Effect of culture time in vitro on maturity of induced pluripotent stem cell-derived cardiomyocytes

doi:10.12307/2025.517

Abstract

Abstract: BACKGROUND: It has been proved that induced pluripotent stem cells can differentiate into cardiomyocytes, but there are few reports on the maturity of differentiated cardiomyocytes.
OBJECTIVE: To explore the effect of prolonging the induced differentiation time on the morphology, sarcomere length, binuclear cell content, cardiac gene expression, cardiac protein expression, and mitochondrial function of cardiomyocytes derived from induced pluripotent stem cells.
METHODS: Bone morphogenetic protein 4, CHIR 99021, and IWR1 were used to induce pluripotent stem cells to differentiate into cardiomyocytes, and differentiated cardiomyocytes were collected on days 20 and 40 respectively. The expression levels of cardiac genes and proteins in differentiated cardiomyocytes were detected by RT-PCR and immunofluorescence, respectively. LAS X image analysis software was used to analyze the morphology and sarcomere length of differentiated cardiomyocytes. MitoTracker Green FM mitochondrial staining was used to detect total mitochondria. JC-1 mitochondrial staining was used to detect mitochondrial membrane potential.
RESULTS AND CONCLUSION: Differentiated cardiomyocytes on day 40 had longer cell circumference and sarcomere length, and larger cell area than those on day 20 (P < 0.05). The proportion of multinucleated cells rose sharply from about 16% on day 20 to about 29% on day 40 (P < 0.05). Differentiated cardiomyocytes on day 40 had gene expression levels that were more similar to those of the primary cardiomyocytes, and the expression levels of SERCA2A, Cx-43, and α-MHC genes were significantly higher than on day 20 (P < 0.05). Compared with the differentiated cardiomyocytes on day 20, the expression density of TNNT2 and α-MHC protein was relatively higher, the distribution density of mitochondria was larger, and the number of functional mitochondria was greater on day 40 (P < 0.05). The results show that prolonging the induced differentiation time can increase the maturity of differentiated cardiomyocytes by increasing the length of sarcomere and the number of functional mitochondria, as well as improving the expression levels of cardiac genes and proteins.

Key words: induced pluripotent stem cell, differentiated cardiomyocyte, mitochondria, cardiac gene, cardiac protein, maturity

CLC Number:

Xiong Tinglin, Zhang Lisha, Wang Dewei, Cao Haiping, Yang Yan. Effect of culture time in vitro on maturity of induced pluripotent stem cell-derived cardiomyocytes[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(25): 5304-5310.

Figures/Tables 2

2.1 不同时间点分化心肌细胞形态比较 LAS X图像分析软件分析第20天和第40天的分化心肌细胞免疫荧光图像的形态差异，共测量70个细胞。平均细胞周长从第20天时的(152.55±18.48) μm增加至第40天时的(182.71±21.59) μm，差异有显著性意义(P < 0.05)。同样，随着培养时间的延长，平均细胞面积从第20天时的(1 146.09±194.51) μm2显著增加到第40天时的(1 752.46±254.77) μm2 (P < 0.05)，见图2。 2.2 不同时间点分化心肌细胞肌节长度比较通过α-actin免疫荧光染色和LAS X图像分析软件来测量分化心肌细胞的肌节长度。第40天时分化心肌细胞有更高的密度和更明显的肌原纤维条纹排列，然而第20天时分化心肌细胞具有不规则的亚细胞组织和较低的肌原纤维密度。更成熟的分化心肌细胞进一步证据是肌节长度的显著延长，从第20天的(1.58±0.22) μm延长到第40天时的(1.96± 0.28) μm，差异有显著性意义(P < 0.05)，见图3。 2.3 不同时间点分化心肌细胞中多核细胞含量比较在成熟过程中，分化心肌细胞逐渐失去增殖能力，因此DNA复制不再与细胞分裂(胞质分裂)相关，更多的分化心肌细胞变成多核并通过间隙连接，形成功能性合胞体。α-actin和Hoechst 34580免疫荧光染色后随机观察200个分化心肌细胞，多核细胞比例从第20天的16%左右大幅上升至第40天的29%左右(P < 0.05)，见图4。 2.4 不同时间点分化心肌细胞中基因表达见图5。第20天和第40天时分化心肌细胞中TNNT2、Nkx2.5基因表达水平没有差异，其表达水平虽明显高于诱导多能干细胞(P < 0.05)，但明显低于原代心肌细胞(P < 0.05)，其中TNNT2对心肌细胞的自发搏动至关重要。 TNNI1基因在诱导多能干细胞和原代心肌细胞在几乎不表达，其在第40天的分化心肌细胞中的表达水平明显低于第20天的分化心肌细胞(P < 0.05)；而TNNI3基因在原代心肌细胞中的表达水平明显高于各时间点的分化心肌细胞和诱导多能干细胞(P < 0.05)，其在第40天的分化心肌细胞中的表达水平稍高于第20天的分化心肌细胞，但差异无显著性意义(P > 0.05)。随着培养时间的延长，β-MHC基因的表达水平逐渐下降，其在第20天分化心肌细胞中的表达水平明显高于第40天分化心肌细胞和原代心肌细胞(P < 0.05)；然而α-MHC基因表达水平出现了完全相反的结果，在原代心肌细胞中的表达水平明显高于各时间点的分化心肌细胞和诱导多能干细胞(P < 0.05)，第40天分化心肌细胞中的表达水平明显高于第20天分化心肌细胞(P < 0.05)。体外细胞分化培养过程中，检测到编码内向整流钾通道Kir2.1(KCNJ2)的基因表达水平升高，但差异无显著性意义(P > 0.05)，原代心肌细胞中的表达量明显高于分化心肌细胞和诱导多能干细胞(P < 0.05)。诱导多能干细胞和第20天分化心肌细胞的Cx-43基因表达水平几乎相同且明显低于第40天分化心肌细胞和原代心肌细胞(P < 0.05)，表明经过1个多月的心肌分化后，分化心肌细胞中的Cx-43表达量增加了2倍多，达到了原代心肌细胞的表达水平。肌浆网Ca2+-ATP酶基因(SERCA2a)在心肌细胞钙稳态的调节中起重要作用，其在原代心肌细胞的表达水平明显高于分化心肌细胞和诱导多能干细胞(P < 0.05)，随着体外培养时间的延长，其表达水平也明显升高(P < 0.05)。 2.5 不同时间点分化心肌细胞中心脏蛋白表达体外培养第20天和第40天的分化心肌细胞均阳性表达NKX2.5、TNNT2、α-MHC和CX-43心脏蛋白。NKX2.5和CX-43蛋白的表达水平在两个时间点都是相似的，而TNNT2和α-MHC蛋白的表达水平在第40天时相对较高，见图6。"

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