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
). Despite seemingly similar results, our study differs from their work in several important ways. Foremost, they differentiated hESC using serum-free medium conditioned by visceral endoderm-like mouse cells, whereas we used serum induction, a frequently employed method for spontaneous hESC differentiation (2
). Second, they studied HES2, HES3, HES3-GFP hESC lines, while we carried out our studies using another extensively characterized (H9) hESC line. The increase in cardiogenesis observed irrespective of differentiation protocol across three hESC lines [our work and (20
)] suggests that the effects of p38MAPK inhibition are not hESC line-specific. Third, the previously reported study examined cardiogenesis in a dose-dependent manner only, while we evaluated the effect of SB203580 in a developmental stage-specific manner. We show that the combination of timing and dose has an additive effect on cardiogenic enrichment. Our results emphasize the need for identifying the appropriate time-points during differentiation in culture to achieve a maximal effect. Each study adds significant details to the primary observation of p38MAPK inhibition as promoting hESC cardiogenesis, and the similar results using different hESC lines and different culture conditions (feeder-free serum replacement versus visceral endoderm co-culture) suggest that p38MAPK activity is important during hESC differentiation, and its manipulation may be useful in directing the differentiation of other mesoderm-derived tissues as well.
Others have observed that p38MAPK controls an early switch between commitment to mesoderm versus ectoderm during mouse embryonic stem cell (mESC) differentiation (19
). There appear to be two waves of p38MAPK activity, one peaking at an early time-point between days 2 and 5 of mESC differentiation, and one at a later time-point between days 12 and 16. The early activity appears to control the decision between mesoderm and ectoderm (29
), which is consistent with our finding that inhibition of p38MAPK at an early time-point (days 4–6) shifts differentiation toward cardiogenesis.
The early up-regulation of cardiac-specific genes in p38MAPK-inhibited hEB prompted our investigation into the ultrastructural maturity of treated versus untreated hCM. Surprisingly, we observed more mature sarcomere organization in the treated hEB. Previous studies have shown a progressive increase in the amount and organization of sarcomere structures coinciding with the developmental stage of hEB (3
) or differentiation conditions (30
). While we observed more extensive myofibrillar arrangements and evidence of nascent sarcomere formation earlier in treated cells than in untreated cells, we did not see characteristics of fully mature hCM in any of our samples. This variability in ultrastructural maturity between cultures, and even compared with fetal hearts, has been noted by others and thought to be because of differences between individual culture systems (3
). This variability emphasizes the need to specify the differences between hESC differentiation protocols, the use of various culture additives, and the role of different culture systems, such as MEF and visceral endoderm-like cells (30
In summary, we show that inhibition of p38MAPK during differentiation of hESC in co-culture with MEF results in accelerated differentiation of hCM and a 2.1-fold increase in hCM yield. We demonstrate that this enrichment is dependent on inhibiting p38MAPK at the time of ectoderm–mesoendoderm discrimination. We show that p38MAPK inhibition does not abolish pluripotency, as treated hESC remain competent at forming tissues from all three embryonic germ layers, and that the resulting hCM are genetically stable, can be purified by established methods (Percoll gradient) and are viable upon in vivo transplantation into mouse myocardium. These studies highlight further the utility of this method for producing hCM for therapeutic purposes.