Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology · 2019
Nkx2.5 Based Ventricular Programming of Murine ESC-Derived Cardiomyocytes.
СтатьяНазвание и аннотация: Перевод в очереди
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Thiele Frauke, Voelkner Christin, Krebs Vivien, Müller Paula, Jung Julia Jeannine, Rimmbach Christian, Steinhoff Gustav, Noack Thomas, David Robert, Lemcke Heiko
Аннотация
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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSION: 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.