猪骨髓间充质干细胞体外向心肌细胞分化的实验研究
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摘要
目的:探讨猪骨髓间充质干细胞(mesenchymal stem cells,MSCs)自发分化和5-氮胞苷诱导分化为心肌细胞的潜能,以及体外向心肌细胞分化的规律性。
方法:抽取猪髂骨骨髓,密度梯度离心法分离单个核细胞,贴壁培养法筛选和培养MSCs,选取第9,10,11,12,15和20代MSCs,研究其向心肌样细胞自发分化潜能和5-氮胞苷诱导后4周能否分化为心肌细胞,并探索猪MSCs体外向心肌细胞分化的规律性。各代MSCs采用连续显微镜下观察、流式细胞仪、免疫细胞化学染色法、Real Time-PCR、透射电镜检测相关指标。
结果:体外自然培养条件下,P9,P11,P12,P15,P20和大部分P10 MSCs形态一致,呈梭形成纤维细胞样形态,但部分P10细胞多核、胞浆延长,转变为肌管样形态。5-氮胞苷诱导1周后,部分P9,P11,P12,P15和P20MSCs表现为较长的胞浆形态和多核化,4周后更多细胞相互连接形成不规则的管样结构,而P10MSCs则形成相对规则的肌管样结构。
流式法显示,P10MSCs的G0/G1期比例显著增多(88.00±3.90)%,P10与P9相比有显著性差异(P＜0.0001),出现细胞生长停滞。贴壁细胞CD90、CD29阳性,而CD34、CD45阴性,说明贴壁细胞为MSCs。
免疫细胞化学染色显示,自然培养和诱导4周后各代MSCs均表达心肌特异性结构蛋白Cx43和α-sarcomeric actin,而诱导后P10MSCs分化率与其他各代相比有显著性差异(P=0.011)。
Real time-PCR结果表明,自然培养和诱导4周后的各代MSCs均有心肌特异性转录因子GATA4和结构基因MLC、α-SKA、Cx43、cTNI基因表达,而P10MSCs自然培养和诱导后4周,各基因的表达丰度均高于其他代在相同条件下的分化细胞,而α-SKA诱导后增高显著(P＜0.0001),而P20MSCs表达的基因减少。
透射电镜显示在自然培养状态下和诱导后4周,P9,P10,P11,P12,P15 MSCs均有较多不规则细肌丝,而P10MSCs经诱导后出现较多不规则排列粗肌丝,P20MSCs肌丝减少。
结论:1.猪骨髓MSCs在体外自然培养条件下,各代形态、增殖能力和分化潜能是不均一的,传代次数的增加MSCs增殖逐渐变慢、部分细胞自发分化为心肌样细胞。2.MSCs于第10代出现细胞生长停滞,但并没有丧失多向分化潜能。3,在体外长期培养过程中,MSCs出现转化能力。4.5-氮胞苷可诱导MSCs分化为心肌样细胞,P10MSCs分化更具有成熟心肌细胞的特异性表型。4.结合本实验研究组前期的结果,推测P10可能是猪MSCs体外分化为心肌细胞过程中的拐点。
Objectives:Mesenchymal stem cells(MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation.The aim of this study was to explore the proliferative potential and cardiomyogenic phenotype development of porcine bone marrow- derived MSCs before or after 5-azacytidine treatment,and find out the regularity of MSCs differentiation in vitro.
Methods:Bone marrow samples were aspirated from Chinese mini-swine.The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in high-glucose DMEM containing 10%fetal bovine serum.After primary culture,relatively purified MSCs were obtained by using the method of adherence screening.MSCs at passage9,10,11,12,15 and 20 were examined for their proliferation and differentiation ability before or after 5-azacytidine(10μM) treatment in vitro by FACS,immunocytochemistry, transmission electrical microscopy and real time-PCR.
Results:In normal growth medium,porcine bone marrow-derived MSCs were positive for CD90 and CD29,negative for CD34 and CD45.The growth property analysis demenstrated P10 pMSCs showed a growth-arrest appearance,while other passages displayed an exponential growth pattern.MSCs at every passage in vitro expressed cardimyocyte-specific genes and proteins.However,only passage 10 took on the formation of myotube structure.
After 5- azacytidine treatment,pMSCs at every passage in vitro took on cardiomyocyte -like phenotype and formation of myotube structure,and expressed cardimyocyte-specific genes and proteins.However,MSCs at passage 10 expressed cardimyocyte-specific genes higher than any other passages,especiallyα-SKA,P10 vs P9,P10vs P11,P10vs P12,P10vsP15,P10vs P20(P＜0.0001).
