This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells. A fusion gene consisting of the ␣ -cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures ( Ͼ 99% pure) were highly differentiated. G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice.

Fetal cardiomyocytes isolated from transgenic mice carrying a fusion gene of the alpha-cardiac myosin heavy chain promoter with a beta-galactosidase reporter were examined for their ability to form stable intracardiac grafts. Embryonic day 15 transgenic cardiomyocytes delivered directly into the myocardium of syngeneic hosts formed stable grafts, as identified by nuclear beta-galactosidase activity. Grafted cardiomyocytes were observed as long as 2 months after implantation, the latest date assayed. Intracardiac graft formation did not induce overtly negative effects on the host myocardium and was not associated with chronic immune rejection. Electron microscopy revealed the presence of nascent intercalated disks connecting the engrafted fetal cardiomyocytes and the host myocardium. These results suggest that intracardiac grafting might provide a useful approach for myocardial repair, provided that the grafted cells can contribute to myocardial function.

A novel member of the winged helix (formerly HNF-3/ Forkhead) transcriptional regulatory family, termed Genesis, was isolated and characterized. Putative translation of the complete cDNA revealed the winged helix DNA binding domain to be centrally located within the protein, with regions on either side that contain known transcriptional regulatory motifs. Extensive Northern analysis of Genesis found that the message was exclusively expressed in embryonic stem cells or their malignant equivalent, embryonal carcinoma cells. The Genesis transcript was down-regulated when these cells were stimulated to differentiate. DNA sequences that Genesis protein would interact with were characterized and were found to contain a consensus similar to that found in an embryonic stem cell enhancer sequence. Co-transfection experiments revealed that Genesis is a transcriptional repressor. Genesis mapped to mouse chromosome 4 in a region syntenic with human chromosome 1p31, a site of nonrandom abnormalities in germ cell neoplasia, neuroblastoma, and acute lymphoblastic leukemia. Genesis is a candidate for regulating the phenotype of normal or malignant embryonic stem cells.

We have assessed the ability of skeletal myoblasts to form long-term, differentiated grafts in ventricular myocardium.C2C12 myoblasts were grafted directly into the heart of syngeneic mice. Viable grafts were observed as long as 3 mo after implantation. Immunohistological analyses revealed the presence of differentiated myotubes that stably expressed the skeletal myosin heavy chain isoform. Thymidine uptake studies indicated that virtually all of the grafted skeletal myocytes were withdrawn from the cell cycle by 14 d after grafting. Graft myocytes exhibited ultrastructural characteristics typical of differentiated myotubes. Graft formation and the associated myocardial remodeling did not induce overt cardiac arrhythmia. This study indicates that the myocardium can serve as a stable platform for skeletal myoblast grafts. The long-term survival, differentiated phenotype, and absence of sustained proliferative activity observed in myoblast grafts raise the possibility that similar grafting approaches may be used to replace diseased myocardium. Furthermore, the genetic tractability of myoblasts could provide a useful means for the local delivery of recombinant molecules to the heart. (J. Clin. Invest. 1993.