment epithelium using a transwell system. Cell Physiol Biochem, 2013, 31(4-5):601-613.
18.	陈盼盼, 张丽华, 董为人, 等. 脂肪组织源性干细胞分步诱导分化为神经元样细胞. 南方医科大学学报, 2011, 31(3):512-517.
19.	Ning H, Lin G, Lue TF, et al. Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells. Differentiation, 2006, 74(9-10):510-518.
20.	Mahmood A, Lu D, Chopp M. Marrow stromal cell transplantation after traumatic brain injury promotes cellular proliferation within the brain. Neurosurgery, 2004, 55(5):1185-1193.
21.	郭再玉, 姜晓丹, 徐如祥, 等. 骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究. 中华神经医学杂志, 2005, 4(6):545-550.

1. Kaengkan P, Baek SE, Kim JY, et al. Administration of mesenchymal stem cells and ziprasidone enhanced amelioration of ischemic brain damage in rats. Mol Cells, 2013, 36(6):534-541.
2. Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res, 2000, 61(4):364-370.
3. Zurita M, Vaquero J, Oya S, et al. Schwann cells induce neuronal differentiation of bone marrow stromal cells. Neuroreport, 2005, 16(5):505-508.
4. Ito R, Morimoto N, Liem PH, et al. Adipogenesis using human adipose tissue-derived stromal cells combined with a collagen/gelatin sponge sustaining release of basic fibroblast growth factor. J Tissue Eng Regen Med, 2014, 8(12):1000-1008.
5. Fraser JK, Wulur I, Alfonso Z, et al. Fat tissue:an underappreciated source of stem cells for biotechnology. Trends Biotechnol, 2006, 24(4):150-154.
6. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
7. Hiwatashi N, Hirano S, Mizuta M, et al. Adipose-derived stem cells versus bone marrow-derived stem cells for vocal fold regeneration. Laryngoscope, 2014, 124(12):E461-E469.
8. 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.
9. Armati PJ, Mathey EK. Clinical implications of Schwann cell biology. J Peripher Nerv Syst, 2014, 19(1):14-23.
10. Xiaojie L, Dapeng L, Ping G, et al. Neural differentiation of adiposederived stem cells by indirect co-culture with Schwann cells. Arch Biol Sci, 2009, 61(4):703-711.
11. Kingham PJ, Kalbermatten DF, Mahay D, et al. Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neurol, 2007, 207(2):267-274.
12. Ni WF, Wu AM, Li QL, et al. Induced human bone marrow stromal cells differentiate into neural cells by bFGF and cocultured with olfactory ensheathing cells. Curr Stem Cell Res Ther, 2014, 9(4):291-296.
13. Jang S, Cho HH, Cho YB, et al. Functional neural differentiation of human adipose tissue-derived stem cells using bFGF and forskolin. BMC Cell Biol, 2010, 11:25.
14. Qian DX, Zhang HT, Ma X, et al. Comparison of the efficiencies of three neural induction protocols in human adipose stromal cells. Neurochem Res, 2010, 35(4):572-579.
15. Wrage PC, Tran T, To K, et al. The neuro-glial properties of adiposederived adult stromal (ADAS) cells are not regulated by Notch 1 and are not derived from neural crest lineage. PLoS One, 2008, 3(1):e1453.
16. Lu P, Blesch A, Tuszynski MH. Induction of bone marrow stromal cells to neurons:differentiation, transdifferentiation, or artifact? J Neurosci Res, 2004, 77(2):174-191.
17. Duan P, Xu H, Zeng Y, et al. Human bone marrow stromal cells can differentiate to a retinal pigment epithelial phenotype when co-cultured with pig retinal pigment epithelium using a transwell system. Cell Physiol Biochem, 2013, 31(4-5):601-613.
18. 陈盼盼, 张丽华, 董为人, 等. 脂肪组织源性干细胞分步诱导分化为神经元样细胞. 南方医科大学学报, 2011, 31(3):512-517.
19. Ning H, Lin G, Lue TF, et al. Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells. Differentiation, 2006, 74(9-10):510-518.
20. Mahmood A, Lu D, Chopp M. Marrow stromal cell transplantation after traumatic brain injury promotes cellular proliferation within the brain. Neurosurgery, 2004, 55(5):1185-1193.
21. 郭再玉, 姜晓丹, 徐如祥, 等. 骨髓基质细胞源性神经干细胞体外分化及电生理特性的研究. 中华神经医学杂志, 2005, 4(6):545-550.

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Chinese Journal of Reparative and Reconstructive Surgery

ADIPOSE-DERIVED STEM CELLS DIFFERENTIATION INTO NEURON-LIKE CELLS INDUCED BY CO-CULTURE WITH SCHWANN CELLS

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