Before or after 5- azacytidine treatment,MSCs at passage 9,11,12,15 and 20 all had cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However,only MSCs at passage 10 took on relatively mature cardiomyocyte ultrastructure,including myofilaments.MSCs had no observation of sarcomeres, transverse striations,intercalated discs in vitro.
Conclusion:Cardiomyogenic potential of porcine bone Marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine in vitro is dependent upon the endogenous gene expression and passage-restricted pattern.These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.
方法:抽取猪髂骨骨髓,密度梯度离心法分离单个核细胞,贴壁培养法筛选和培养MSCs,选取第9,10,11,12,15和20代MSCs,研究其向心肌样细胞自发分化潜能和5-氮胞苷诱导后4周能否分化为心肌细胞,并探索猪MSCs体外向心肌细胞分化的规律性。各代MSCs采用连续显微镜下观察、流式细胞仪、免疫细胞化学染色法、Real Time-PCR、透射电镜检测相关指标。
结果:体外自然培养条件下,P9,P11,P12,P15,P20和大部分P10 MSCs形态一致,呈梭形成纤维细胞样形态,但部分P10细胞多核、胞浆延长,转变为肌管样形态。5-氮胞苷诱导1周后,部分P9,P11,P12,P15和P20MSCs表现为较长的胞浆形态和多核化,4周后更多细胞相互连接形成不规则的管样结构,而P10MSCs则形成相对规则的肌管样结构。
流式法显示,P10MSCs的G0/G1期比例显著增多(88.00±3.90)%,P10与P9相比有显著性差异(P＜0.0001),出现细胞生长停滞。贴壁细胞CD90、CD29阳性,而CD34、CD45阴性,说明贴壁细胞为MSCs。
免疫细胞化学染色显示,自然培养和诱导4周后各代MSCs均表达心肌特异性结构蛋白Cx43和α-sarcomeric actin,而诱导后P10MSCs分化率与其他各代相比有显著性差异(P=0.011)。
Real time-PCR结果表明,自然培养和诱导4周后的各代MSCs均有心肌特异性转录因子GATA4和结构基因MLC、α-SKA、Cx43、cTNI基因表达,而P10MSCs自然培养和诱导后4周,各基因的表达丰度均高于其他代在相同条件下的分化细胞,而α-SKA诱导后增高显著(P＜0.0001),而P20MSCs表达的基因减少。
透射电镜显示在自然培养状态下和诱导后4周,P9,P10,P11,P12,P15 MSCs均有较多不规则细肌丝,而P10MSCs经诱导后出现较多不规则排列粗肌丝,P20MSCs肌丝减少。
结论:1.猪骨髓MSCs在体外自然培养条件下,各代形态、增殖能力和分化潜能是不均一的,传代次数的增加MSCs增殖逐渐变慢、部分细胞自发分化为心肌样细胞。2.MSCs于第10代出现细胞生长停滞,但并没有丧失多向分化潜能。3,在体外长期培养过程中,MSCs出现转化能力。4.5-氮胞苷可诱导MSCs分化为心肌样细胞,P10MSCs分化更具有成熟心肌细胞的特异性表型。4.结合本实验研究组前期的结果,推测P10可能是猪MSCs体外分化为心肌细胞过程中的拐点。
Objectives:Mesenchymal stem cells(MSCs) are a group of heterogenous stem cells with multipotency in growth and differentiation.The aim of this study was to explore the proliferative potential and cardiomyogenic phenotype development of porcine bone marrow- derived MSCs before or after 5-azacytidine treatment,and find out the regularity of MSCs differentiation in vitro.
Methods:Bone marrow samples were aspirated from Chinese mini-swine.The mononuclear cells were isolated by density gradient centrifugation through 1.077g/ml Percoll and then cultured in high-glucose DMEM containing 10%fetal bovine serum.After primary culture,relatively purified MSCs were obtained by using the method of adherence screening.MSCs at passage9,10,11,12,15 and 20 were examined for their proliferation and differentiation ability before or after 5-azacytidine(10μM) treatment in vitro by FACS,immunocytochemistry, transmission electrical microscopy and real time-PCR.
Results:In normal growth medium,porcine bone marrow-derived MSCs were positive for CD90 and CD29,negative for CD34 and CD45.The growth property analysis demenstrated P10 pMSCs showed a growth-arrest appearance,while other passages displayed an exponential growth pattern.MSCs at every passage in vitro expressed cardimyocyte-specific genes and proteins.However,only passage 10 took on the formation of myotube structure.
