人脐带间充质干细胞向神经元样细胞诱导分化:两种方法的比较
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摘要
背景:干细胞因其可向神经元样细胞诱导分化而在临床神经损伤疾病治疗中具有广阔的前景,但干细胞向神经元样细胞诱导分化的效率不高,为此作者对传统的诱导方法进行了改良。目的:探讨2种不同方法诱导人脐带间充质干细胞向神经元样细胞分化的潜能及2种诱导方法的优劣。方法:采用组织块法分离培养人脐带间充质干细胞,显微镜下观察其形态并利用流式细胞仪进行干细胞鉴定,在神经营养因子诱导方案下,采用相同的诱导剂(2%B27、20μg/L碱性成纤维细胞生长因子、20μg/L表皮生长因子),2种不同的诱导方法将其向神经元样细胞诱导分化(传统方法:将B27、碱性成纤维细胞生长因子、表皮生长因子及青链霉素合剂加入DMEM/F12培养基中进行诱导,3 d后加入全反式维甲酸,共培养10 d;改良方法:将B27、碱性成纤维细胞生长因子、表皮生长因子、青链霉素合剂及胎牛血清加入DMEM/F12培养基中进行诱导,6d后用胰酶消化后重新接种于共聚焦小皿内,次日去除B27和胎牛血清,并加入全反式维甲酸,共培养14 d)。应用免疫荧光染色对诱导分化后细胞表面神经标志物——神经丝蛋白(NF-M)和胶质纤维酸性蛋白(GFAP)进行检测,比较2种方法诱导后细胞表面神经标志物的阳性表达率。结果与结论:(1)成功分离出人脐带间充质干细胞,经流式细胞仪鉴定其高表达CD29、CD105,低表达CD34、CD45,符合干细胞特性;(2)2种方法诱导后,镜下观察到的细胞均可呈现神经细胞样改变,免疫荧光检测提示2种方法诱导后细胞中神经丝蛋白与胶质纤维酸性蛋白均呈阳性表达;(3)传统方法组神经丝蛋白与胶质纤维酸性蛋白的阳性率分别为18.73%和18.01%,改良方法组二者的阳性率分别为27.48%和29.24%,两组比较差异有显著性意义(P <0.05);(4)结果表明,2种诱导方法均可将人脐带间充质干细胞诱导分化为神经元样细胞;但在诱导分化后的细胞形态、神经标志物(神经丝蛋白与胶质纤维酸性蛋白)的阳性表达率方面,改良方法更具有优势。
BACKGROUND: Stem cells are a promising treatment for nerve injury diseases due to its ability of differentiating into neuron-like cells. However, the differentiation efficacy is low, so we have make an advance based on the traditional induction method. OBJECTIVE: To explore the potentiality of human umbilical cord mesenchymal stem cells differentiating into neuron-like cells induced by two methods and their merits. METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured using tissue explant method. The cell morphology was observed under microscope, and the cultured cells were identified by flow cytometry. Human umbilical cord mesenchymal stem cells were differentiated into neuron-like cell under the neurotrophic factor inducing scheme. Two different methods but using the same inducers(2% B27, 20 μg/L basic fibroblast growth factor and 20 μg/L epidermal growth factor) were utilized. Traditional method: B27, basic fibroblast growth factor, epidermal growth factor and penicillin streptomycin combination were added in the DMEM/F12 medium, 3 days later all-trans retinoic acid was added, followed by 10 days of culture. Advanced method: B27, basic fibroblast growth factor, epidermal growth factor, penicillin streptomycin combination and fetal bovine serum were added in the DMEM/F12 medium, 6 days later the cells underwent trypsinization and re-seeded in the confocal capsule, B27 and fetal bovine serum were removed next day, and all-trans retinoic acid was added, cultured for 14 days. Then the differentiated nerve cells were identified by immunofluorescence detection of neurofilament protein and glial fibrillary acidic protein. Meanwhile, the positive rate of the neuromarkers in the cells was compared between two methods. RESULTS AND CONCLUSION: Primary human umbilical cord mesenchymal stem cells were isolated and cultured successfully, and highly expressed CD29 and CD105 whereas lowly expressed CD34 and CD45, which were the characteristics of the stem cell. The differentiated cells were presented as nerve cells in the morphology under microscope in both inducing methods. The results of the immunofluorescence indicated that the neurofilament protein and the glial fiber acidic protein in the differentiated cells were all expressed positive in both methods. The positive rates of neurofilament protein and glial fiber acidic protein were 18.73% and 18.01% for the traditional method, while 27.48% and 29.24% for the advanced methods, respectively(P < 0.05). In summary, human umbilical cord mesenchymal stem cells can differentiate into the neuron-like cells induced by two methods. Moreover, the advanced method is better than the traditional method in the morphology of the differentiated cells and the positive rate of neurofilament protein and glial fiber acidic protein.
