长期传代培养对人脐带间充质干细胞生物学特性的影响
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摘要
该课题研究长期体外传代培养对人脐带间充质干细胞重要生物学属性的影响。采用组织块法分离人脐带间充质干细胞,体外反复传代培养后比较第5、10、20代细胞的主要生物学属性:采用细胞计数法与流式细胞仪检测不同代次细胞增殖活性和表面免疫标志物;染色体常规核型分析和微阵列分析检测其基因稳定性;成脂、成骨诱导分化检测其多向分化潜能;定量RT-PCR检测端粒酶反转录基因hTERT的表达; SA-β-gal活性确定细胞老化状态。第5、10、20代脐带间充质干细胞呈现相同的增殖曲线,各代次干细胞的增殖速率无显著性差异。不同培养代次的脐带间充质干细胞表面均呈现CD105、CD90、CD44、CD73高表达, CD19、CD34、CD45及HLA-DR不表达或低表达。基因稳定性分析证实三个代次干细胞均为正常二倍体核型,染色体基因组未发生基因拷贝数缺失、重复和大片段纯合子现象。成脂和成骨分化潜能以及hTERT表达均未见显著性差异。SA-β-gal活性检测显示,随培养代次的增加,脐带间充质干细胞开始呈现衰老征象,尤以第20代明显。体外反复传代长期培养对人脐带间充质干细胞的基本生物学属性、基因稳定性及其多向分化潜能均无显著性影响。过度长期培养有可能导致干细胞老化,活性降低。
The purpose of this study is to evaluate the effects of long-term in vitro expansion on the stability and functions of human umbilical cord mesenchymal stem cells(hUC-MSCs).The hUC-MSCs were isolated from Wharton's jelly and in vitro cultured till passage 20.The hUC-MSCs at passage 5,10,20 were selected to compare their proliferation capacity,immunophenotype,genomic stability,multi-differentiation potency,telomerase activity and senescence.Proliferation capacity of the hUC-MSCs at passage 5,10,20 remained no significant change.Longterm expansion of hUC-MSCs had no effect on their immunophenotype determined by expression levels of the cell surface markers,karyotype and chromosomal microarray analysis showed genomic stability,and h TERT expression showed similar telomerase activity.Furthermore,the multi-differentiation potency of the hUC-MSCs still remained strong after long-term culture,demonstrated by adipogenic and osteogenic induction.However,an increase of senescence-associated β-galactosidase activity was observed over time.All the major biological characters and functions of hUC-MSCs remain unchanged after long-term in vitro expansion.However,hUC-MSCs cultured over 10 passages may have a reduced cell activity due to increasing senescence,therefore,hUC-MSCs cultured less than 10 passages are suitable for clinical research/therapy.
The purpose of this study is to evaluate the effects of long-term in vitro expansion on the stability and functions of human umbilical cord mesenchymal stem cells(hUC-MSCs).The hUC-MSCs were isolated from Wharton's jelly and in vitro cultured till passage 20.The hUC-MSCs at passage 5,10,20 were selected to compare their proliferation capacity,immunophenotype,genomic stability,multi-differentiation potency,telomerase activity and senescence.Proliferation capacity of the hUC-MSCs at passage 5,10,20 remained no significant change.Longterm expansion of hUC-MSCs had no effect on their immunophenotype determined by expression levels of the cell surface markers,karyotype and chromosomal microarray analysis showed genomic stability,and h TERT expression showed similar telomerase activity.Furthermore,the multi-differentiation potency of the hUC-MSCs still remained strong after long-term culture,demonstrated by adipogenic and osteogenic induction.However,an increase of senescence-associated β-galactosidase activity was observed over time.All the major biological characters and functions of hUC-MSCs remain unchanged after long-term in vitro expansion.However,hUC-MSCs cultured over 10 passages may have a reduced cell activity due to increasing senescence,therefore,hUC-MSCs cultured less than 10 passages are suitable for clinical research/therapy.
引文
1 Spees JL,Lee RH,Gregory CA.Mechanisms of mesenchymal stem/stromal cell function.Stem Cell Res Ther 2016;7(1):125.
2 Ding DC,Chang YH,Shyu WC,Lin SZ.Human umbilical cord mesenchymal stem cells:a new era for stem cell therapy.Cell Transplant 2015;24(3):339-47.
3 Arutyunyan I,Elchaninov A,Makarov A,Fatkhudinov T.Umbilical cord as prospective source for mesenchymal stem cellbased therapy.Stem Cells Int 2016;2016:6901286.