After 5- azacytidine treatment,pMSCs at every passage in vitro took on cardiomyocyte -like phenotype and formation of myotube structure,and expressed cardimyocyte-specific genes and proteins.However,MSCs at passage 10 expressed cardimyocyte-specific genes higher than any other passages,especiallyα-SKA,P10 vs P9,P10vs P11,P10vs P12,P10vsP15,P10vs P20(P＜0.0001).
Before or after 5- azacytidine treatment,MSCs at passage 9,11,12,15 and 20 all had cardiomyocyte-like ultrastructure such as irregular myofilaments in vitro. However,only MSCs at passage 10 took on relatively mature cardiomyocyte ultrastructure,including myofilaments.MSCs had no observation of sarcomeres, transverse striations,intercalated discs in vitro.
Conclusion:Cardiomyogenic potential of porcine bone Marrow-derived MSCs spontaneously or in response to stimulation of 5-azacytidine in vitro is dependent upon the endogenous gene expression and passage-restricted pattern.These findings reveal a novel regulation on the transdifferentiation of MSCs and provide useful information for exploiting the clinical therapeutic potential of MSCs.
引文
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1.Pfeffer,M.A.and E.Braunwald,Ventricular remodeling after myocardial infarction.Experimental observations and clinical implications.Circulation,1990.81(4):p.1161-72.
2.Beltrami,A.P.,et al.,Evidence that human cardiac myocytes divide after myocardial infarction.N Engl J Med,2001.344(23):p.1750-7.
3.Makino,S.,et al.,Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest,1999.103(5):p.697-705.
4.Jiang,Y.,et al.,Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,2002.415(6893):p.41-9.
5.Le Blanc,K.,et al.,Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet,2004.363(9419):p.1439-41.
6.Pittenger,M.F.,et al.,Multilineage potential of adult human mesenchymal stem cells.Science,1999.284(5411):p.143-7.
7.Orlic,D.,et al.,Bone marrow cells regenerate infarcted myocardium.Nature,2001.410(6829):p.701-5.
8.Alvarez-Dolado,M.,et al.,Fusion of bone-marrow-derived cells with Purkinje neurons,cardiomyocytes and hepatocytes.Nature,2003.425(6961):p.968-73.
9.Liu,Y.,et al.,Growth and differentiation of rat bone marrow stromal cells:does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003.55(2):p.460-8.
10.Reyes,M.,et al.,Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002.109(3):p.337-46.
11.Harris,R.G.,et al.,Lack of a fusion requirement for development of bone marrow-derived epithelia.Science,2004.305(5680):p.90-3.
12.Tomita,S.,et al.,Autologous transplantation of bone marrow cells improves damaged heart function.Circulation,1999.100(19 Suppl):p.Ⅱ247-56.
13.Kadivar,M.,et al.,In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells.Biochem Biophys Res Commun,2006.340(2):p.639-47.
14.Xu,W.,et al.,Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro.Exp Biol Med(Maywood),2004.229(7):p.623-31.
15.Xu,M.,et al.,Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes.Circulation,2004.110(17):p.2658-65.
16.Planat-Benard,V.,et al.,Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells.Circ Res,2004.94(2):p.223-9.
17.Grinnemo,K.H.,et al.,Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel.Ann Med,2006.38(2):p.144-53.
18.Sartore,S.,et al.,Amniotic mesenchymal cells autotransplanted in a porcine model of cardiac ischemia do not differentiate to cardiogenic phenotypes.Eur J Cardiothorac Surg,2005.28(5):p.677-84.
19.Minguell,J.J.,A.Erices,and P.Conget,Mesenchymal stem cells.Exp Biol Med(Maywood),2001.226(6):p.507-20.
20.Gronthos,S.,et al.,Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow.J Cell Sci,2003.116(Pt 9):p.1827-35.
21.Dennis,J.E.and P.Charbord,Origin and differentiation of human and murine stroma.Stem Ceils,2002.20(3):p.205-14.
22.Prockop,D.J.,Marrow stromal cells as stem cells for nonhematopoietic tissues.Science,1997.276(5309):p.71-4.
23.Conget,P.A.and J.J.Minguell,Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells.J Cell Physiol,1999.181(1):p.67-73.
24.Moscoso,I.,et al.,Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.Transplant Proc,2005.37(1):p.481-2.
25.Muraglia,A.,R.Cancedda,and R.Quarto,Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model.J Cell Sci,2000.113(Pt 7):p.1161-6.
26.Dennis,J.E.,et al.,A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.J Bone Miner Res,1999.14(5):p.700-9.
27.Rao,S.G.and G.Dravid,Expansion of haematopoietic stem cells in vitro:a challenge to stem cell biologists.Indian J Exp Biol,1999.37(11):p.1051-2.