BACKGROUND: Stem cells are a promising treatment for nerve injury diseases due to its ability of differentiating into neuron-like cells. However, the differentiation efficacy is low, so we have make an advance based on the traditional induction method. OBJECTIVE: To explore the potentiality of human umbilical cord mesenchymal stem cells differentiating into neuron-like cells induced by two methods and their merits. METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured using tissue explant method. The cell morphology was observed under microscope, and the cultured cells were identified by flow cytometry. Human umbilical cord mesenchymal stem cells were differentiated into neuron-like cell under the neurotrophic factor inducing scheme. Two different methods but using the same inducers(2% B27, 20 μg/L basic fibroblast growth factor and 20 μg/L epidermal growth factor) were utilized. Traditional method: B27, basic fibroblast growth factor, epidermal growth factor and penicillin streptomycin combination were added in the DMEM/F12 medium, 3 days later all-trans retinoic acid was added, followed by 10 days of culture. Advanced method: B27, basic fibroblast growth factor, epidermal growth factor, penicillin streptomycin combination and fetal bovine serum were added in the DMEM/F12 medium, 6 days later the cells underwent trypsinization and re-seeded in the confocal capsule, B27 and fetal bovine serum were removed next day, and all-trans retinoic acid was added, cultured for 14 days. Then the differentiated nerve cells were identified by immunofluorescence detection of neurofilament protein and glial fibrillary acidic protein. Meanwhile, the positive rate of the neuromarkers in the cells was compared between two methods. RESULTS AND CONCLUSION: Primary human umbilical cord mesenchymal stem cells were isolated and cultured successfully, and highly expressed CD29 and CD105 whereas lowly expressed CD34 and CD45, which were the characteristics of the stem cell. The differentiated cells were presented as nerve cells in the morphology under microscope in both inducing methods. The results of the immunofluorescence indicated that the neurofilament protein and the glial fiber acidic protein in the differentiated cells were all expressed positive in both methods. The positive rates of neurofilament protein and glial fiber acidic protein were 18.73% and 18.01% for the traditional method, while 27.48% and 29.24% for the advanced methods, respectively(P < 0.05). In summary, human umbilical cord mesenchymal stem cells can differentiate into the neuron-like cells induced by two methods. Moreover, the advanced method is better than the traditional method in the morphology of the differentiated cells and the positive rate of neurofilament protein and glial fiber acidic protein.
引文
[1]Pop DM, Sori??u O,?u?man S, et al. Potential of placental-derived human mesenchymal stem cells for osteogenesis and neurogenesis.Rom J Morphol Embryol. 2015;56(3):989-996.
[2]Nitkin CR, Bonfield TL. Concise Review:Mesenchymal Stem Cell Therapy for Pediatric Disease:Perspectives on Success and Potential Improvements. Stem Cells Transl Med. 2017;6(2):539-565.
[3]Nekanti U, Mohanty L, Venugopal P, et al. Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications.Stem Cell Res. 2010;5(3):244-254.
[4]Gao F, Chiu SM, Motan DA, et al. Mesenchymal stem cells and immunomodulation:current status and future prospects. Cell Death Dis.2016;7:e2062.
[5]Du WJ, Chi Y, Yang ZX, et al. Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue,umbilical cord, and placenta. Stem Cell Res Ther. 2016;7(1):163.
[6]Antunes MA, Laffey JG, Pelosi P, et al. Mesenchymal stem cell trials for pulmonary diseases. J Cell Biochem. 2014;115(6):1023-1032.
[7]Latifpour M, Shakiba Y, Amidi F, et al. Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells.Avicenna J Med Biotechnol. 2014;6(4):218-227.
[8]Tao R, Sun TJ, Han YQ, et al. Epimorphin-induced differentiation of human umbilical cord mesenchymal stem cells into sweat gland cells.Eur Rev Med Pharmacol Sci. 2014;18(9):1404-1410.
