人胎盘间充质干细胞的扩增与鉴定
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摘要
背景:作为组织工程的种子细胞,胎盘间充质干细胞因具有多向分化、低免疫原性、免疫调节等作用,被广泛用于临床疾病的治疗及临床科研中。目的:根据间充质干细胞的定义来鉴定从宁夏医科大学总医院干细胞研究所获得的人胎盘间充质干细胞,为后续的实验提供可靠的保障。方法:培养从宁夏医科大学总医院干细胞研究所获得的人胎盘间充质干细胞,观察其在塑料培养皿或培养瓶内的贴壁情况,并间断观察细胞形态发生的变化。将培养的第3代人胎盘间充质干细胞通过流式细胞技术检测细胞表面特异性抗原,并且用成脂、成骨、成软骨诱导培养基分别诱导21 d后,采用油红O、茜素红、甲苯胺蓝进行细胞染色观察人胎盘间充质干细胞向脂肪、骨,软骨细胞的分化能力。实验方案得到宁夏医科大学总医院医学科研伦理审查委员会批准(编号:2018-047)。结果与结论:(1)经过体外培养的第3代人胎盘间充质干细胞呈漩涡状贴壁生长,单个细胞呈梭状,形似纤维细胞;(2)人胎盘间充质干细胞具有特殊的细胞免疫学表型:流式细胞术检测CD105、CD90、CD73呈强阳性表达;造血标记物CD34、CD45、CD14以及HLA-DR呈阴性表达;(3)人胎盘间充质干细胞在成脂、成骨、成软骨诱导培养21 d后,油红O染色、茜素红染色及甲苯胺蓝染色均呈阳性。提示:根据间充质干细胞的定义,来自宁夏医科大学总医院干细胞研究所的人胎盘间充质干细胞符合间充质干细胞的特性,为后续实验提供良好保证。
BACKGROUND: As a seed cell for tissue engineering, placental mesenchymal stem cells are widely used in the treatment of clinical diseases and clinical research due to their multi-directional differentiation, low immunogenicity, and immunomodulation. OBJECTIVE: To identify human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital according to the definition of mesenchymal stem cells, and to provide reliable protection for subsequent experiments. METHODS: The human placental mesenchymal stem cells were obtained from the Stem Cell Research Institute of Ningxia Medical University General Hospital. Cells attached to plastic culture dishes or culture flasks were detected, and cell morphology was observed intermittently. The cultured third-generation human placental mesenchymal stem cells were tested for cell surface specific antigen by flow cytometry, and were cultured in adipogenic, osteogenic and chondrogenic induction media, respectively. After 21 days of induction, Oil red O, alizarin red and toluidine blue were used for cell staining to observe the differentiation ability of human placental mesenchymal stem cells into fat, bone and chondrocytes. The study protocol was approved by the Ethics Committee of Ningxia Medical University General Hospital(approval No. 2018-047). RESULTS AND CONCLUSION: The third-generation human placental mesenchymal stem cells cultured in vitro showed a vortex-like adherent growth. The single cell was fusiform with fibroblast-like appearance. Human placental mesenchymal stem cells had a special cellular immunological phenotype: CD105, CD90, and CD73 were positively expressed, and CD34, CD45, CD14 and HLA-DR were negatively expressed. The human placental mesenchymal stem cells were induced to differentiate in vitro after 21 days, and were positive for alizarin red, oil red O, and toluidine blue staining. As per the definition of mesenchymal stem cells, human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital meet the characteristics of mesenchymal stem cells, providing good guarantee for further experiments.
