人诱导多能干细胞分化的心肌细胞电生理鉴定
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摘要
本文旨在研究人诱导多能干细胞分化的心肌细胞(human induced pluripotent stem cell-derived cardiomyocytes,hiPSC-CMs)的电生理特性。采用时序性短暂激活/短暂抑制Wnt信号通路的方法将未分化的IMR90-4细胞系定向诱导分化为心肌细胞。用免疫荧光染色法和流式细胞术检测心肌肌钙蛋白T(cardiac troponin T,cTnT)蛋白表达,计算hiPSC-CMs的分化率。用膜片钳技术记录hiPSC-CMs的动作电位,根据动作电位的表现对心肌细胞进行分类,并进一步分析电生理特征。结果显示,hiPSC-CMs的纯度大于95%。根据动作电位的表现,hiPSC-CMs可分为心房肌样细胞、心室肌样细胞和窦房结样细胞。其中,心室肌样细胞的动作电位时程(action potential duration,APD)、动作电位幅度(action potential amplitude,APA)和最大去极化速率(dV/dt_(max))均大于心房肌样细胞和窦房结样细胞,窦房结样细胞的d V/dt_(max)低于心室肌样细胞和心房肌样细胞。以上结果表明hiPSC-CMs纯度高,并且分化出的三种不同类型的心肌细胞具有和成熟心肌细胞相似的动作电位特征。
The present study was aimed to characterize the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs). IMR90-4 cells were induced to differentiate into cardiomyocytes by temporal modulation of regulators of canonical Wnt signaling. The protein expression of cardiac troponin T(cTnT) was detected by immunofluorescence staining and flow cytometry, and the differentiation rate of hiPSC-CMs was calculated. The action potentials(APs) of hiPSC-CMs were recorded by patch clamp and used to classify different types of cardiomyocytes. The electrophysiological characteristics of hiPSC-CMs were further analyzed. The results showed that the c Tn T positive rate of hiPSC-CMs was above 95%. hiPSC-CMs were differentiated into 3 types of cardiomyocytes based on the properties of AP: ventricular-, atrial-and nodal-like cells. In comparison with the other two types of cells, the APs of ventricular-like cells exhibited longer duration, higher amplitude and higher d V/dt_(max). The nodal-like cells had the lowest d V/dt_(max) among all the three types. These results indicate that hiPSC can be differentiated into the cardiomyocytes with high purity and the differentiated hiPSC-CMs have similar electrophysiological characteristics to adult cardiomyocytes.
The present study was aimed to characterize the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs). IMR90-4 cells were induced to differentiate into cardiomyocytes by temporal modulation of regulators of canonical Wnt signaling. The protein expression of cardiac troponin T(cTnT) was detected by immunofluorescence staining and flow cytometry, and the differentiation rate of hiPSC-CMs was calculated. The action potentials(APs) of hiPSC-CMs were recorded by patch clamp and used to classify different types of cardiomyocytes. The electrophysiological characteristics of hiPSC-CMs were further analyzed. The results showed that the c Tn T positive rate of hiPSC-CMs was above 95%. hiPSC-CMs were differentiated into 3 types of cardiomyocytes based on the properties of AP: ventricular-, atrial-and nodal-like cells. In comparison with the other two types of cells, the APs of ventricular-like cells exhibited longer duration, higher amplitude and higher d V/dt_(max). The nodal-like cells had the lowest d V/dt_(max) among all the three types. These results indicate that hiPSC can be differentiated into the cardiomyocytes with high purity and the differentiated hiPSC-CMs have similar electrophysiological characteristics to adult cardiomyocytes.
引文
1 Yang X,Pabon L,Murry CE.Engineering adolescence:maturation of human pluripotent stem cell-derived cardiomyocytes.Circ Res 2014;114(3):511-523.
2 Cao N,Liu Z,Chen Z,Wang J,Chen T,Zhao X,Ma Y,Qin L,Kang J,Wei B,Wang L,Jin Y,Yang HT.Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells.Cell Res 2012;22(1):219-236.
3 Mummery CL,Zhang J,Ng ES,Elliott DA,Elefanty AG,Kamp TJ.Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes:a methods overview.Circ Res 2012;111(3):344-358.
4 Lian X,Zhang J,Azarin SM,Zhu K,Hazeltine LB,Bao X,Hsiao C,Kamp TJ,Palecek SP.Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions.Nat Protoc 2013;8(1):162-175.
5 Sharma A,Li G,Rajarajan K,Hamaguchi R,Burridge PW,Wu SM.Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.J Vis Exp 2015;97:52628.
6 Yang X,Rodriguez M,Pabon L,Fischer KA,Reinecke H,Regnier M,Sniadecki NJ,Ruohola-Baker H,Murry CE.Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.J Mol Cell Cardiol 2014;72:296-304.
7 Lee JH,Protze SI,Laksman Z,Backx PH,Keller GM.Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations.Cell Stem Cell 2017;21(2):179-194.e4.
8 Conrath CE,Opthof T.Ventricular repolarization:an overview of(patho)physiology,sympathetic effects and genetic aspects.Prog Biophys Mol Biol 2006;92(3):269-307.
9 Sanchez C,Bueno-Orovio A,Wettwer E,Loose S,Simon J,Ravens U,Pueyo E,Rodriguez B.Inter-subject variability in human atrial action potential in sinus rhythm versus chronic atrial fibrillation.PLo S One 2014;9(8):e105897.
