To measure the biological activity of the MN spheroids and muscle differentiation, total RNA was isolated from tissues with TRIzol reagent (Life Science, Waltham, MA, USA). Reverse transcription (RT) was performed using a SuperScript VILO cDNA Synthesis Kit (Invitrogen, Waltham, MA, USA). The primer sequences are shown in table S1. RT-PCR was performed with a 7900HT Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) using SYBR Premix Ex Taq (Takara, Kusatsu, Japan). The mRNA level of GAPDH (a housekeeping gene) was set to 100% and used as the internal standard in all experiments. The RT-PCR experiment was repeated at least three times for cDNA prepared from at least three batches.

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