BACKGROUND: As a seed cell for tissue engineering, placental mesenchymal stem cells are widely used in the treatment of clinical diseases and clinical research due to their multi-directional differentiation, low immunogenicity, and immunomodulation. OBJECTIVE: To identify human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital according to the definition of mesenchymal stem cells, and to provide reliable protection for subsequent experiments. METHODS: The human placental mesenchymal stem cells were obtained from the Stem Cell Research Institute of Ningxia Medical University General Hospital. Cells attached to plastic culture dishes or culture flasks were detected, and cell morphology was observed intermittently. The cultured third-generation human placental mesenchymal stem cells were tested for cell surface specific antigen by flow cytometry, and were cultured in adipogenic, osteogenic and chondrogenic induction media, respectively. After 21 days of induction, Oil red O, alizarin red and toluidine blue were used for cell staining to observe the differentiation ability of human placental mesenchymal stem cells into fat, bone and chondrocytes. The study protocol was approved by the Ethics Committee of Ningxia Medical University General Hospital(approval No. 2018-047). RESULTS AND CONCLUSION: The third-generation human placental mesenchymal stem cells cultured in vitro showed a vortex-like adherent growth. The single cell was fusiform with fibroblast-like appearance. Human placental mesenchymal stem cells had a special cellular immunological phenotype: CD105, CD90, and CD73 were positively expressed, and CD34, CD45, CD14 and HLA-DR were negatively expressed. The human placental mesenchymal stem cells were induced to differentiate in vitro after 21 days, and were positive for alizarin red, oil red O, and toluidine blue staining. As per the definition of mesenchymal stem cells, human placental mesenchymal stem cells from the Stem Cell Research Institute of Ningxia Medical University General Hospital meet the characteristics of mesenchymal stem cells, providing good guarantee for further experiments.
引文
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[14]Jo CH,Lim HJ,Yoon KS.Characterization of Tendon-Specific Markers in Various Human Tissues,Tenocytes and Mesenchymal Stem Cells.Tissue Eng Regen Med.2019;16(2):151-159.
[15]Wang Q,Yang Q,Wang Z,et al.Comparative analysis of human mesenchymal stem cells from fetal-bone marrow,adipose tissue,and Warton''s jelly as sources of cell immunomodulatory therapy.Hum Vaccin Immunother.2016;12(1):85-96.
[16]Le Blanc K,Gtherstrm C,Ringdn O,et al.Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta.Transplantation.2005;79(11):1607-1614.
[17]Murabayashi D,Mochizuki M,Tamaki Y,et al.Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia:implications for regenerative medicine.Human Cell.2017;30(3):169-180.
[18]Gentile P,Scioli MG,Bielli A,et al.Stem cells from human hair follicles:first mechanical isolation for immediate autologous clinical use in androgenetic alopecia and hair loss[J].Stem Cell Investigation,2017,4(7):58-58.
[19]Khosravi M,Azarpira N,Shamdani S,et al.Differentiation of umbilical cord derived mesenchymal stem cells to hepatocyte cells by transfection of miR-106a,miR-574-3p,and miR-451.Gene.2018;667:1-9.
[20]Lu Y,Wang Z,Chen L,et al.The in vitro differentiation of GDNF gene-engineered amniotic fluid-derived stem cells into renal tubular epithelial-like cells.Stem Cells Dev.2018;27(9):590-599.
[21]Brooke G,Rossetti T,Pelekanos R,et al.Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials.Br J Haematol.2009;144(4):571-579.
[22]Tran TC,Kimura K,Nagano M,et al.Identification of human placenta-derived mesenchymal stem cells involved in re-endothelialization.J Cell Physiol.2011;226(1):224-235.
[23]Semenov OV,Koestenbauer S,Riegel M,et al.Multipotent mesenchymal stem cells from human placenta:critical parameters for isolation and maintenance of stemness after isolation.Am J Obstet Gynecol.2010;202(2):193.e1-193.e13.
[24]Katsiani E,Garas A,Skentou C,et al.Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing.Cell Tissue Bank.2016;17(3):517-529.
[25]易桥,卢燕勤,黄宏宇,等.间充质干细胞在免疫调节过程中的作用与应用进展[J].中国组织工程研究,2016,20(41):6216-6224.
[26]Li Y,Li Y,Li H,et al.Placenta derived mesenchymal stem cells improve airway hyperresponsiveness and inflammation in asthmatic rats by modulating the Th17/Treg balance.Molecular Medicine Reports.2017;16(6):8137-8145.
[27]González PL,Carvajal C,Cuenca J,et al.Chorion Mesenchymal Stem Cells Show Superior Differentiation,Immunosuppressive,and Angiogenic Potentials in Comparison With Haploidentical Maternal Placental Cells.Stem Cells Transl Med.2015;4(10):1109-1121.
