ISL-1诱导脂肪干细胞向起搏样细胞分化
|
摘要
目的探讨过表达胰岛素基因增强子结合蛋白1(insulin gene enhancer binding protein 1,ISL-1)的慢病毒在体外转染脂肪干细胞(adipose-derived stem cells,ADSCs),能否诱导ADSCs向起搏样细胞分化。方法取第3～5代ADSCs,随机分成Bank、m Cherry和胰岛素基因增强子结合蛋白1(insulin gene enhancer binding protein 1,ISL-1) 3组,按分组分别转染病毒,经荧光强度和流式分析确定最适感染复数,与乳鼠心室肌细胞(neonatal rat cardiomyocytes,NRVMs)共培养7天后进行实时荧光定量聚合酶式反应(quantitative real time polymerase chain reaction,qRT-PCR)、蛋白质印迹、免疫荧光检测分析,期间观察细胞形态和搏动频率变化,并用膜片钳技术记录细胞内电流活动。结果分离贴壁后的ADSCs呈长梭形,慢病毒转染ADSCs的最适感染复数为50。ISL-1组ADSCs形态呈多样化,窦房结特异性基因HCN4、Cx45和Tbx3的mRNA表达水平上调,而工作心肌特异性基因Nkx2. 5下调,组间比较差异有统计学意义(P <0. 05)。ISL-1组大多数细胞可检测到HCN4表达并且可记录到超极化内向电流。结论经ISL-1基因修饰的ADSCs通过体外心肌微环境诱导,产生了一定的高表达窦房结标志性基因并具有细胞内典型超极化电活动的起搏样细胞。
Objective To investigate whether the lentivirus over-expressing insulin gene enhancer binding protein( ISL-1) can induce adipose-derived stem cells( ADSCs) to differentiate into pacemaker-like cells in vitro. Methods The 3-5 th generation of ADSCs were randomly divided into three groups( Bank,GFP and ISL-1) and were transfected lentivirus by group. By the fluorescence intensity and flow analysis,we selected the optimal multiplicity of infection( MOI) to make subsequent experiments. After co-cultured with neonatal rat ventricular cardiomyocytes( NRVMs) in vitro for 7 days,we estimated the outcome of morphological and beat frequency by quantitative real time polymerase chain reaction( qRT-PCR),Western blot,immunofluorescence and we recorded cell current activity by patch clamp technique. Results The most of adherent ADSCs existed long-spindle shape. The optimum MOI value of transfection was50. The morphological of ADSCs transfected ISL-1 was diversified. In group ISL-1,the mRNA expression levels of the specific sinoatrial node specific genes of HCN4,Cx45 and Tbx3 were up-regulated,meanwhile the working myocardium specific gene of Nkx2. 5,was down-regulated. There were significant differences between the groups( P < 0. 05). Most ADSCs in the group ISL-1 could detect the expression of HCN4 and record the hyperpolarization inward current. Conclusion ADSCs transfected with ISL-1 in the myocardial microenvironment can be successfully differentiated into pacemaker-like cells,which expressed highly the sinus node-like genes and typical hyperpolarized electrical activity.
Objective To investigate whether the lentivirus over-expressing insulin gene enhancer binding protein( ISL-1) can induce adipose-derived stem cells( ADSCs) to differentiate into pacemaker-like cells in vitro. Methods The 3-5 th generation of ADSCs were randomly divided into three groups( Bank,GFP and ISL-1) and were transfected lentivirus by group. By the fluorescence intensity and flow analysis,we selected the optimal multiplicity of infection( MOI) to make subsequent experiments. After co-cultured with neonatal rat ventricular cardiomyocytes( NRVMs) in vitro for 7 days,we estimated the outcome of morphological and beat frequency by quantitative real time polymerase chain reaction( qRT-PCR),Western blot,immunofluorescence and we recorded cell current activity by patch clamp technique. Results The most of adherent ADSCs existed long-spindle shape. The optimum MOI value of transfection was50. The morphological of ADSCs transfected ISL-1 was diversified. In group ISL-1,the mRNA expression levels of the specific sinoatrial node specific genes of HCN4,Cx45 and Tbx3 were up-regulated,meanwhile the working myocardium specific gene of Nkx2. 5,was down-regulated. There were significant differences between the groups( P < 0. 05). Most ADSCs in the group ISL-1 could detect the expression of HCN4 and record the hyperpolarization inward current. Conclusion ADSCs transfected with ISL-1 in the myocardial microenvironment can be successfully differentiated into pacemaker-like cells,which expressed highly the sinus node-like genes and typical hyperpolarized electrical activity.