[9]Li W, Ye B, Cai XY, et al. Differentiation of human umbilical cord mesenchymal stem cells into prostate-like epithelial cells in vivo. PLoS One. 2014;9(7):e102657.
[10] Bai J, Hu Y, Wang YR, et al. Comparison of human amniotic fluid-derived and umbilical cord Wharton's Jelly-derived mesenchymal stromal cells:Characterization and myocardial differentiation capacity. J Geriatr Cardiol.2012;9(2):166-171.
[11] Hu Y, Liang J, Cui H, et al. Wharton's jelly mesenchymal stem cells differentiate into retinal progenitor cells. Neural Regen Res. 2013;8(19):1783-1792.
[12] Hang H, Yu Y, Wu N, et al. Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4αtransduction. PLoS One. 2014;9(8):e104133.
[13] Qu H, Liu X, Ni Y, et al. Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells. J Transl Med. 2014;12:135.
[14] Doan CC, Le TL, Hoang NS, et al. Differentiation of umbilical cord lining membrane-derived mesenchymal stem cells into endothelial-like cells.Iran Biomed J. 2014;18(2):67-75.
[15] Paldino E, Cenciarelli C, Giampaolo A, et al. Induction of dopaminergic neurons from human Wharton's jelly mesenchymal stem cell by forskolin.J Cell Physiol. 2014;229(2):232-244.
[16] Alves da Silva ML, Costa-Pinto AR, Martins A, et al. Conditioned medium as a strategy for human stem cells chondrogenic differentiation. J Tissue Eng Regen Med. 2015;9(6):714-723.
[17] Ali H, Al-Yatama MK, Abu-Farha M, et al. Multi-lineage differentiation of human umbilical cord Wharton's Jelly Mesenchymal Stromal Cells mediates changes in the expression profile of stemness markers. PLoS One. 2015;10(4):e0122465.
[18] Bárcia RN, Santos JM, Filipe M, et al. What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells.Stem Cells Int. 2015;2015:583984.
[19] Hu SL, Luo HS, Li JT, et al. Functional recovery in acute traumatic spinal cord injury after transplantation of human umbilical cord mesenchymal stem cells. Crit Care Med. 2010;38(11):2181-2189.
[20] Pang KM, Sung MA, Alrash-dan MS, et al. Tans-plantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration. Neural Regen Res. 2010;5(11):838-845.
[21] Cheng H, Liu X, Hua R, et al. Clinical observation of umbilical cord mesenchymal stem cell transplantation in treatment for sequelae of thoracolumbar spinal cord injury. J Transl Med. 2014;12:253.
[22] Chen J, Venkat P, Zacharek A, et al. Neurorestorative therapy for stroke.Front Hum Neurosci. 2014;8:382.
[23]张飞,王一雄,武忠炎,等.人脐带间充质干细胞生物特性比较:胰酶冷消化和组织块法体外培养[J].中国组织工程研究, 2014,18(41):6614-6619.
[24] McElreavey KD, Irvine AI, Ennis KT, et al. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord. Biochem Soc Trans. 1991;19(1):29S.
[25]王武,张飞,李贵才,等.以Ed U体外标记人脐带间充质干细胞:5,10μmol/L是其最适浓度[J].中国组织工程研究, 2015,19(32):5167-5171.
[26] Ianus A, Holz GG, Theise ND, et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest. 2003;111(6):843-850.
[27] Ra JC, Shin IS, Kim SH, et al. Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans.Stem Cells Dev. 2011;20(8):1297-1308.
[28] Nazarov I, Lee JW, Soupene E, et al. Multipotent stromal stem cells from human placenta demonstrate high therapeutic potential. Stem Cells Transl Med. 2012;1(5):359-372.
[29] Rohban R, Pieber TR. Mesenchymal Stem and Progenitor Cells in Regeneration:Tissue Specificity and Regenerative Potential. Stem Cells Int. 2017;2017:5173732.
[30] Guan LX, Guan H, Li HB, et al. Therapeutic efficacy of umbilical cord-derived mesenchymal stem cells in patients with type 2 diabetes.Exp Ther Med. 2015;9(5):1623-1630.
[31] Zhao XF, Xu Y, Zhu ZY, et al. Clinical observation of umbilical cord mesenchymal stem cell treatment of severe systolic heart failure. Genet Mol Res. 2015;14(2):3010-3017.
