Elsevier

Cytotherapy

Volume 12, Issue 6, October 2010, Pages 807-817
Cytotherapy

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

https://doi.org/10.3109/14653249.2010.491821Get rights and content

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.

Introduction

Human cardiomyocytes (hCM) differentiated from various human embryonic stem cell (hESC) lines have been shown to exhibit properties similar to endogenous hCM, including cardiac-specific gene expression (1), sarcomere ultrastructure (2,3) and characteristic action potentials (4., 5., 6.). Successful engraftment of isolated beating clusters into various models of myocardial disease has generated enthusiasm for hESC as a source of new hCM for myocardial cell therapy (1,5,7,8).

The differentiation of hESC into hCM, however, is a low-yield process, as most groups have relied on spontaneous differentiation in the presence of serum (9., 10., 11.). For an effective therapy, the cardiac cells need to be generated in large numbers and with sufficient purity to avoid the formation of other tissues (11., 12., 13.). Xu et al. (10) reported increased cardiogenesis with the addition of the de-methylation agent azacytidine, followed by Percoll gradient separation. Mummery et al. (14,15) subsequently showed an increased cardiomyocyte yield by co-culturing hESC with a mouse endoderm-like cell line with or without serum. Other groups have since reported increased cardiogenesis by the addition of various culture additives, such as activin A and bone morphogenetic proteins 2 and 4 (16,17).

Pathways regulating the activity of p38 mitogen-activated protein kinase (p38MAPK) have been implicated in regulating mammalian hCM proliferation (18) and neural differentiation (19). Recently, Graichen et al. (20) detected the expression of p38MAPK in hESC as well as differentiating hEB and suggested that its inhibition could induce cardiogenesis. They reported enhanced cardiogenesis from hESC grown in co-culture with visceral endoderm cells in the presence of a p38MAPK inhibitor (20).

In this study, we used SB203580, a small-molecule inhibitor of p38MAPK, to study the dynamics of cardiogenesis with p38MAPK inhibition. We report that inhibition of p38MAPK during the stage of hESC differentiation that coincides with ectoderm/mesoendoderm divergence results in directed, accelerated differentiation of hCM, and that the resulting hCM maintain properties, such as genomic stability and survival in vivo, that are essential for cell transplant therapy.

Section snippets

hESC culture and differentiation

All hESC procedures were approved by the Stem Cell Research Oversight Committee at the University of California (San Francisco, CA, USA). The H9 hESC line (WA09) expressing enhanced green fluorescent protein (GFP) under the control of the ubiquitin C promoter (21) was maintained on irradiated CF1 mouse embryonic fibroblasts (MEF), as described previously (22). All reagents were purchased from Invitrogen, Carlsbad, CA, USA, except where indicated. hESC were cultured between passages 35–90 in KSR

Inhibition of p38MAPK directs differentiation of hESC-derived hCM in a stage- and dose-specific manner

To determine whether inhibition of p38MAPK alters the dynamics of hESC differentiation, we exposed hEB to the p38MAPK inhibitor, SB203580, for sequential time periods during in vitro differentiation (Figure 1A) and counted the number of beating hEB on day 21, based on earlier reports that contractile activity peaks by this time-point (9). We observed a significant increase in hCM formation, as evidenced by an increase in the percentage of hEB with spontaneous contractile activity, with exposure

Discussion

In the current study, we have shown that treatment of hESC with an inhibitor of p38MAPK (a) accelerated the directed differentiation of hESC into hCM in a developmental stage-specific manner, (b) did not affect hESC pluripotency, (c) did not induce genomic instability and (d) maintained viability of transplanted hCM in mouse myocardium.

During the course of our experiments, another group reported increased cardiogenesis after p38MAPK inhibition in endoderm co-cultures using hESC lines (20).

Acknowledgments

This work was supported by a Comprehensive Research Grant from the California Institute for Regenerative Medicine (RC1-00104), a Public Health Service Grant (HL085377) from NHLBI, and a gift from the Pollin Foundation to HSB; and funds from the UCSF Cardiac Stem Cell Foundation to YY. We thank Frank King for technical advice and Angela Feraco for assistance with statistical analysis.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the

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    These authors contributed equally to this work.

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