引文
1 Boink GJ,Christoffels VM,Robinson RB,et al. The past,present,and future of pacemaker therapies[J]. Trends Cardiovasc Med,2015,25(8):661-673
2 Ionta V,Liang W,Kim EH,et al. SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells,improving biological pacing ability[J]. Stem Cell Rep,2015,4(1):129-142
3 Yang M,Zhang GG,Wang T,et al. TBX18 gene induces adipose-derived stem cells to differentiate into pacemaker-like cells in the myocardial microenvironment[J]. Int J Mol Med,2016,38(5):1403-1410
4 Yechikov S,Copaciu R,Gluck J M,et al. Same-single-cell analysis of pacemaker-specific markers in human induced pluripotent stem cell-derived cardiomyocyte subtypes classified by electrophysiology[J]. Stem Cells,2016,34(11):2670-2680
5 Dmitrieva RI,Minullina IR,Bilibina AA,et al. Bone marrow-and subcutaneous adipose tissue-derived mesenchymal stem cells:differences and similarities[J]. Cell Cycle,2012,11(2):377-383
6 Joo HJ,Kim JH,Hong SJ. Adipose tissue-derived stem cells for myocardial regeneration[J]. Korean Circ J,2017,47(2):151-159
7 Rangappa S,Fen C,Lee EH,et al. Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes[J].Ann Thorac Surg,2003,75(3):775-779
8 Planat-Benard V,Menard C,Andre M,et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells[J]. Circ Res,2004,94(2):223-229
9 Chen L,Deng ZJ,Zhou JS,et al. Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells[J]. Mol Cell Biochem,2017,433(1-2):61-77
10 Vedantham V,Galang G,Evangelista M,et al. RNA sequencing of mouse sinoatrial node reveals an upstream regulatory role for Islet-1in cardiac pacemaker cells[J]. Circ Res,2015,116(5):797-803
11 Dorn T,Goedel A,Lam J T,et al. Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity[J]. Stem Cells,2015,33(4):1113-1129
12 Liang X,Zhang Q,Cattaneo P,et al. Transcription factor ISL1 is essential for pacemaker development and function[J]. J Clin Invest,2015,125(8):3256-3268
13 Boink GJ,Christoffels VM,Robinson RB,et al. The past,present,and future of pacemaker therapies[J]. Trends Cardiovasc Med,2015,25(8):661-673
14 Shen H,Wang Y,Zhang Z,et al. Mesenchymal stem cells for cardiac regenerative therapy:optimization of cell differentiation strategy[J].Stem Cells Int,2015,2015(3):524756
15 Zhu Y,Liu T,Song K,et al. ADSCs differentiated into cardiomyocytes in cardiac microenvironment[J]. Mol Cell Biochem,2009,324(1-2):117-129
16 Choi YS,Dusting GJ,Stubbs S,et al. Differentiation of human adipose-derived stem cells into beating cardiomyocytes[J]. J Cell Mol Med,2010,14(4):878-889
17 Brioschi C,Micheloni S,Tellez Jo,et al. Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node[J]. J Mol Cell Cardiol,2009,47(2):221-227
18 Boyett MR,Inada S,Yoo S,et al. Connexins in the sinoatrial and atrioventricular nodes[J]. Adv Cardiol,2006,42:175-197
19 Ye WG,Yue B,Aoyama H,et al. Junctional delay,frequency,and direction-dependent uncoupling of human heterotypic Cx45/Cx43 gap junction channels[J]. J Mol Cell Cardiol,2017,111:17-26
20 Ye W,Song Y,Huang Z,et al. Genetic regulation of sinoatrial node development and pacemaker program in the venous pole[J]. J Cardiovasc Dev Dis,2015,2(4):282-298
21 Mommersteeg MT,Dominguez JN,Wiese C,et al. The sinus venosus progenitors separate and diversify from the first and second heart fields early in development[J]. Cardiovasc Res,2010,87(1):92-101
22 Weinberger F,Mehrkens D,Friedrich FW,et al. Localization of Islet-1 -positive cells in the healthy and infarcted adult murine heart[J].Circ Res,2012,110(10):1303-1310
23 Wiese C,Grieskamp T,Airik R,et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3[J]. Circ Res,2009,104(3):388-397
24 Hoogaars WM,Engel A,Brons JF,et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria[J].Genes Dev,2007,21(9):1098-1112
25 Espinoza-Lewis RA,Liu H,Sun C,et al. Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice[J]. Dev Biol,2011,356(2):359-369
26 Hausburg F,Jung JJ,David R. Specific cell(Re-)programming:approaches and perspectives[J]. Adv Biochem Eng Biotechnol,2018,163:71-115