[32] D'souza N, Rossignoli F, Golinelli G, et al. Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies. BMC Med. 2015;13:186.
[33] Hendijani F, Sadeghi-Aliabadi H, Haghjooy Javanmard S. Comparison of human mesenchymal stem cells isolated by explant culture method from entire umbilical cord and Wharton's jelly matrix. Cell Tissue Bank.2014;15(4):555-565.
[34] Zhou X, Gu J, Gu Y, et al. Human Umbilical Cord-Derived Mesenchymal Stem Cells Improve Learning and Memory Function in Hypoxic-Ischemic Brain-Damaged Rats via an IL-8-Mediated Secretion Mechanism Rather than Differentiation Pattern Induction. Cell Physiol Biochem.2015;35(6):2383-2401.
[35]álvarez D, Levine M, Rojas M. Regenerative medicine in the treatment of idiopathic pulmonary fibrosis:current position. Stem Cells Cloning.2015;8:61-65.
[36] Rismanchi N, Floyd CL, Berman RF, et al. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells:a comparison of protocols. Brain Res. 2003;991(1-2):46-55.
[37]孙丽,于丽,张华芳,等.人脐带间充质干细胞的富集及向神经细胞的诱导分化[J].神经解剖杂志,2011,27(2):185-190.
[38]郜元军,钱伟,汪涉海,等.大鼠骨髓基质干细胞诱导分化为肠神经细胞的体外研究[J].中华消化杂志,2006,26(10):661-665.
[2]Nitkin CR, Bonfield TL. Concise Review:Mesenchymal Stem Cell Therapy for Pediatric Disease:Perspectives on Success and Potential Improvements. Stem Cells Transl Med. 2017;6(2):539-565.
[3]Nekanti U, Mohanty L, Venugopal P, et al. Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications.Stem Cell Res. 2010;5(3):244-254.
[4]Gao F, Chiu SM, Motan DA, et al. Mesenchymal stem cells and immunomodulation:current status and future prospects. Cell Death Dis.2016;7:e2062.
[5]Du WJ, Chi Y, Yang ZX, et al. Heterogeneity of proangiogenic features in mesenchymal stem cells derived from bone marrow, adipose tissue,umbilical cord, and placenta. Stem Cell Res Ther. 2016;7(1):163.
[6]Antunes MA, Laffey JG, Pelosi P, et al. Mesenchymal stem cell trials for pulmonary diseases. J Cell Biochem. 2014;115(6):1023-1032.
[7]Latifpour M, Shakiba Y, Amidi F, et al. Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells.Avicenna J Med Biotechnol. 2014;6(4):218-227.
[8]Tao R, Sun TJ, Han YQ, et al. Epimorphin-induced differentiation of human umbilical cord mesenchymal stem cells into sweat gland cells.Eur Rev Med Pharmacol Sci. 2014;18(9):1404-1410.
[9]Li W, Ye B, Cai XY, et al. Differentiation of human umbilical cord mesenchymal stem cells into prostate-like epithelial cells in vivo. PLoS One. 2014;9(7):e102657.
[10] Bai J, Hu Y, Wang YR, et al. Comparison of human amniotic fluid-derived and umbilical cord Wharton's Jelly-derived mesenchymal stromal cells:Characterization and myocardial differentiation capacity. J Geriatr Cardiol.2012;9(2):166-171.
[11] Hu Y, Liang J, Cui H, et al. Wharton's jelly mesenchymal stem cells differentiate into retinal progenitor cells. Neural Regen Res. 2013;8(19):1783-1792.
[12] Hang H, Yu Y, Wu N, et al. Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4αtransduction. PLoS One. 2014;9(8):e104133.
[13] Qu H, Liu X, Ni Y, et al. Laminin 411 acts as a potent inducer of umbilical cord mesenchymal stem cell differentiation into insulin-producing cells. J Transl Med. 2014;12:135.
[14] Doan CC, Le TL, Hoang NS, et al. Differentiation of umbilical cord lining membrane-derived mesenchymal stem cells into endothelial-like cells.Iran Biomed J. 2014;18(2):67-75.
[15] Paldino E, Cenciarelli C, Giampaolo A, et al. Induction of dopaminergic neurons from human Wharton's jelly mesenchymal stem cell by forskolin.J Cell Physiol. 2014;229(2):232-244.