4 Todeschi MR,El Backly R,Capelli C,Daga A,Patrone E,Introna M,et al.Transplanted umbilical cord mesenchyma stem cells modify the in vivo microenvironment enhancing angiogenesis and leading to bone regeneration.Stem Cells Dev 2015;24(13):1570-81.
5 Lamm N,Ben-David U,Golan-Lev T,StorchováZ,Benvenisty N,Kerem B.Genomic instability in human pluripotent stem cells arises from replicative stress and chromosome condensation defects.Cell Stem Cell 2016;18(2):253-61.
6 Kim JS,Park YS,Kim JY,Kim YG,Kim YJ,Lee HK,et al.Inhibition of human pancreatic tumor growth by cytokine-induced killer cells in nude mouse xenograft model.Immune Netw2012;12(6):247-52.
7 Pan QZ,Pan K,Wang QJ,Weng DS,Zhao JJ,Zheng HX,et al.Annexin A3 as a potential target for immunotherapy of liver cancer stem-like cells.Stem Cells 2015;33(2):354-66.
8姚惟琦,武栋成.一种人脐带间充质干细胞分离培养方法.中国(Yao Weiqi,Wu Dongcheng.A method for isolation of human umbilical cord mesenchymal stem cell.China)201110261543.5[P].2016-12-14.
9 Li S,Wang Y,Guan L,Ji M.Characteristics of human umbilical cord mesenchymal stem cells during ex vivo expansion.Mol Med Rep 2015;12(3):4320-5.
10张立新,陈亚宝,叶军,李林,林梅,黄俊星,等.脐带间充质干细胞染色体核型制备及安全评估.海南医学(Li Xin,Chen Yabo,YeJun,Li Lin,Lin Mei,Huang Junxing,et al.Preparation and safety assessment of the chromosome karyotype of umbilical mesenchymal stem cells.Hainan Medical Journal)2012;23(13):11-2.
11 Bao C,Chen W,Weir MD,Thein-Han W,Xu HH.Effects of electrospun submicron fibers in calcium phosphate cement scaffold on mechanical properties and osteogenic differentiation of umbilical cord stem cells.Acta Biomater 2011;7(11):4037-44.
12 Bièche I1,Noguès C,Paradis V,Olivi M,Bedossa P,Lidereau R,et al.Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay.Clin Cancer Res 2000;6(2):452-9.
13 Dimri GP,Lee X,Basile G,Acosta M,Scott G,Roskelley C,et al.A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc Natl Acad Sci USA 1995;92(20):9363-7.
14 Dominici M,Le Blanc K,Mueller I,Slaper-Cortenbach I,Marini F,Krause D,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.Cytotherapy 2006;8(4):315-7.
15 Grade M,Difilippantonio MJ,Camps J.Patterns of chromosomal aberrations in solid tumors.Recent Results Cancer Res2015;200:115-42.
16 Kim DW,Staples M,Shinozuka K,Pantcheva P,Kang SD,Borlongan CV.Wharton’s Jelly-derived mesenchymal stem cells:phenotypic characterization and optimizing their therapeutic potential for clinical applications.Int J Mol Sci 2013;14(6):11692-712.
17 Wang Y,Zhang Z,Chi Y,Zhang Q,Xu F,Yang Z,et al.Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation.Cell Death Dis 2013;4:e950.
18 De Witte SFH,Lambert EE,Merino A,Strini T,Douben HJCW,O’Flynn L,et al.Aging of bone marrow-and umbilical cord-derived mesenchymal stromal cells during expansion.Cytotherapy 2017;19(7):798-807.
19王有为.脐带间充质干细胞临床前安全性研究(博士论文).北京协和医学院(Wang Youwei.Doctoral dissertation of preclinical safety study of umbilical cord mesenchymal stem cells.Peking Union Medical College),2013.
20 Counter CM,Meyerson M,Eaton EN,Ellisen LW,Caddle SD,Haber DA,et al.Telomerase activity is restored in human cells by ectopic expression of hTERT,the catalytic subunit of telomerase.Oncogene 1998;16(9):1217-22.
2 Ding DC,Chang YH,Shyu WC,Lin SZ.Human umbilical cord mesenchymal stem cells:a new era for stem cell therapy.Cell Transplant 2015;24(3):339-47.