[28]Zhang Y,Zhang W,Wang H,et al.miR-21 Contributes to Human Amniotic Membrane-Derived Mesenchymal Stem Cell Growth and Human Amniotic Membrane-Derived Mesenchymal Stem Cell-Induced Immunoregulation.Genet Test Mol Biomarkers.2018;22(12):665-673..
[29]施剑明,邬亚华,耿书国,等.间充质干细胞增殖、衰老及分化:Wnt信号通路经典与非经典的调控作用[J].中国组织工程研究,2014,18(41):6719-6724.
[30]章毅,许惠利,王锋,等.不同代次人胎盘间充质干细胞的生物活性比较研究[J].中国医药生物技术,2016,11(5):400-406.
[31]白金萍,李秀英,李雪,等.胎盘间充质干细胞传代后的增殖能力[J].中国组织工程研究,2014,18(10:1591-1596.
[32]Liu Y,Goldberg AJ,Dennis JE,et al.One-step derivation of mesenchymal stem cell(MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating.PLoS One.2012;7(3):e33225.
[33]Chen YS,Pelekanos RA,Ellis RL,et al.Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells.Stem Cells Transl Med.2012;1(2):83-95.
[34]Ziadlou R,Shahhoseini M,Safari F,et al.Comparative analysis of neural differentiation potential in human mesenchymal stem cells derived from chorion and adult bone marrow.Cell Tissue Res.2015;362(2):367-377.
[2]Pham H,Tonai R,Wu M,et al.CD73,CD90,CD105 and Cadherin-11 RT-PCR Screening for Mesenchymal Stem Cells from Cryopreserved Human Cord Tissue.Int J Stem Cells.2018;11(1):26-38.
[3]Huang YC,Yang ZM,Chen XH,et al.Isolation of mesenchymal stem cells from human placental decidua basalis and resistance to hypoxia and serum deprivation.Stem Cell Rev.2009;5(3):247-255.
[4]Barlow S,Brooke G,Chatterjee K,et al.Comparison of human placenta-and bone marrow-derived multipotent mesenchymal stem cells.Stem Cells Dev.2008;17(6):1095-1107.
[5]Antoniadou E,David AL.Placental stem cells.Best Pract Res Clin Obstet Gynaecol.2016;31:13-29.
[6]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.Cytotherapy.2006;8(4):315-317.
[7]Sousa BR,Parreira RC,Fonseca EA,et al.Human adult stem cells from diverse origins:An overview from multiparametric immunophenotyping to clinical applications.Cytometry A.2014;85(1):43-77.
[8]Kobolak J,Dinnyes A,Memic A,et al.Mesenchymal stem cells:Identification,phenotypic characterization,biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche.Methods.2016;99:62-68.
[9]Bharti D,Shivakumar SB,Park JK,et al.Comparative analysis of human Wharton’s jelly mesenchymal stem cells derived from different parts of the same umbilical cord.Cell Tissue Res.2018;372(1):51-65.
[10]朱永朝,马海滨,刘婷,等.干扰素-γ对人胎盘胎儿来源间充质干细胞免疫调节功能的影响[J].细胞与分子免疫学杂志,2012,28(10):1058-1061.
[11]Xu M,Liu G,Jia Y,et al.Transplantation of human placenta mesenchymal stem cells reduces the level of inflammatory factors in lung tissues of mice with acute lung injury.Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi.2018;34(2):105-109.
[12]Horn P,Bork S,Wagner W.Standardized Isolation of Human Mesenchymal Stromal Cells with Red Blood Cell Lysis.Methods Mol Biol.2011;698:23-35.
[13]Tuan RS,Boland G,Tuli R.Adult mesenchymal stem cells and cell-based tissue engineering.Arthritis Res Ther.2003;5(1):32-45.
[14]Jo CH,Lim HJ,Yoon KS.Characterization of Tendon-Specific Markers in Various Human Tissues,Tenocytes and Mesenchymal Stem Cells.Tissue Eng Regen Med.2019;16(2):151-159.
