Increased cardiomyocyte differentiation from human embryonic stem cells in serum-free cultures.
Human embryonic stem cells (hESCs) can differentiate into cardiomyocytes, but the efficiency of this process is low. We routinely induce cardiomyocyte differentiation of the HES-2 cell line by coculture with a visceral endoderm-like cell line, END-2, in the presence of 20% fetal calf serum (FCS). In this study, we demonstrate a striking inverse relationship between cardiomyocyte differentiation and the concentration of FCS during HES-2-END-2 coculture. The number of beating areas in the cocultures was increased 24-fold in the absence of FCS compared with the presence of 20% FCS. An additional 40% increase in the number of beating areas was observed when ascorbic acid was added to serum-free cocultures. The increase in serum-free cocultures was accompanied by increased mRNA and protein expression of cardiac markers and of Isl1, a marker of cardiac progenitor cells. The number of beating areas increased up to 12 days after initiation of coculture of HES-2 with END-2 cells. However, the number of alpha-actinin-positive cardiomyocytes per beating area did not differ significantly between serum-free cocultures (503 +/- 179; mean +/- standard error of the mean) and 20% FCS cocultures (312 +/- 227). The stimulating effect of serum-free coculture on cardiomyocyte differentiation was observed not only in HES-2 but also in the HES-3 and HES-4 cell lines. To produce sufficient cardiomyocytes for cell replacement therapy in the future, upscaling cardiomyocyte formation from hESCs is essential. The present data provide a step in this direction and represent an improved in vitro model, without interfering factors in serum, for testing other factors that might promote cardiomyocyte differentiation.
Passier
Robert
R
Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, Netherlands.
Oostwaard
Dorien Ward-van
DW
Snapper
Jolanda
J
Kloots
Jantine
J
Hassink
Rutger J
RJ
Kuijk
Ewart
E
Roelen
Bernard
B
de la Riviere
Aart Brutel
AB
Mummery
Christine
C
D000185:Actinin / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008856:Microscopy, Fluorescence; D032383:Myocytes, Cardiac / Q000166:cytology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*
D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D000185:Actinin