28.Shahdadfar,A.,et al.,In vitro expansion of human mesenchymal stem cells:choice of serum is a determinant of cell proliferation,differentiation,gene expression,and transcriptome stability.Stem Cells,2005.23(9):p.1357-66.
29.Zhang,F.B.,et al.,Passage-restricted differentiation potential of mesenchymal stem cells into cardiomyocyte-like cells.Biochem Biophys Res Commun,2005.336(3):p.784-92.
30.Bruder,S.P.,N.Jaiswai,and S.E.Haynesworth,Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem,1997.64(2):p.278-94.
31.Naruse,K.,et al.,Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation.Bone,2004.35(4):p.850-8.
32.Yamada,Y.,et al.,Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells.Exp Cell Res,2007.313(4):p.698-706.
33.Heng,B.C.,et al.,Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro.Cardiovasc Res,2004.62(1):p.34-42.
34.Fukuda,K.,Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes.C R Biol,2002.325(10):p.1027-38.
35.Tomoyuki Saito,J.E.D.,Donald P.Lennon,Randell G.Young,Arnold I.Caplan.,Myogenic Expression of Mesenchymal Stem Cells within Myotubes of mdx Mice in Vitro and in Vivo.Tissue Engineering,1995.1(4):p.327.
36.Wakitani,S.,T.Saito,and A.I.Caplan,Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.Muscle Nerve,1995.18(12):p.1417-26.
37.Fukuda,K.,Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering.Artif Organs,2001.25(3):p.187-93.
38.Antonitsis,P.,et al.,Cardiomyogenic potential of human adult bone marrow mesenchymal stem cells in vitro.Thorac Cardiovasc Surg,2008.56(2):p.77-82.
39.Carrier,L.,et al.,Expression of the sarcomeric actin isogenes in the rat heart with development and senescence.Circ Res,1992.70(5):p.999-1005.
40.Schwartz,K.,et al.,Alpha-skeletal muscle actin mRNA's accumulate in hypertrophied adult rat hearts.Circ Res,1986.59(5):p.551-5.
41.Schwartz,K.,et al.,Regulation of myosin heavy chain and actin isogenes during cardiac growth and hypertrophy.Symp Soc Exp Biol,1992.46:p.265-72.
42.Winegrad,S.,C.Wisnewsky,and K.Schwartz,Effect of thyroid hormone on the accumulation of mRNA for skeletal and cardiac alpha-actin in hearts from normal and hypophysectomized rats.Proc Natl Acad Sci U S A,1990.87(7):p.2456-60.
43.Ruzicka,D.L.and R.J.Schwartz,Sequential activation of alpha-actin genes during avian cardiogenesis:vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.J Cell Biol,1988.107(6 Pt 2):p.2575-86.
44.Woodcock-Mitchell,J.,et al.,Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles.Differentiation,1988.39(3):p.161-6.
45.Vandekerckhove,J.,G.Bugaisky,and M.Buckingham,Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells.A quantitative determination of the two actin isoforms.J Biol Chem,1986.261(4):p.1838-43.
46.Hewett,T.E.,Grupp,I.L.,Grupp,G.and Robbins,J.,a-skeletal actin is associated with increased contractility in the mouse heart.Circ.Res,1994.74:p.7.
47.Clement,S.,et al.,Expression and function of alpha-smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation.J Cell Sci,2007.120(Pt 2):p. 229-38.
48.Bayes-Genis,A.,et al.,Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cells.Transplant Proc,2005.37(9):p.4077-9.
49.Seshi,B.,S.Kumar,and D.King,Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis.Blood Cells Mol Dis,2003.31(2):p.268-85.
50.Konieczny,S.F.and C.P.Emerson,Jr.,5-Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells:evidence for regulatory genes controlling determination.Cell,1984.38(3):p.791-800.
51.Taylor,S.M.,5-Aza-2'-deoxycytidine:cell differentiation and DNA methylation.Leukemia,1993.7 Suppl 1:p.3-8.
52.Zuscik,M.J.,et al.,5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes.J Cell Biochem,2004.92(2):p.316-31.
53.Choi,S.C.,et al.,5-azacytidine induces cardiac differentiation of P19 embryonic stem cells.Exp Mol Med,2004.36(6):p.515-23.
54.Ye,N.S.,et al.,Proteomic profiling of rat bone marrow mesenchymal stem cells induced by 5-azacytidine.Stem Cells Dev,2006.15(5):p.665-76.
55.Pochampally,R.R.,et al.,Serum deprivation of human marrow stromal cells(hMSCs) selects for a subpopulation of early progenitor cells with enhanced expression of OCT-4 and other embryonic genes.Blood,2004.103(5):p.1647-52.
56.Majumdar,M.K.,et al.,Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs) and stromal cells.J Cell Physiol,1998.176(1):p.57-66.