3 Arutyunyan I,Elchaninov A,Makarov A,Fatkhudinov T.Umbilical cord as prospective source for mesenchymal stem cellbased therapy.Stem Cells Int 2016;2016:6901286.
4 Todeschi MR,El Backly R,Capelli C,Daga A,Patrone E,Introna M,et al.Transplanted umbilical cord mesenchyma stem cells modify the in vivo microenvironment enhancing angiogenesis and leading to bone regeneration.Stem Cells Dev 2015;24(13):1570-81.
5 Lamm N,Ben-David U,Golan-Lev T,StorchováZ,Benvenisty N,Kerem B.Genomic instability in human pluripotent stem cells arises from replicative stress and chromosome condensation defects.Cell Stem Cell 2016;18(2):253-61.
6 Kim JS,Park YS,Kim JY,Kim YG,Kim YJ,Lee HK,et al.Inhibition of human pancreatic tumor growth by cytokine-induced killer cells in nude mouse xenograft model.Immune Netw2012;12(6):247-52.
7 Pan QZ,Pan K,Wang QJ,Weng DS,Zhao JJ,Zheng HX,et al.Annexin A3 as a potential target for immunotherapy of liver cancer stem-like cells.Stem Cells 2015;33(2):354-66.
8姚惟琦,武栋成.一种人脐带间充质干细胞分离培养方法.中国(Yao Weiqi,Wu Dongcheng.A method for isolation of human umbilical cord mesenchymal stem cell.China)201110261543.5[P].2016-12-14.
9 Li S,Wang Y,Guan L,Ji M.Characteristics of human umbilical cord mesenchymal stem cells during ex vivo expansion.Mol Med Rep 2015;12(3):4320-5.
10张立新,陈亚宝,叶军,李林,林梅,黄俊星,等.脐带间充质干细胞染色体核型制备及安全评估.海南医学(Li Xin,Chen Yabo,YeJun,Li Lin,Lin Mei,Huang Junxing,et al.Preparation and safety assessment of the chromosome karyotype of umbilical mesenchymal stem cells.Hainan Medical Journal)2012;23(13):11-2.
11 Bao C,Chen W,Weir MD,Thein-Han W,Xu HH.Effects of electrospun submicron fibers in calcium phosphate cement scaffold on mechanical properties and osteogenic differentiation of umbilical cord stem cells.Acta Biomater 2011;7(11):4037-44.
12 Bièche I1,Noguès C,Paradis V,Olivi M,Bedossa P,Lidereau R,et al.Quantitation of hTERT gene expression in sporadic breast tumors with a real-time reverse transcription-polymerase chain reaction assay.Clin Cancer Res 2000;6(2):452-9.
13 Dimri GP,Lee X,Basile G,Acosta M,Scott G,Roskelley C,et al.A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc Natl Acad Sci USA 1995;92(20):9363-7.
14 Dominici M,Le Blanc K,Mueller I,Slaper-Cortenbach I,Marini F,Krause D,et al.Minimal criteria for defining multipotent mesenchymal stromal cells.Cytotherapy 2006;8(4):315-7.
15 Grade M,Difilippantonio MJ,Camps J.Patterns of chromosomal aberrations in solid tumors.Recent Results Cancer Res2015;200:115-42.
16 Kim DW,Staples M,Shinozuka K,Pantcheva P,Kang SD,Borlongan CV.Wharton’s Jelly-derived mesenchymal stem cells:phenotypic characterization and optimizing their therapeutic potential for clinical applications.Int J Mol Sci 2013;14(6):11692-712.
17 Wang Y,Zhang Z,Chi Y,Zhang Q,Xu F,Yang Z,et al.Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation.Cell Death Dis 2013;4:e950.
18 De Witte SFH,Lambert EE,Merino A,Strini T,Douben HJCW,O’Flynn L,et al.Aging of bone marrow-and umbilical cord-derived mesenchymal stromal cells during expansion.Cytotherapy 2017;19(7):798-807.
19王有为.脐带间充质干细胞临床前安全性研究(博士论文).北京协和医学院(Wang Youwei.Doctoral dissertation of preclinical safety study of umbilical cord mesenchymal stem cells.Peking Union Medical College),2013.
20 Counter CM,Meyerson M,Eaton EN,Ellisen LW,Caddle SD,Haber DA,et al.Telomerase activity is restored in human cells by ectopic expression of hTERT,the catalytic subunit of telomerase.Oncogene 1998;16(9):1217-22.