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

Department of Medicine, University of California, San Francisco, California, USA.
Cytotherapy (Impact Factor: 3.29). 10/2010; 12(6):807-17. DOI: 10.3109/14653249.2010.491821
Source: PubMed


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.
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.
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.
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.

Download full-text


Available from: Richard E Sievers, Oct 02, 2015
20 Reads
  • Source
    • "SB203580-treated hEBs display an increase in expression of both early mesoderm markers (Brachyury T, Tbx6, and Mesp1) and cardiac αMHC as well as increased cardiomyocyte numbers. Gaur and colleagues [38] subsequently showed that p38MAPK inhibition occurs in a dose- and stage-dependent manner, that it also causes the accelerated differentiation of hESC-derived cardiomyocytes by using the standard hEB formation method, and that it appears to act at the ectoderm/mesoendoderm branch point during hESC differentiation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Human embryonic stem cells have the capacity for self-renewal and pluripotency and thus are a primary candidate for tissue engineering and regenerative therapies. These cells also provide an opportunity to study the development of human tissues ex vivo. To date, numerous human embryonic stem cell lines have been derived and characterized. In this review, we will detail the strategies used to direct tissue-specific differentiation of embryonic stem cells. We also will discuss how these strategies have produced new sources of tissue-specific progenitor cells. Finally, we will describe the next generation of methods being developed to identify and select stem cell-derived tissue precursors for experimental study and clinical use.
    Stem Cell Research & Therapy 05/2012; 3(3):17. DOI:10.1186/scrt108 · 3.37 Impact Factor
  • Source
    • "No data are available regarding hCSC. However, a role played by p38MAPK inhibition in inducing myocyte differentiation of embryonic stem cells has been reported(Gaur, et al., 2010). "
    Senescence, 02/2012; , ISBN: 978-953-51-0144-4
  • Source
    • "This protocol can be enhanced by adding BMP4 during d0-d4 (Takei et al., 2009), by the addition of WNT3A during d0-d2 (Tran et al., 2009), and via small molecule inhibition of WNT signaling by IWR-1 during d4-d6 (Ren et al., 2011). FBS-and insulin-free media can support EB cardiac differentiation, the efficiency of which can be enhanced by adding prostaglandin I 2 and the MAP kinase inhibitor SB203580 (Xu et al., 2008), specifically between d4-d6 (Gaur et al., 2010). Factors such as WNT3A (Bu et al., 2009), G-CSF (Shimoji et al., 2010), and L–ascorbic acid (Cao et al., 2011) can improve cardiac differentiation by enhancing cardiac progenitor cell proliferation, whereas IGF1 and IGF2 can enhance hPSC-derived cardiomyocyte proliferation (McDevitt et al., 2005). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cardiovascular disease is a leading cause of death worldwide. The limited capability of heart tissue to regenerate has prompted methodological developments for creating de novo cardiomyocytes, both in vitro and in vivo. Beyond uses in cell replacement therapy, patient-specific cardiomyocytes may find applications in drug testing, drug discovery, and disease modeling. Recently, approaches for generating cardiomyocytes have expanded to encompass three major sources of starting cells: human pluripotent stem cells (hPSCs), adult heart-derived cardiac progenitor cells (CPCs), and reprogrammed fibroblasts. We discuss state-of-the-art methods for generating de novo cardiomyocytes from hPSCs and reprogrammed fibroblasts, highlighting potential applications and future challenges.
    Cell stem cell 01/2012; 10(1):16-28. DOI:10.1016/j.stem.2011.12.013 · 22.27 Impact Factor
Show more