57.Rubio,D.,et al.,Spontaneous human adult stem cell transformation.Cancer Res,2005.65(8):p.3035-9.
58.Nagy,R.D.,et al.,Stem cell transplantation as a therapeutic approach to organ failure.J Surg Res,2005.129(1):p.152-60.
59.Kotev-Emeth,S.,et al.,Effect of maturation on the osteogenic response of cultured stromal bone marrow cells to basic fibroblast growth factor.Bone,2000.27(6):p.777-83.
60.Service,R.F.,Tissue engineers build new bone.Science,2000.289(5484):p.1498-500.
61.Weintraub,H.,The MyoD family and myogenesis:redundancy,networks,and thresholds.Cell,1993.75(7):p.1241-4.
62.Rhodes,S.J.and S.F.Konieczny,Identification of MRF4:a new member of the muscle regulatory factor gene family.Genes Dev,1989.3(12B):p.2050-61.
63.Seidman,C.E.,et al.,Nucleotide sequences of the human and mouse atrial natriuretic factor genes.Science,1984.226(4679):p.1206-9.
64.Sudoh,T.,et al.,A new natriuretic peptide in porcine brain.Nature,1988.332(6159):p.78-81.
65.Rangappa,S.,et al.,Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype.J Thorac Cardiovasc Surg,2003.126(1):p.124-32.
66.Shim,W.S.,et al.,Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells.Biochem Biophys Res Commun,2004.324(2):p.481-8.
67.Banfi,A.,et al.,Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells:Implications for their use in cell therapy.Exp Hematol,2000.28(6):p.707-15.
68.Derubeis,A.R.and R.Cancedda,Bone marrow stromal cells(BMSCs) in bone engineering:limitations and recent advances.Ann Biomed Eng,2004.32(1):p.160-5.
69.Vacanti,V.,et al.,Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture.J Cell Physiol,2005.205(2):p.194-201.
70.Izadpanah,R.,et al.,Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue.J Cell Biochem,2006.99(5):p.1285-97.
71.Molkentin,J.D.,The zinc finger-containing transcription factors GATA-4,-5,and-6.Ubiquitously expressed regulators of tissue-specific gene expression.J Biol Chem,2000.275(50):p.38949-52.
72.Crisostomo,P.R.,et al.,High passage number of stem cells adversely affects stem cell activation and myocardial protection.Shock,2006.26(6):p.575-80.
73.Tang,Y.L.,et al.,Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium.Regul Pept,2004.117(1):p.3-10.
74.Hiasa,K.,et al.,Bone marrow mononuclear cell therapy limits myocardial infarct size through vascular endothelial growth factor.Basic Res Cardiol,2004.99(3):p.165-72.
75.Bonab,M.M.,et al.,Aging of mesenchymal stem cell in vitro.BMC Cell Biol,2006.7:p.14.
2.Beltrami,A.P.,et al.,Evidence that human cardiac myocytes divide after myocardial infarction.N Engl J Med,2001.344(23):p.1750-7.
3.Makino,S.,et al.,Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest,1999.103(5):p.697-705.
4.Jiang,Y.,et al.,Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,2002.418(6893):p.41-9.
5.Le Blanc,K.,et al.,Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet,2004.363(9419):p.1439-41.
6.Pittenger,M.F.,et al.,Multilineage potential of adult human mesenchymal stem cells.Science,1999.284(5411):p.143-7.
7.Orlic,D.,et al.,Bone marrow cells regenerate infarcted myocardium.Nature,2001.410(6829):p.701-5.
8.Alvarez-Dolado,M.,et al.,Fusion of bone-marrow-derived cells with Purkinje neurons,cardiomyocytes and hepatocytes.Nature,2003.425(6961):p.968-73.
9.Liu,Y.,et al.,Growth and differentiation of rat bone marrow stromal cells:does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003.58(2):p.460-8.
10.Reyes,M.,et al.,Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002.109(3):p.337-46.
11.Harris,R.G.,et al.,Lack of a fusion requirement for development of bone marrow-derived epithelia.Science,2004.305(5680):p.90-3.
12.Tomita,S.,et al.,Autologous transplantation of bone marrow cells improves damaged heart function.Circulation,1999.100(19 Suppl):p.Ⅱ247-56.
13.Kadivar,M.,et al.,In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells.Biochem Biophys Res Commun,2006.340(2):p.639-47.
14.Xu,W.,et al.,Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro.Exp Biol Med(Maywood),2004.229(7):p.623-31.
15.Xu,M.,et al.,Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes.Circulation,2004.110(17):p.2658-65.
