Retinoic acid promotes metabolic maturation of human Embryonic Stem Cell-derived Cardiomyocytes

Abstract

Cardiomyocytes differentiated from human embryonic stem cells (hESCs) represent a promising cell source for heart repair, disease modeling and drug testing. However, improving the differentiation efficiency and maturation of hESC-derived cardiomyocytes (hESC-CMs) is still a major concern. Retinoic acid (RA) signaling plays multiple roles in heart development. However, the effects of RA on cardiomyocyte differentiation efficiency and maturation are still unknown. Methods: RA was added at different time intervals to identify the best treatment windows for cardiomyocyte differentiation and maturation. The efficiency of cardiomyocyte differentiation was detected by quantitative real-time PCR and flow cytometry. Cardiomyocytes maturation was detected by immunofluorescence staining, metabolic assays and patch clamp to verify structural, metabolic and electrophysiological maturation, respectively. RNA sequencing was used for splicing analysis. Results: We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of TNNT2 , NKX2.5 and MYH6 on day 10 of differentiation. In addition, flow cytometry showed that the proportion of differentiated cardiomyocytes in the RA-treated group was significantly higher than that in control group. RA treatment on days 15-20 increased cardiomyocyte area, sarcomere length, multinucleation and mitochondrial copy number. RNA sequencing revealed RA promoted RNA isoform switch to the maturation-related form. Meanwhile, RA promoted electrophysiological maturation and calcium handling of hESC-CMs. Importantly, RA-treated cardiomyocytes showed decreased glycolysis and enhanced mitochondrial oxidative phosphorylation, with the increased utilization of fatty acid and exogenous pyruvate but not glutamine. Conclusion: Our data indicated that RA treatment at an early time window (days 2-4) promotes the efficiency of cardiomyocyte differentiation and that RA treatment post beating (days 15-20) promotes cardiomyocyte maturation. The biphasic effects of RA provide new insights for improving cardiomyocyte differentiation and quality.

Mitochondria and metabolic transitions in cardiomyocytes: lessons from development for stem cell-derived cardiomyocytes

“…Treatment of PSC-CMs with supraphysiologic levels of ATRA (1 μmol/L) during early differentiation (days 2-4, mesoderm stage) increased expression of cardiac markers TNNT2, NKX2.5, and MYH6 by day 10, while treatment with retinoic acid from days 15-20 of differentiation enhanced cardiomyocyte size, increased action potential duration, and increased mitochondrial content and maximal respiration capacity [109]. Duration and dose of retinoic acid appear to be important factors in directing cardiomyocytes toward their desired phenotype, as treatment with 500 nmol/L ATRA from days 3-12 of differentiation leads to specification of an atrial cardiomyocyte phenotype rather than ventricular cardiomyocyte maturation [110].…”

Intermittent Starvation Promotes Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes

“…Thus, combinatorial approaches might be more efficient and necessary to accurately control the maturation of hPSC-CMs to meet different requirements in disease-modeling and clinical applications. Our recent study has indicated that retinoid acid (RA) treatment could also enhance hPSC-CM maturation (Miao et al, 2020), and in the future the combined treatment is expected to further optimize the protocol for hPSC-CM maturation.…”

“…The hESCs at 80% confluence were passaged using 0.5 mM EDTA (Sigma, United States), and 2 µM thiazovivin (Selleck Chemicals, United States) was supplemented for 24 h after cell passage. Cardiomyocyte differentiation was induced in the chemically defined medium CDM3 which is made of RPMI1640 (Thermo Fisher, United States), bovine serum albumin (Sigma-Aldrich, United States) and L-ascorbic acid 2-phosphate (Sigma-Aldrich, United States) as previously described (Miao et al, 2020). Briefly, hESCs at 95% confluence were first cultured in CDM3 supplemented with 5 µM CHIR99021 (Sigma-Aldrich, United States) for 2 days to induce mesoderm differentiation, and the medium was then refreshed with CDM3.…”

“…Many approaches such as long-term culture, biophysical, biochemical, and bioelectrical stimulations, have been developed to enhance the maturity of hPSC-CMs (Hirt et al, 2014;Yang et al, 2014). For instance, we and others have reported that retinoic acid and fatty acid treatment could enhance the structural, metabolic and electrophysiological maturation of hPSC-CMs (Yang et al, 2019;Miao et al, 2020). In addition, tissue engineering, 3D culture and miRNAs were also used to promote hPSC-CMs maturation (Lee et al, 2015;Sun and Nunes, 2017).…”

Regenerating Damaged Myocardium: A Review of Stem-Cell Therapies for Heart Failure

“…PSC-CMs also more closely resemble embryonic than adult cardiomyocytes [79,80], which has led researchers to seek methods for improving PSC-CM maturity. Treatment with retinoic acid led to increases in hESC-CM yield when added 2-4 days after differentiation was initiated and to improvements in maturity (e.g., increases in surface area, sarcomere length, multinucleation, and mitochondrial copy number) when added after the cells had begun beating [81]; however, retinoic acid also appears to direct cardiomyocyte differentiation toward an atrial-like phenotype and may promote the reentry-like conduction disorders associated with atrial arrhythmia [82]. Cardiac fibroblasts, which are the primary producers of cardiac extracellular matrix (ECM) proteins, also improved maturation when included in cultures of hiPSC-CMs [83], and for engineered heart tissues, efforts to improve PSC-CM maturity have led to the development of techniques that recreate the electromechanical properties of native myocardium.…”