Appropriate Exogenous Expression Stoichiometry of GATA4 as an Important Factor for Cardiac Reprogramming of Human Dermal Fibroblasts
Appropriate Exogenous Expression Stoichiometry of GATA4 as an Important Factor for Cardiac Reprogramming of Human Dermal Fibroblasts
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GATA4 的适当外源表达化学计量是人真皮成纤维细胞心脏重编程的重要因素
DOI：
10.1089/cell.2022.0014
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发表时间：
2022-06-28
影响因子：
1.6
通讯作者：
Kang，Kai
中科院分区：
文献类型：
--
作者：
Zhang，Xiangyu;Zhang，Qi;Kang，Kai
将人皮肤成纤维细胞（HDF）重编程为诱导的心肌样细胞（iCM）代表了人类心脏再生的一种有前途的策略。心脏转录因子的不同混合物可以将HDF转化为iCM，尽管效率低且表型不成熟。在这里，GATA 4，MEF 2C，TBX 5，MESP 1和MYOCD（简称GMTMeMy）被用来通过逆转录病毒感染重编程HDF。我们发现GATA 4的外源表达化学计量（GATA 4化学计量）显著影响重编程效率。以心肌基因表达水平、心肌肌钙蛋白T和α-心肌肌球蛋白重链免疫阳性细胞百分比以及与小鼠CM共培养时出现钙振荡或同步搏动的iCM数量为指标，采用1/8剂量的GATA 4病毒（GATA 4剂量）+MTMeMy时，重编程效率较编码各因子的平均合并病毒明显提高。此外，我们还利用H9人胚胎干细胞系的条件维持液（CMM）分化制备了条件维持液。结果发现，与传统的维持液相比，CMM使iCM更早地显示出组织良好的肌节形成和特征性的钙振荡波。这些发现表明，适当的GATA 4化学计量对于心脏重编程是必不可少的，并且CMM中的一些组分对于iCM的成熟是重要的。
Reprogramming of human dermal fibroblasts (HDFs) into induced cardiomyocyte-like cells (iCMs) represents a promising strategy for human cardiac regeneration. Different cocktails of cardiac transcription factors can convert HDFs into iCMs, although with low efficiency and immature phenotype. Here, GATA4, MEF2C, TBX5, MESP1, and MYOCD (GMTMeMy for short) were used to reprogram HDFs by retrovirus infection. We found that the exogenous expression stoichiometry of GATA4 (GATA4 stoichiometry) significantly affected reprogramming efficiency. When 1/8 dosage of GATA4 virus (GATA4 dosage) plus MTMeMy was used, the reprogramming efficiency was obviously improved compared with average pooled virus encoding each factor, which measured, by the expression level of cardiac genes, the percentage of cardiac troponin T and alpha-cardiac myosin heavy-chain immunopositive cells and the numbers of iCMs showing calcium oscillation or beating synchronously in co-culture with mouse CMs. In addition, we prepared conditioned maintenance medium (CMM) by CM differentiation of H9 human embryonic stem cell line. We found that compared with traditional maintenance medium (TMM), CMM made iCMs show well-organized sarcomere formation and characteristic calcium oscillation wave earlier. These findings demonstrated that appropriate GATA4 stoichiometry was essential for cardiac reprogramming and some components in CMM were important for maturation of iCMs.