[16] Alves da Silva ML, Costa-Pinto AR, Martins A, et al. Conditioned medium as a strategy for human stem cells chondrogenic differentiation. J Tissue Eng Regen Med. 2015;9(6):714-723.
[17] Ali H, Al-Yatama MK, Abu-Farha M, et al. Multi-lineage differentiation of human umbilical cord Wharton's Jelly Mesenchymal Stromal Cells mediates changes in the expression profile of stemness markers. PLoS One. 2015;10(4):e0122465.
[18] Bárcia RN, Santos JM, Filipe M, et al. What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells.Stem Cells Int. 2015;2015:583984.
[19] Hu SL, Luo HS, Li JT, et al. Functional recovery in acute traumatic spinal cord injury after transplantation of human umbilical cord mesenchymal stem cells. Crit Care Med. 2010;38(11):2181-2189.
[20] Pang KM, Sung MA, Alrash-dan MS, et al. Tans-plantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration. Neural Regen Res. 2010;5(11):838-845.
[21] Cheng H, Liu X, Hua R, et al. Clinical observation of umbilical cord mesenchymal stem cell transplantation in treatment for sequelae of thoracolumbar spinal cord injury. J Transl Med. 2014;12:253.
[22] Chen J, Venkat P, Zacharek A, et al. Neurorestorative therapy for stroke.Front Hum Neurosci. 2014;8:382.
[23]张飞,王一雄,武忠炎,等.人脐带间充质干细胞生物特性比较:胰酶冷消化和组织块法体外培养[J].中国组织工程研究, 2014,18(41):6614-6619.
[24] McElreavey KD, Irvine AI, Ennis KT, et al. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord. Biochem Soc Trans. 1991;19(1):29S.
[25]王武,张飞,李贵才,等.以Ed U体外标记人脐带间充质干细胞:5,10μmol/L是其最适浓度[J].中国组织工程研究, 2015,19(32):5167-5171.
[26] Ianus A, Holz GG, Theise ND, et al. In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest. 2003;111(6):843-850.
[27] Ra JC, Shin IS, Kim SH, et al. Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans.Stem Cells Dev. 2011;20(8):1297-1308.
[28] Nazarov I, Lee JW, Soupene E, et al. Multipotent stromal stem cells from human placenta demonstrate high therapeutic potential. Stem Cells Transl Med. 2012;1(5):359-372.
[29] Rohban R, Pieber TR. Mesenchymal Stem and Progenitor Cells in Regeneration:Tissue Specificity and Regenerative Potential. Stem Cells Int. 2017;2017:5173732.
[30] Guan LX, Guan H, Li HB, et al. Therapeutic efficacy of umbilical cord-derived mesenchymal stem cells in patients with type 2 diabetes.Exp Ther Med. 2015;9(5):1623-1630.
[31] Zhao XF, Xu Y, Zhu ZY, et al. Clinical observation of umbilical cord mesenchymal stem cell treatment of severe systolic heart failure. Genet Mol Res. 2015;14(2):3010-3017.
[32] D'souza N, Rossignoli F, Golinelli G, et al. Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies. BMC Med. 2015;13:186.
[33] Hendijani F, Sadeghi-Aliabadi H, Haghjooy Javanmard S. Comparison of human mesenchymal stem cells isolated by explant culture method from entire umbilical cord and Wharton's jelly matrix. Cell Tissue Bank.2014;15(4):555-565.
[34] Zhou X, Gu J, Gu Y, et al. Human Umbilical Cord-Derived Mesenchymal Stem Cells Improve Learning and Memory Function in Hypoxic-Ischemic Brain-Damaged Rats via an IL-8-Mediated Secretion Mechanism Rather than Differentiation Pattern Induction. Cell Physiol Biochem.2015;35(6):2383-2401.
[35]álvarez D, Levine M, Rojas M. Regenerative medicine in the treatment of idiopathic pulmonary fibrosis:current position. Stem Cells Cloning.2015;8:61-65.
[36] Rismanchi N, Floyd CL, Berman RF, et al. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells:a comparison of protocols. Brain Res. 2003;991(1-2):46-55.
[37]孙丽,于丽,张华芳,等.人脐带间充质干细胞的富集及向神经细胞的诱导分化[J].神经解剖杂志,2011,27(2):185-190.
[38]郜元军,钱伟,汪涉海,等.大鼠骨髓基质干细胞诱导分化为肠神经细胞的体外研究[J].中华消化杂志,2006,26(10):661-665.