[15]Wang Q,Yang Q,Wang Z,et al.Comparative analysis of human mesenchymal stem cells from fetal-bone marrow,adipose tissue,and Warton''s jelly as sources of cell immunomodulatory therapy.Hum Vaccin Immunother.2016;12(1):85-96.
[16]Le Blanc K,Gtherstrm C,Ringdn O,et al.Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta.Transplantation.2005;79(11):1607-1614.
[17]Murabayashi D,Mochizuki M,Tamaki Y,et al.Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia:implications for regenerative medicine.Human Cell.2017;30(3):169-180.
[18]Gentile P,Scioli MG,Bielli A,et al.Stem cells from human hair follicles:first mechanical isolation for immediate autologous clinical use in androgenetic alopecia and hair loss[J].Stem Cell Investigation,2017,4(7):58-58.
[19]Khosravi M,Azarpira N,Shamdani S,et al.Differentiation of umbilical cord derived mesenchymal stem cells to hepatocyte cells by transfection of miR-106a,miR-574-3p,and miR-451.Gene.2018;667:1-9.
[20]Lu Y,Wang Z,Chen L,et al.The in vitro differentiation of GDNF gene-engineered amniotic fluid-derived stem cells into renal tubular epithelial-like cells.Stem Cells Dev.2018;27(9):590-599.
[21]Brooke G,Rossetti T,Pelekanos R,et al.Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials.Br J Haematol.2009;144(4):571-579.
[22]Tran TC,Kimura K,Nagano M,et al.Identification of human placenta-derived mesenchymal stem cells involved in re-endothelialization.J Cell Physiol.2011;226(1):224-235.
[23]Semenov OV,Koestenbauer S,Riegel M,et al.Multipotent mesenchymal stem cells from human placenta:critical parameters for isolation and maintenance of stemness after isolation.Am J Obstet Gynecol.2010;202(2):193.e1-193.e13.
[24]Katsiani E,Garas A,Skentou C,et al.Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing.Cell Tissue Bank.2016;17(3):517-529.
[25]易桥,卢燕勤,黄宏宇,等.间充质干细胞在免疫调节过程中的作用与应用进展[J].中国组织工程研究,2016,20(41):6216-6224.
[26]Li Y,Li Y,Li H,et al.Placenta derived mesenchymal stem cells improve airway hyperresponsiveness and inflammation in asthmatic rats by modulating the Th17/Treg balance.Molecular Medicine Reports.2017;16(6):8137-8145.
[27]González PL,Carvajal C,Cuenca J,et al.Chorion Mesenchymal Stem Cells Show Superior Differentiation,Immunosuppressive,and Angiogenic Potentials in Comparison With Haploidentical Maternal Placental Cells.Stem Cells Transl Med.2015;4(10):1109-1121.
[28]Zhang Y,Zhang W,Wang H,et al.miR-21 Contributes to Human Amniotic Membrane-Derived Mesenchymal Stem Cell Growth and Human Amniotic Membrane-Derived Mesenchymal Stem Cell-Induced Immunoregulation.Genet Test Mol Biomarkers.2018;22(12):665-673..
[29]施剑明,邬亚华,耿书国,等.间充质干细胞增殖、衰老及分化:Wnt信号通路经典与非经典的调控作用[J].中国组织工程研究,2014,18(41):6719-6724.
[30]章毅,许惠利,王锋,等.不同代次人胎盘间充质干细胞的生物活性比较研究[J].中国医药生物技术,2016,11(5):400-406.
[31]白金萍,李秀英,李雪,等.胎盘间充质干细胞传代后的增殖能力[J].中国组织工程研究,2014,18(10:1591-1596.
[32]Liu Y,Goldberg AJ,Dennis JE,et al.One-step derivation of mesenchymal stem cell(MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating.PLoS One.2012;7(3):e33225.
[33]Chen YS,Pelekanos RA,Ellis RL,et al.Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells.Stem Cells Transl Med.2012;1(2):83-95.
[34]Ziadlou R,Shahhoseini M,Safari F,et al.Comparative analysis of neural differentiation potential in human mesenchymal stem cells derived from chorion and adult bone marrow.Cell Tissue Res.2015;362(2):367-377.