16.Planat-Benard,V.,et al.,Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells.Circ Res,2004.94(2):p.223-9.
17.Grinnemo,K.H.,et al.,Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel.Ann Med,2006.38(2):p.144-53.
18.Sartore,S.,et al.,Amniotic mesenchymal cells autotransplanted in a porcine model of cardiac ischemia do not differentiate to cardiogenic phenotypes.Eur J Cardiothorac Surg,2005.28(5):p.677-84.
19.Minguell,J.J.,A.Erices,and P.Conget,Mesenchymal stem cells.Exp Biol Med (Maywood),2001.226(6):p.507-20.
20.Gronthos,S.,et al.,Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow.J Cell Sci,2003.116(Pt 9):p.1827-35.
21.Dennis,J.E.and P.Charbord,Origin and differentiation of human and murine stroma.Stem Cells,2002.20(3):p.205-14.
22.Prockop,D.J.,Marrow stromal cells as stem cells for nonhematopoietic tissues.Science,1997.276(5309):p.71-4.
23.Conger,P.A.and J.J.Minguell,Phenotypical and functional properties of human bone marrow mesenchymalprogenitor cells.J Cell Physiol,1999.181(1):p.67-73.
24.Moscoso,I.,et al.,Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.Transplant Proc,2005.37(1):p.481-2.
25.Muraglia,A.,R.Cancedda,and R.Quarto,Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model.J Cell Sci,2000.113(Pt 7):p.1161-6.
26.Dennis,J.E.,et al.,A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.J Bone Miner Res,1999.14(5):p.700-9.
27.Rao,S.G.and G.Dravid,Expansion of haematopoietic stem cells in vitro:a challenge to stem cell biologists.Indian J Exp Biol,1999.37(11):p.1051-2.
28.Shahdadfar,A.,et al.,In vitro expansion of human mesenchymal stem cells:choice of serum is a determinant of cell proliferation,differentiation,gene expression,and transcriptome stability.Stem Cells,2005.23(9):p.1357-66.
29.Zhang,F.B.,et al.,Passage-restricted differentiation potential of mesenchymai stem cells into cardiomyocyte-like cells.Biochem Biophys Res Commun,2005.336(3):p.784-92.
30.Bruder,S.P.,N.Jaiswal,and S.E.Haynesworth,Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation.J Cell Biochem,1997.64(2):p.278-94.
31.Naruse,K.,et al.,Spontaneous differentiation of mesenchymal stem cells obtained from fetal rat circulation.Bone,2004.35(4):p.850-8.
32.Yamada,Y.,et al.,Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells.Exp Cell Res,2007.313(4):p.698-706.
33.Heng,B.C.,et al.,Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro.Cardiovasc Res,2004.62(1):p.34-42.
34.Fukuda,K.,Reprogramming of bone marrow mesenchymal stem cells into cardiomyocytes.C R Biol,2002.325(10):p.1027-38.
35.Tomoyuki Saito,J.E.D.,Donald P.Lennon,Randell G.Young,Arnold I.Caplan.,Myogenic Expression of Mesenchymal Stem Cells within Myotubes of mdx Mice in Vitro and in Vivo.Tissue Engineering,1995.1(4):p.327.
36.Wakitani,S.,T.Saito,and A.I.Caplan,Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine.Muscle Nerve,1995.18(12):p.1417-26.
37.Fukuda,K.,Development of regenerative cardiomyocytes from mesenchymal stem cells for cardiovascular tissue engineering.Artif Organs,2001.25(3):p.187-93.
38.Antonitsis,P.,et al.,Cardiomyogenic potential of human adult bone marrow mesenchymal stem cells in vitro.Thorac Cardiovasc Surg,2008.56(2):p.77-82.
39.Carrier,L.,et al.,Expression of the sarcomeric actin isogenes in the rat heart with development and senescence.Circ Res,1992.70(5):p.999-1005.
40.Schwartz,K.,et al.,Alpha-skeletal muscle actin mRNA's accumulate in hypertrophied adult rat hearts.Circ Res,1986.59(5):p.551-5.
41.Schwartz,K.,et al.,Regulation of myosin heavy chain and actin isogenes during cardiac growth and hypertrophy.Symp Soc Exp Biol,1992.46:p.265-72.
42.Winegrad,S.,C.Wisnewsky,and K.Schwartz,Effect of thyroid hormone on the accumulation of mRNA for skeletal and cardiac alpha-actin in hearts from normal and hypophysectomized rats.Proc Natl Acad Sci U S A,1990.87(7):p.2456-60.
43.Ruzicka,D.L.and R.J.Schwartz,Sequential activation of alpha-actin genes during avian cardiogenesis:vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation.J Cell Biol,1988.107(6 Pt 2):p.2575-86.
