Enhanced cardiomyogenesis of human embryonic stem cells by a small molecular inhibitor of p38 MAPK.
Human embryonic stem cells (hESC) can differentiate to cardiomyocytes in vitro but with generally poor efficiency. Here, we describe a novel method for the efficient generation of cardiomyocytes from hESC in a scalable suspension culture process. Differentiation in serum-free medium conditioned by the cell line END2 (END2-CM) readily resulted in differentiated cell populations with more than 10% cardiomyocytes without further enrichment. By screening candidate molecules, we have identified SB203580, a specific p38 MAP kinase inhibitor, as a potent promoter of hESC-cardiogenesis. SB203580 at concentrations <10 microM, induced more than 20% of differentiated cells to become cardiomyocytes and increased total cell numbers, so that the overall cardiomyocyte yield was approximately 2.5-fold higher than controls. Gene expression indicated that early mesoderm formation was favored in the presence of SB203580. Accordingly, transient addition of the inhibitor at the onset of differentiation only was sufficient to determine the hESC fate. Patch clamp electrophysiology showed that the distribution of cardiomyocyte phenotypes in the population was unchanged by the compound. Interestingly, cardiomyogenesis was strongly inhibited at SB203580 concentrations > or =15 microM. Thus, modulation of the p38MAP kinase pathway, in combination with factors released by END2 cells, plays an essential role in early lineage determination in hESC and the efficiency of cardiomyogenesis. Our findings contribute to transforming human cardiomyocyte generation from hESC into a robust and scalable process.
Graichen
Ralph
R
ES Cell International Pte Ltd., 11 Biopolis Way Singapore 138667, Singapore.
Xu
Xiuqin
X
Braam
Stefan R
SR
Balakrishnan
Thavamalar
T
Norfiza
Siti
S
Sieh
Shirly
S
Soo
Set Yen
SY
Tham
Su Chin
SC
Mummery
Christine
C
Colman
Alan
A
Zweigerdt
Robert
R
Davidson
Bruce P
BP
D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016895:Culture Media, Serum-Free; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006321:Heart / Q000196:embryology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*
D016895:Culture Media, Serum-Free; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D048051:p38 Mitogen-Activated Protein Kinases; C093642:SB 203580