10 Ma J,Guo L,Fiene SJ,Anson BD,Thomson JA,Kamp TJ,Kolaja KL,Swanson BJ,January CT.High purity humaninduced pluripotent stem cell-derived cardiomyocytes:electrophysiological properties of action potentials and ionic currents.Am J Physiol Heart Circ Physiol 2011;301(5):H2006-H2017.
11 He JQ,Ma Y,Lee Y,Thomson JA,Kamp TJ.Human embryonic stem cells develop into multiple types of cardiac myocytes:action potential characterization.Circ Res 2003;93(1):32-39.
12 Jonsson MK,Duker G,Tropp C,Andersson B,Sartipy P,Vos MA,van Veen TA.Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes.Stem Cell Res 2010;4(3):189-200.
13 Karakikes I,Ameen M,Termglinchan V,Wu JC.Human induced pluripotent stem cell-derived cardiomyocytes:insights into molecular,cellular,and functional phenotypes.Circ Res 2015;117(1):80-88.
14 Priori SG,Napolitano C,Di Pasquale E,Condorelli G.Induced pluripotent stem cell-derived cardiomyocytes in studies of inherited arrhythmias.J Clin Invest 2013;123(1):84-91.
15 Paci M,Hyttinen J,Rodriguez B,Severi S.Human induced pluripotent stem cell-derived versus adult cardiomyocytes:an in silico electrophysiological study on effects of ionic current block.Br J Pharmacol 2015;172(21):5147-5160.
16 Kim JJ,Yang L,Lin B,Zhu X,Sun B,Kaplan AD,Bett GC,Rasmusson RL,London B,Salama G.Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells.J Mol Cell Cardiol 2015;81:81-93.
17 Du DT,Hellen N,Kane C,Terracciano CM.Action potential morphology of human induced pluripotent stem cell-derived cardiomyocytes does not predict cardiac chamber specificity and is dependent on cell density.Biophys J 2015;108(1):1-4.
2 Cao N,Liu Z,Chen Z,Wang J,Chen T,Zhao X,Ma Y,Qin L,Kang J,Wei B,Wang L,Jin Y,Yang HT.Ascorbic acid enhances the cardiac differentiation of induced pluripotent stem cells through promoting the proliferation of cardiac progenitor cells.Cell Res 2012;22(1):219-236.
3 Mummery CL,Zhang J,Ng ES,Elliott DA,Elefanty AG,Kamp TJ.Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes:a methods overview.Circ Res 2012;111(3):344-358.
4 Lian X,Zhang J,Azarin SM,Zhu K,Hazeltine LB,Bao X,Hsiao C,Kamp TJ,Palecek SP.Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/beta-catenin signaling under fully defined conditions.Nat Protoc 2013;8(1):162-175.
5 Sharma A,Li G,Rajarajan K,Hamaguchi R,Burridge PW,Wu SM.Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.J Vis Exp 2015;97:52628.
6 Yang X,Rodriguez M,Pabon L,Fischer KA,Reinecke H,Regnier M,Sniadecki NJ,Ruohola-Baker H,Murry CE.Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.J Mol Cell Cardiol 2014;72:296-304.
7 Lee JH,Protze SI,Laksman Z,Backx PH,Keller GM.Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations.Cell Stem Cell 2017;21(2):179-194.e4.
8 Conrath CE,Opthof T.Ventricular repolarization:an overview of(patho)physiology,sympathetic effects and genetic aspects.Prog Biophys Mol Biol 2006;92(3):269-307.
9 Sanchez C,Bueno-Orovio A,Wettwer E,Loose S,Simon J,Ravens U,Pueyo E,Rodriguez B.Inter-subject variability in human atrial action potential in sinus rhythm versus chronic atrial fibrillation.PLo S One 2014;9(8):e105897.
10 Ma J,Guo L,Fiene SJ,Anson BD,Thomson JA,Kamp TJ,Kolaja KL,Swanson BJ,January CT.High purity humaninduced pluripotent stem cell-derived cardiomyocytes:electrophysiological properties of action potentials and ionic currents.Am J Physiol Heart Circ Physiol 2011;301(5):H2006-H2017.
11 He JQ,Ma Y,Lee Y,Thomson JA,Kamp TJ.Human embryonic stem cells develop into multiple types of cardiac myocytes:action potential characterization.Circ Res 2003;93(1):32-39.
12 Jonsson MK,Duker G,Tropp C,Andersson B,Sartipy P,Vos MA,van Veen TA.Quantified proarrhythmic potential of selected human embryonic stem cell-derived cardiomyocytes.Stem Cell Res 2010;4(3):189-200.
13 Karakikes I,Ameen M,Termglinchan V,Wu JC.Human induced pluripotent stem cell-derived cardiomyocytes:insights into molecular,cellular,and functional phenotypes.Circ Res 2015;117(1):80-88.
14 Priori SG,Napolitano C,Di Pasquale E,Condorelli G.Induced pluripotent stem cell-derived cardiomyocytes in studies of inherited arrhythmias.J Clin Invest 2013;123(1):84-91.
15 Paci M,Hyttinen J,Rodriguez B,Severi S.Human induced pluripotent stem cell-derived versus adult cardiomyocytes:an in silico electrophysiological study on effects of ionic current block.Br J Pharmacol 2015;172(21):5147-5160.
16 Kim JJ,Yang L,Lin B,Zhu X,Sun B,Kaplan AD,Bett GC,Rasmusson RL,London B,Salama G.Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells.J Mol Cell Cardiol 2015;81:81-93.
17 Du DT,Hellen N,Kane C,Terracciano CM.Action potential morphology of human induced pluripotent stem cell-derived cardiomyocytes does not predict cardiac chamber specificity and is dependent on cell density.Biophys J 2015;108(1):1-4.