title: Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells.
abstract: BACKGROUND
  Human pluripotent stem cells (hPSCs) are being applied in regenerative medicine and for the in vitro modeling of human intractable disorders. In particular, neural cells derived from disease-specific human induced pluripotent stem cells (hiPSCs) established from patients with neurological disorders have been used as in vitro disease models to recapitulate in vivo pathogenesis because neural cells cannot be usually obtained from patients themselves.
  
  
  RESULTS
  In this study, we established a rapid, efficient, and simple method for efficiently deriving motor neurons from hPSCs that is useful for pathophysiological analysis and the development of drugs to treat motor neuron diseases. Treatment with GSK3β inhibitors during the initial phase of differentiation in combination with dual SMAD inhibition was sufficient to induce PAX6 (+) and SOX1 (+) neural progenitors within 1 week, and subsequent treatment with retinoic acid (RA) and purmorphamine, which activates sonic hedgehog (SHH) signaling, resulted in the highly efficient induction of HB9(+) and ISL-1(+) motor neurons within 2 weeks. After 4 weeks of monolayer differentiation in motor neuron maturation medium, hPSC-derived motor neurons were shown to mature, displaying larger somas and clearer staining for the mature motor neuron marker choline acetyltransferase (ChAT). Moreover, hPSC-derived motor neurons were able to form neuromuscular junctions with human myotubes in vitro and induced acetylcholine receptor (AChR) clustering, as detected by Alexa 555-conjugated α-Bungarotoxin (α-BTX), suggesting that these hPSC-derived motor neurons formed functional contacts with skeletal muscles. This differentiation system is simple and is reproducible in several hiPSC clones, thereby minimizing clonal variation among hPSC clones. We also established a system for visualizing motor neurons with a lentiviral reporter for HB9 (HB9 (e438) ::Venus). The specificity of this reporter was confirmed through immunocytochemistry and quantitative RT-PCR analysis of high-positive fractions obtained via fluorescence-activated cell sorting (FACS), suggesting its applicability for motor neuron-specific analysis.
  
  
  CONCLUSIONS
  Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.|Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Shimojo
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.|Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Kazunari
authors.initials: K
authors.lastname: Onodera
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Doi-Torii
authors.affiliation: Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Ishihara
authors.affiliation: Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
authors.forename: Chinatsu
authors.initials: C
authors.lastname: Hattori
authors.affiliation: Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
authors.forename: Yukino
authors.initials: Y
authors.lastname: Miwa
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.|Department of Orthopedic Surgery, Nagoya University Graduate School of Medicine, Nagoya, 466-8550, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Tanaka
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.|Division of Regenerative Medicine, Jikei University School of Medicine, Tokyo, 105-8461, Japan.
authors.forename: Rina
authors.initials: R
authors.lastname: Okada
authors.affiliation: Department of Dermatology, Keio University School of Medicine, Tokyo, 160-8582, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Ohyama
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa, 251-8555, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Shoji
authors.affiliation: Integrated Technology Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Kanagawa, 251-8555, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nakanishi
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.
authors.forename: Manabu
authors.initials: M
authors.lastname: Doyu
authors.affiliation: Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan. hidokano@a2.keio.jp.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Okano
authors.affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan. yohei@aichi-med-u.ac.jp.|Department of Physiology, Keio University School of Medicine, Tokyo, 160-8582, Japan. yohei@aichi-med-u.ac.jp.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Okada
mesh_terms: D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D017930:Genes, Reporter; D000066449:Human Embryonic Stem Cells / Q000166:cytology; D006801:Humans; D016086:Lentivirus / Q000378:metabolism; D008297:Male; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009046:Motor Neurons / Q000166:cytology*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology*; D054852:Small Molecule Libraries / Q000494:pharmacology; D055815:Young Adult
chemical_list: D054852:Small Molecule Libraries