Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells

Daisuke Shimojo, Kazunari Onodera, Yukiko Doi-Torii, Yasuharu Ishihara, Chinatsu Hattori, Yukino Miwa, Satoshi Tanaka, Rina Okada, Manabu Ohyama, Masanobu Shoji, Atsushi Nakanishi, Manabu Doyu, Hideyuki Okano, Yohei Okada

Research output: Contribution to journalArticle

18 Citations (Scopus)

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.

Original languageEnglish
Article number79
JournalMolecular Brain
Volume8
Issue number1
DOIs
Publication statusPublished - 2015 Dec 1

Fingerprint

Pluripotent Stem Cells
Motor Neurons
Induced Pluripotent Stem Cells
Motor Neuron Disease
Clone Cells
Venus
Bungarotoxins
Lentivirus
Regenerative Medicine
Choline O-Acetyltransferase
Neuromuscular Junction
Skeletal Muscle Fibers
Carisoprodol
Cholinergic Receptors
Tretinoin
Nervous System Diseases
Cluster Analysis
Flow Cytometry
Skeletal Muscle
Immunohistochemistry

Keywords

  • Human embryonic stem cells
  • Human induced pluripotent stem cells
  • Lentiviral reporter
  • Long-term culture of motor neurons
  • Motor neurons

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Molecular Biology

Cite this

Shimojo, D., Onodera, K., Doi-Torii, Y., Ishihara, Y., Hattori, C., Miwa, Y., ... Okada, Y. (2015). Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. Molecular Brain, 8(1), [79]. https://doi.org/10.1186/s13041-015-0172-4

Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. / Shimojo, Daisuke; Onodera, Kazunari; Doi-Torii, Yukiko; Ishihara, Yasuharu; Hattori, Chinatsu; Miwa, Yukino; Tanaka, Satoshi; Okada, Rina; Ohyama, Manabu; Shoji, Masanobu; Nakanishi, Atsushi; Doyu, Manabu; Okano, Hideyuki; Okada, Yohei.

In: Molecular Brain, Vol. 8, No. 1, 79, 01.12.2015.

Research output: Contribution to journalArticle

Shimojo, D, Onodera, K, Doi-Torii, Y, Ishihara, Y, Hattori, C, Miwa, Y, Tanaka, S, Okada, R, Ohyama, M, Shoji, M, Nakanishi, A, Doyu, M, Okano, H & Okada, Y 2015, 'Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells', Molecular Brain, vol. 8, no. 1, 79. https://doi.org/10.1186/s13041-015-0172-4
Shimojo, Daisuke ; Onodera, Kazunari ; Doi-Torii, Yukiko ; Ishihara, Yasuharu ; Hattori, Chinatsu ; Miwa, Yukino ; Tanaka, Satoshi ; Okada, Rina ; Ohyama, Manabu ; Shoji, Masanobu ; Nakanishi, Atsushi ; Doyu, Manabu ; Okano, Hideyuki ; Okada, Yohei. / Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells. In: Molecular Brain. 2015 ; Vol. 8, No. 1.
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AU - Onodera, Kazunari

AU - Doi-Torii, Yukiko

AU - Ishihara, Yasuharu

AU - Hattori, Chinatsu

AU - Miwa, Yukino

AU - Tanaka, Satoshi

AU - Okada, Rina

AU - Ohyama, Manabu

AU - Shoji, Masanobu

AU - Nakanishi, Atsushi

AU - Doyu, Manabu

AU - Okano, Hideyuki

AU - Okada, Yohei

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N2 - 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.

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KW - Human induced pluripotent stem cells

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KW - Long-term culture of motor neurons

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