Formation of Postsynaptic-Like Membranes during Differentiation of Embryonic Stem Cellsin Vitro |
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Authors: | Jü rgen Rohwedel,Thomas Kleppisch,Uta Pich,Kaomei Guan,Shan Jin,Werner Zuschratter,Carsten Hopf,Werner Hoch,Jü rgen Hescheler,Veit Witzemann,Anna M. Wobus |
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Affiliation: | aIn Vitro Differentiation Group, IPK Gatersleben, Corrensstrasse 3, D-06466, Gatersleben;bInstitute of Pharmacology and Toxicology, Technical University, D-80802, Munich;cInstitute for Neurobiology, D-39008, Magdeburg;dMax-Planck-Institute of Developmental Biology, D-72076, Tübingen;eInstitute of Neurophysiology, University of Cologne, D-50931, Cologne;fMax-Planck-Institute of Medical Research, D-69028, Heidelberg, Germany |
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Abstract: | To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, α1subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the γ- and ?-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR ?-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiationin vitroneuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by “loss of function” analysisin vitro. |
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Keywords: | embryonic stem cells myogenesis neurogenesis nicotinic acetylcholine receptors neuromuscular junctions. |
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