Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors

Abstract

Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(−) CD108(−) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-β inhibitors. TGF-β inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.

Prostaglandin EP2 receptor downstream of Notch signaling inhibits differentiation of human skeletal muscle progenitors in differentiation conditions

“…Unexpectedly, PGE2 and butaprost, an agonist of EP2, had no significant effects on the fusion index ( Fig. 7), probably because differentiation of hiPSC-derived myogenic cells is severely suppressed by TGF-β signaling, as previously reported 10 . DAPT slightly suppressed the proliferation of hiPSCderived muscle progenitors, but there was no significant difference in cell numbers among the other groups (Fig.…”

“…Human induced pluripotent stem cells (hiPSCs) can be induced to differentiate into skeletal muscle cells even after extensive expansion [6][7][8][9][10] . Therefore, hiPS cells are expected to provide sufficient amounts of muscle progenitors for cell therapy.…”

“…Therefore, hiPS cells are expected to provide sufficient amounts of muscle progenitors for cell therapy. Recently, we reported an improved sphere culture-based protocol for induction of muscle progenitors from hiPSCs 10 . Induced muscle progenitors efficiently formed multinucleated myotubes in vitro and differentiated into myofibers in immune-deficient dystrophin-deficient mdx mice.…”

“…Induced muscle progenitors efficiently formed multinucleated myotubes in vitro and differentiated into myofibers in immune-deficient dystrophin-deficient mdx mice. However, the number of dystrophin-positive myofibers in mdx muscle was not satisfactory 10 , requiring further investigation to clarify why myogenic cells, which differentiate efficiently into myotubes in vitro, do not form myofibers in vivo after engraftment.…”

Stepwise in vitro induction of human somitic mesoderm and its derivatives

“…Protocols for the induction of human skeletal muscle cells from iPSCs have been reported previously 3,13,14 . The protocol described here for skeletal muscle induction from stepwise in vitro induced human DM cells is similar to the protocol published by Chal et al 13 .…”