Thiele Frauke, Voelkner Christin, Krebs Vivien, Müller Paula, Jung Julia Jeannine, Rimmbach Christian, Steinhoff Gustav, Noack Thomas, David Robert, Lemcke Heiko
Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University Medicine Rostock, Rostock, Germany.
Faculty of Interdisciplinary Research, Department Life, Light & Matter, University Rostock, Rostock, Germany.
Cell Physiol Biochem. 2019;53(2):337-354. doi: 10.33594/000000142.
BACKGROUND/AIMS: The availability of truly maturated cardiomyocytic subtypes is a major prerequisite for cardiovascular cell replacement therapies. Pluripotent stem cells provide a suitable source for the development of new strategies to overcome enormous hurdles such as yield, purity and safety of in vitro generated cells.
To address these issues, we have refined existing forward programming protocols by combining forced exogenous overexpression of the early cardiovascular transcription factor Nkx2.5 with a αMHC-promoter-based antibiotic selection step. Additionally, we applied small molecules such as ascorbic acid to enhance cardiomyogenic differentiation efficiency. Subsequently, we evaluated the cell fate of the resulting cardiomyocytes on the mRNA as well as protein levels. The latter was performed using high-resolution confocal microscopy. Furthermore, we examined the response of the cells` beating activities to pharmacological substance administration.
Our results reveal an apparent influence of Nkx2.5 on the cell fate of ESC-derived cardiomyocytes. Resulting single cells exhibit characteristics of early ventricular cardiomyocytes, such as sarcomeric marker expression, spontaneous beating frequency, and distinct L-type calcium channel occurrence.
Therefore, we demonstrate cardiovascular subtype forward programming of ESCs using a combination of transcription factors along with small molecule administration. However, our findings also underline current assumptions, that a terminal maturation of PSC derived cardiomyocytes in vitro is still an unsolved problem which urgently needs to be addressed in the field.
背景/目的：真正成熟的心肌细胞亚型的可得性是心血管细胞替代疗法的主要前提条件。多能干细胞为开发新策略提供了合适的来源，以克服体外生成细胞的产量、纯度和安全性等巨大障碍。
为了解决这些问题，我们通过将早期心血管转录因子Nkx2.5的强制外源性过表达与基于αMHC启动子的抗生素选择步骤相结合，优化了现有的正向编程方案。此外，我们应用了小分子如抗坏血酸来提高心肌分化效率。随后，我们在mRNA和蛋白质水平上评估了所得心肌细胞的细胞命运。后者使用高分辨率共聚焦显微镜进行。此外，我们研究了细胞搏动活动对药物给药的反应。
我们的结果揭示了Nkx2.5对胚胎干细胞衍生心肌细胞的细胞命运有明显影响。所得单细胞表现出早期心室心肌细胞的特征，如肌节标记物表达、自发搏动频率和独特的L型钙通道出现。
因此，我们证明了使用转录因子与小分子给药相结合对胚胎干细胞进行心血管亚型正向编程。然而，我们的研究结果也强调了当前的假设，即体外多能干细胞衍生心肌细胞的终末成熟仍然是一个未解决的问题，该领域迫切需要解决。
