IN-VITRO DIFFERENTIATION OF EMBRYONIC STEM-CELLS INTO CARDIOMYOCYTES OR SKELETAL-MUSCLE CELLS IS SPECIFICALLY MODULATED BY RETINOIC ACID

Pluripotent embryonic stem cells (ES cells) differentiating via embryo-like aggregates (embryoid bodies) into derivatives of the primary germ layers were used as a model system to investigate the time- and concentration-dependent effects of retinoic acid (RA) on the in vitro differentiation pattern. When ES cells, cultivated normally under conditions resulting in cardiomyocyte differentiation, were treated during the first 2 days of embryoid body formation with high RA concentrations (10(-9) to 10(-7) M), strong inhibition of cardiogenesis was found. ES cells differentiating as embryoid bodies and treated with the same RA concentration between the 5th and 7th day resulted in a slight induction of cardiogenesis. In contrast, incubation of embryoid bodies with 10(-8) and 10(-7) M RA between the 2nd and 5th day of embryoid body development resulted in a total inhibition of cardiogenesis but in an induction of myogenesis. This was demonstrated by indirect immunofluorescence and, as shown by reverse transcription-polymerase chain reaction (RT-PCR), by the time- and concentration-dependent inhibition of transcription of cardiac-specific alpha- and beta-cardiac myosin heavy chain (MHC) genes, and the induction of transcription of skeletal muscle-specific myogenin. In addition, using the whole-cell patch-clamp technique, these skeletal myocytes were functionally characterized by the expression of tissue-specific Ca2+ channels and nicotinic cholinoceptors. In summary, a specific effect of RA on ES cell differentiation in the embryoid body resulting in a switch from cardiogenesis to myogenesis and an induction of neuronal cells was found.