2 Ionta V,Liang W,Kim EH,et al. SHOX2 overexpression favors differentiation of embryonic stem cells into cardiac pacemaker cells,improving biological pacing ability[J]. Stem Cell Rep,2015,4(1):129-142
3 Yang M,Zhang GG,Wang T,et al. TBX18 gene induces adipose-derived stem cells to differentiate into pacemaker-like cells in the myocardial microenvironment[J]. Int J Mol Med,2016,38(5):1403-1410
4 Yechikov S,Copaciu R,Gluck J M,et al. Same-single-cell analysis of pacemaker-specific markers in human induced pluripotent stem cell-derived cardiomyocyte subtypes classified by electrophysiology[J]. Stem Cells,2016,34(11):2670-2680
5 Dmitrieva RI,Minullina IR,Bilibina AA,et al. Bone marrow-and subcutaneous adipose tissue-derived mesenchymal stem cells:differences and similarities[J]. Cell Cycle,2012,11(2):377-383
6 Joo HJ,Kim JH,Hong SJ. Adipose tissue-derived stem cells for myocardial regeneration[J]. Korean Circ J,2017,47(2):151-159
7 Rangappa S,Fen C,Lee EH,et al. Transformation of adult mesenchymal stem cells isolated from the fatty tissue into cardiomyocytes[J].Ann Thorac Surg,2003,75(3):775-779
8 Planat-Benard V,Menard C,Andre M,et al. Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells[J]. Circ Res,2004,94(2):223-229
9 Chen L,Deng ZJ,Zhou JS,et al. Tbx18-dependent differentiation of brown adipose tissue-derived stem cells toward cardiac pacemaker cells[J]. Mol Cell Biochem,2017,433(1-2):61-77
10 Vedantham V,Galang G,Evangelista M,et al. RNA sequencing of mouse sinoatrial node reveals an upstream regulatory role for Islet-1in cardiac pacemaker cells[J]. Circ Res,2015,116(5):797-803
11 Dorn T,Goedel A,Lam J T,et al. Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity[J]. Stem Cells,2015,33(4):1113-1129
12 Liang X,Zhang Q,Cattaneo P,et al. Transcription factor ISL1 is essential for pacemaker development and function[J]. J Clin Invest,2015,125(8):3256-3268
13 Boink GJ,Christoffels VM,Robinson RB,et al. The past,present,and future of pacemaker therapies[J]. Trends Cardiovasc Med,2015,25(8):661-673
14 Shen H,Wang Y,Zhang Z,et al. Mesenchymal stem cells for cardiac regenerative therapy:optimization of cell differentiation strategy[J].Stem Cells Int,2015,2015(3):524756
15 Zhu Y,Liu T,Song K,et al. ADSCs differentiated into cardiomyocytes in cardiac microenvironment[J]. Mol Cell Biochem,2009,324(1-2):117-129
16 Choi YS,Dusting GJ,Stubbs S,et al. Differentiation of human adipose-derived stem cells into beating cardiomyocytes[J]. J Cell Mol Med,2010,14(4):878-889
17 Brioschi C,Micheloni S,Tellez Jo,et al. Distribution of the pacemaker HCN4 channel mRNA and protein in the rabbit sinoatrial node[J]. J Mol Cell Cardiol,2009,47(2):221-227
18 Boyett MR,Inada S,Yoo S,et al. Connexins in the sinoatrial and atrioventricular nodes[J]. Adv Cardiol,2006,42:175-197
19 Ye WG,Yue B,Aoyama H,et al. Junctional delay,frequency,and direction-dependent uncoupling of human heterotypic Cx45/Cx43 gap junction channels[J]. J Mol Cell Cardiol,2017,111:17-26
20 Ye W,Song Y,Huang Z,et al. Genetic regulation of sinoatrial node development and pacemaker program in the venous pole[J]. J Cardiovasc Dev Dis,2015,2(4):282-298
21 Mommersteeg MT,Dominguez JN,Wiese C,et al. The sinus venosus progenitors separate and diversify from the first and second heart fields early in development[J]. Cardiovasc Res,2010,87(1):92-101
22 Weinberger F,Mehrkens D,Friedrich FW,et al. Localization of Islet-1 -positive cells in the healthy and infarcted adult murine heart[J].Circ Res,2012,110(10):1303-1310
23 Wiese C,Grieskamp T,Airik R,et al. Formation of the sinus node head and differentiation of sinus node myocardium are independently regulated by Tbx18 and Tbx3[J]. Circ Res,2009,104(3):388-397
24 Hoogaars WM,Engel A,Brons JF,et al. Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria[J].Genes Dev,2007,21(9):1098-1112
25 Espinoza-Lewis RA,Liu H,Sun C,et al. Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice[J]. Dev Biol,2011,356(2):359-369
26 Hausburg F,Jung JJ,David R. Specific cell(Re-)programming:approaches and perspectives[J]. Adv Biochem Eng Biotechnol,2018,163:71-115