Timed inhibition of p38MAPK directs accelerated differentiation of human embryonic stem cells into cardiomyocytes

Abstract

Background aims Heart failure therapy with human embryonic stem cell (hESC)-derived cardiomyocytes (hCM) has been limited by the low rate of spontaneous hCM differentiation. As others have shown that p38 mitogen-activated protein kinase (p38MAPK) directs neurogenesis from mouse embryonic stem cells, we investigated whether the p38MAPK inhibitor, SB203580, might influence hCM differentiation. Methods We treated differentiating hESC with SB203580 at specific time-points, and used flow cytometry, immunocytochemistry, quantitative real-time (RT)–polymerase chain reaction (PCR), teratoma formation and transmission electron microscopy to evaluate cardiomyocyte formation. Results We observed that the addition of inhibitor resulted in 2.1-fold enrichment of spontaneously beating human embryoid bodies (hEB) at 21 days of differentiation, and that 25% of treated cells expressed cardiac-specific α-myosin heavy chain. This effect was dependent on the stage of differentiation at which the inhibitor was introduced. Immunostaining and teratoma formation assays demonstrated that the inhibitor did not affect hESC pluripotency; however, treated hESC gave rise to hCM exhibiting increased expression of sarcomeric proteins, including cardiac troponin T, myosin light chain and α-myosin heavy chain. This was consistent with significantly increased numbers of myofibrillar bundles and the appearance of nascent Z-bodies at earlier time-points in treated hCM. Treated hEB also demonstrated a normal karyotype by array comparative genomic hybridization and viability in vivo following injection into mouse myocardium. Conclusions These studies demonstrate that p38MAPK inhibition accelerates directed hCM differentiation from hESC, and that this effect is developmental stage-specific. The use of this inhibitor should improve our ability to generate hESC-derived hCM for cell-based therapy.

Stem cell therapy for cardiac disease

“…Similarly, 5-azacytidine treatment during hESC differentiation has been shown to enhance CM differentiation, suggesting that DNA demethylation is a key factor in directing tissue-specific differentiation (48). Exposure to SB203580, a small-molecule inhibitor of p38 MAPK , has also been observed to improve the efficiency of CM differentiation from hESCs (49,50) and has implicated p38 MAPK in the regulation of the ectoderm-mesoendoderm switch during early ESC differentiation (50). By mimicking the signaling environment of the early mouse embryo, Yang et al established a three-stage protocol that supports cardiac development at high frequency in differentiating hESC cultures (51).…”

Differentiation of Human Atrial Myocytes From Endothelial Progenitor Cell-Derived Induced Pluripotent Stem Cells

“…However, inhibition of P38-MAPK pathway is required during the later stage of differentiation of cardiac progenitor cells toward immature CMs. Study have shown that inhibition of this pathway 12 days after starting EB cardiac differentiation improves cardiomyogenesis with up to 80% beating cells [269,270]. P38 was shown to regulate the mitosis gene expression such as cyclin A and cyclin B in CM that correlate with the cardiac growth during the development.…”

Differential Effects of Retinoids and Inhibitors of ERK and p38 Signaling on Adipogenic and Myogenic Differentiation of P19 Stem Cells

“…P38 has an antiadipogenic action [21], while its activity appears to be required in myogenesis [17,[22][23][24][25][26]. However, three recent studies have shown that the use of small concentrations of the p38 inhibitor SB203580 ( £ 10 mM) rather promotes than inhibits cardiomyogenesis [27][28][29]. ERK activity is required for adipogenesis [30][31][32][33], but its influence in myogenesis is uncertain.…”

“…It is worth noting that in those cases compared to our study, myogenesis was triggered in the absence of exogenous atRA or retinoid. In addition, the impact of the p38 inhibitor SB203580 on cardiomyogenesis was found to be concentration dependent, stimulating at £ 10 mM and inhibiting at higher concentrations [27][28][29]. Because of our desire to increase the myogenic yields, we used the inhibitor at 10 mM with atRA and found equivalent and even ameliorated yields compared to atRA alone (Fig.…”