MUSCLE-CELL DIFFERENTIATION OF EMBRYONIC STEM-CELLS REFLECTS MYOGENESIS IN-VIVO - DEVELOPMENTALLY-REGULATED EXPRESSION OF MYOGENIC DETERMINATION GENES AND FUNCTIONAL EXPRESSION OF IONIC CURRENTS

The mouse blastocyst-derived embryonic stem cell (ES cell) line BLC6 e fficiently differentiates into myosin heavy chain-, desmin- and myogen in-positive skeletal muscle cells when cultivated in embryo-like aggre gates (embryoid bodies). Here, we show that the muscle-specific determ ination genes myf5, myogenin, myoD, and myf6 are expressed in these em bryoid bodies in a characteristic temporal pattern which precisely ref lects the sequence observed during mouse development in vivo. Myf5 is the first gene to be expressed followed by myogenin, myoD, and myf6, i n this order. In situ hybridization demonstrates transcripts for myoge nin. and myoD accumulating in mono- and multinucleated myogenic cells, while myf5 mRNA is already found in mononucleated myoblasts. The myoc ytes also express functional nicotinic cholinoceptors and exhibit T-ty pe Ca2+ currents and later L-type Ca2+ currents, demonstrating physiol ogical properties of skeletal muscle cells. During myocyte differentia tion the density of L-type Ca2+ channels significantly increases while the density of T-type Ca2+ channels decreases. The effect of external signals on myogenic differentiation of BLC6 cells was demonstrated by cocultivation with visceral endodermal END-2 cells and the activin A- secreting WEHI-3 cells. END-2 cells essentially prevent skeletal muscl e differentiation, whereas basic fibroblast growth factor, transformin g growth factor-beta, and WEHI-3 cells have no or an attenuating effec t, respectively. Our results suggest that ES cells recapitulate closel y the early steps of muscle development in vivo and may serve as an ex cellent in vitro system to study this process. (C) 1994 Academic Press , Inc.