44.Woodcock-Mitchell,J.,et al.,Alpha-smooth muscle actin is transiently expressed in embryonic rat cardiac and skeletal muscles.Differentiation,1988.39(3):p.161-6.
45.Vandekerckhove,J.,G.Bugaisky,and M.Buckingham,Simultaneous expression of skeletal muscle and heart actin proteins in various striated muscle tissues and cells.A quantitative determination of the two actin isoforms.J Biol Chem,1986.261(4):p.1838-43.
46.Hewett,T.E.,Grupp,I.L.,Grupp,G.and Robbins,J.,a-skeletal actin is associated with increased contractility in the mouse heart.Circ.Res,1994.74:p.7.
47.Clement,S.,et al.,Expression and function of alpha-smooth muscle actin during embryonic-stem-cell-derived cardiomyocyte differentiation.J Cell Sci,2007.120(Pt 2):p.229-38.
48.Bayes-Genis,A.,et al.,Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cells.Transplant Proc,2005.37(9):p.4077-9.
49.Seshi,B.,S.Kumar,and D.King,Multilineage gene expression in human bone marrow stromal cells as evidenced by single-cell microarray analysis.Blood Cells Mol Dis,2003.31(2):p.268-85.
50.Konieczny,S.F.and C.P.Emerson,Jr.,5-Azacytidine induction of stable mesodermal stem cell lineages from 10T1/2 cells:evidence for regulatory genes controlling determination.Cell,1984.38(3):p.791-800.
51.Taylor,S.M.,5-Aza-2'-deoxycytidine:cell differentiation and DNA methylation.Leukemia,1993.7 Suppl 1:p.3-8.
52.Zuscik,M.J.,et al.,5-azacytidine alters TGF-beta and BMP signaling and induces maturation in articular chondrocytes.J Cell Biochem,2004.92(2):p.316-31.
53.Choi,S.C.,et al.,5-azacytidine induces cardiac differentiation of P19 embryonic stem cells.Exp Mol Med,2004.36(6):p.515-23.
54.Ye,N.S.,et al.,Proteomic profiling of rat bone marrow mesenchymal stem cells induced by 5-azacytidine.Stem Cells Dev,2006.15(5):p.665-76.
55.Pochampally,R.R.,et al.,Serum deprivation of human marrow stromal cells(hMSCs)selects for a subpopulation of early progenitor cells with enhanced expression of OCT-4 and other embryonic genes.Blood,2004.103(5):p.1647-52.
56.Majumdar,M.K.,et al.,Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells(MSCs) and stromal cells.J Cell Physiol,1998.176(1):p.57-66.
57.Rubio,D.,et al.,Spontaneous human adult stem cell transformation.Cancer Res,2005.65(8):p.3035-9.
58.Nagy,R.D.,et al.,Stem cell transplantation as a therapeutic approach to organ failure.J Surg Res,2005.129(1):p.152-60.
59.Kotev-Emeth,S.,et al.,Effect of maturation on the osteogenic response of cuitured stromal bone marrow cells to basic fibroblast growth factor.Bone,2000.27(6):p.777-83.
60.Service,R.F.,Tissue engineers build new bone.Science,2000.289(5484):p.1498-500.
61.Weintraub,H.,The MyoD family and myogenesis:redundancy,networks,and thresholds.Cell,1993.75(7):p.1241-4.
62.Rhodes,S.J.and S.F.Konieczny,Identification of MRF4:a new member of the muscle regulatory factor gene family.Genes Dev,1989.3(12B):p.2050-61.
63.Seidman,C.E.,et al.,Nucleotide sequences of the human and mouse atrial natriuretic factor genes.Science,1984.226(4679):p.1206-9.
64.Sudoh,T.,et al.,A new natriuretic peptide in porcine brain.Nature,1988.332(6159):p.78-81.
65.Rangappa,S.,et al.,Cardiomyocyte-mediated contact programs human mesenchymal stem cells to express cardiogenic phenotype.J Thorac Cardiovasc Surg,2003.126(1):p.124-32.
66.Shim,W.S.,et al.,Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells.Biochem Biophys Res Commun,2004.324(2):p.481-8.
67.Banfi,A.,et al.,Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells:Implications for their use in cell therapy.Exp Hematol,2000.28(6):p.707-15.
68.Derubeis,A.R.and R.Cancedda,Bone marrow stromal cells(BMSCs) in bone engineering:limitations and recent advances.Ann Biomed Eng,2004.32(1):p.160-5.
69.Vacanti,V.,et al.,Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture.J Cell Physiol,2005.205(2):p.194-201.
