L'invention concerne une methode propre a induire une differenciation de cellules souches en cellules myocardiques, caracterisee en ce que cette differenciation s'obtient par culture des cellules souches en presence d'une substance qui inhibe le transfert du signal BMP, ce qui permet d'induire efficacement et selectivement la differenciation en cellules myocardiques.

Methode d'induction d'une differenciation de cellules souches en cellules myocardiques

Background Despite the pluripotency of embryonic stem (ES) cells, the specific control of their cardiomyogenic differentiation remains difficult. The aim of the present study was to investigate whether growth factors may efficiently enhance the in vitro cardiac differentiation of ES cells. Methods and Results Recombinant growth factors at various concentrations or their inhibitors were added according to various schedules during the cardiomyogenic differentiation of ES cells. Cardiomyogenic differentiation was assessed by mRNA and protein expressions of several cardiomyocyte-specific genes. Basic fibroblast growth factor-2 (FGF-2) and/or bone morphogenetic protein-2 (BMP-2) efficiently enhanced the cardiomyogenic differentiation, but only when they were added at the optimal concentration (1.0 ng/ml in FGF-2 and 0.2 ng/ml in BMP-2; relatively lower than expected in both cases) for the first 3 days. Inhibition of FGF-2 and/or BMP-2 drastically suppressed the cardiomyogenic differentiation. Conclusion FGF-2 and BMP-2 play a crucial role in early cardiomyogenesis. The achievement of efficient cardiac differentiation using both growth factors may facilitate ES cell-derived cell therapy for heart diseases as well as contribute to developmental studies of the heart. (Circ J 2004; 68: 691 - 702)

Efficient Cardiomyogenic Differentiation of Embryonic Stem Cell by Fibroblast Growth Factor 2 and Bone Morphogenetic Protein 2

Bone morphogenetic proteins (BMPs) have been shown to induce ectopic expression of cardiac transcription factors and beating cardiomyocytes in nonprecardiac mesodermal cells in chicks, suggesting that BMPs are inductive signaling molecules that participate in the development of the heart. However, the precise molecular mechanisms by which BMPs regulate cardiac development are largely unknown. In the present study, we examined the molecular mechanisms by which BMPs induce cardiac differentiation by using the P19CL6 in vitro cardiomyocyte differentiation system, a clonal derivative of P19 embryonic teratocarcinoma cells. We established a permanent P19CL6 cell line, P19CL6noggin, which constitutively overexpresses the BMP antagonist noggin. Although almost all parental P19CL6 cells differentiate into beating cardiomyocytes when treated with 1% dimethyl sulfoxide, P19CL6noggin cells did not differentiate into beating cardiomyocytes nor did they express cardiac transcription factors or contractile protein genes. The failure of differentiation was rescued by overexpression of BMP-2 or addition of BMP protein to the culture media, indicating that BMPs were indispensable for cardiomyocyte differentiation in this system. Overexpression of TAK1, a member of the mitogen-activated protein kinase kinase kinase superfamily which transduces BMP signaling, restored the ability of P19CL6noggin cells to differentiate into cardiomyocytes and concomitantly express cardiac genes, whereas overexpression of the dominant negative form of TAK1 in parental P19CL6 cells inhibited cardiomyocyte differentiation. Overexpression of both cardiac transcription factors Csx/Nkx-2.5 and GATA-4 but not of Csx/Nkx-2.5 or GATA-4 alone also induced differentiation of P19CL6noggin cells into cardiomyocytes. These results suggest that TAK1, Csx/Nkx-2.5, and GATA-4 play a pivotal role in the cardiogenic BMP signaling pathway.

Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.

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References

Efficient Cardiomyogenic Differentiation of Embryonic Stem Cell by Fibroblast Growth Factor 2 and Bone Morphogenetic Protein 2

Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4.