70.Izadpanah,R.,et al.,Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue.J Cell Biochem,2006.99(5):p.1285-97.
71.Molkentin,J.D.,The zinc finger-containing transcription factors GATA-4,-5,and-6.Ubiquitously expressed regulators of tissue-specific gene expression.J Biol Chem,2000.275(50):p.38949-52.
72.Crisostomo,P.R.,et al.,High passage number of stem cells adversely affects stem cell activation and myocardial protection.Shock,2006.26(6):p.575-80.
73.Tang,Y.L.,et al.,Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium.Regul Pept,2004.117(1):p.3-10.
74.Hiasa,K.,et al.,Bone marrow mononuclear cell therapy limits myocardial infarct size through vascular endothelial growth factor.Basic Res Cardiol,2004.99(3):p.165-72.
75.Bonab,M.M.,et al.,Aging of mesenchymal stem cell in vitro.BMC Cell Biol,2006.7:p.14.
1.Pfeffer,M.A.and E.Braunwald,Ventricular remodeling after myocardial infarction.Experimental observations and clinical implications.Circulation,1990.81(4):p.1161-72.
2.Beltrami,A.P.,et al.,Evidence that human cardiac myocytes divide after myocardial infarction.N Engl J Med,2001.344(23):p.1750-7.
3.Makino,S.,et al.,Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest,1999.103(5):p.697-705.
4.Jiang,Y.,et al.,Pluripotency of mesenchymal stem cells derived from adult marrow.Nature,2002.415(6893):p.41-9.
5.Le Blanc,K.,et al.,Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells.Lancet,2004.363(9419):p.1439-41.
6.Pittenger,M.F.,et al.,Multilineage potential of adult human mesenchymal stem cells.Science,1999.284(5411):p.143-7.
7.Orlic,D.,et al.,Bone marrow cells regenerate infarcted myocardium.Nature,2001.410(6829):p.701-5.
8.Alvarez-Dolado,M.,et al.,Fusion of bone-marrow-derived cells with Purkinje neurons,cardiomyocytes and hepatocytes.Nature,2003.425(6961):p.968-73.
9.Liu,Y.,et al.,Growth and differentiation of rat bone marrow stromal cells:does 5-azacytidine trigger their cardiomyogenic differentiation? Cardiovasc Res,2003.55(2):p.460-8.
10.Reyes,M.,et al.,Origin of endothelial progenitors in human postnatal bone marrow.J Clin Invest,2002.109(3):p.337-46.
11.Harris,R.G.,et al.,Lack of a fusion requirement for development of bone marrow-derived epithelia.Science,2004.305(5680):p.90-3.
12.Tomita,S.,et al.,Autologous transplantation of bone marrow cells improves damaged heart function.Circulation,1999.100(19 Suppl):p.Ⅱ247-56.
13.Kadivar,M.,et al.,In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells.Biochem Biophys Res Commun,2006.340(2):p.639-47.
14.Xu,W.,et al.,Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro.Exp Biol Med(Maywood),2004.229(7):p.623-31.
15.Xu,M.,et al.,Differentiation of bone marrow stromal cells into the cardiac phenotype requires intercellular communication with myocytes.Circulation,2004.110(17):p.2658-65.
16.Planat-Benard,V.,et al.,Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells.Circ Res,2004.94(2):p.223-9.
17.Grinnemo,K.H.,et al.,Human mesenchymal stem cells do not differentiate into cardiomyocytes in a cardiac ischemic xenomodel.Ann Med,2006.38(2):p.144-53.
18.Sartore,S.,et al.,Amniotic mesenchymal cells autotransplanted in a porcine model of cardiac ischemia do not differentiate to cardiogenic phenotypes.Eur J Cardiothorac Surg,2005.28(5):p.677-84.
19.Minguell,J.J.,A.Erices,and P.Conget,Mesenchymal stem cells.Exp Biol Med(Maywood),2001.226(6):p.507-20.
20.Gronthos,S.,et al.,Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow.J Cell Sci,2003.116(Pt 9):p.1827-35.
21.Dennis,J.E.and P.Charbord,Origin and differentiation of human and murine stroma.Stem Ceils,2002.20(3):p.205-14.
22.Prockop,D.J.,Marrow stromal cells as stem cells for nonhematopoietic tissues.Science,1997.276(5309):p.71-4.
23.Conget,P.A.and J.J.Minguell,Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells.J Cell Physiol,1999.181(1):p.67-73.
24.Moscoso,I.,et al.,Differentiation "in vitro" of primary and immortalized porcine mesenchymal stem cells into cardiomyocytes for cell transplantation.Transplant Proc,2005.37(1):p.481-2.
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