TY - JOUR
T1 - Rapid, efficient, and simple motor neuron differentiation from human pluripotent stem cells
AU - Shimojo, Daisuke
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
N1 - Funding Information:
H.O. is a paid member of the Scientific Advisory Board of SanBio Co., Ltd. We are grateful to Dr. Hirofumi Suemori for providing KhES1 hESCs, Dr. Shinya Yamanaka for 201B7 hiPSCs, Dr. Wado Akamatsu for supporting iPSC establishment, Dr. Steven Goldman for the HB9e438 enhancer element, Dr. Hiroyuki Miyoshi for lentiviral vectors, and Drs Hiroki Shinkai, Satoshi Niwa, Kazuko Kondoh, and Ayako Suzuki for technical and administrative assistance. We are also grateful to the members of the Okano and Okada laboratories for their encouragement and kind support. This work was supported by the Kanrinmaru-Project at Keio University; grants from the Practical Research Project for Intractable Diseases of the Ministry of Health, Labour and Welfar (MHLW) and the Japan Agency for Medical Research and development (AMED); a Grant-in-Aid for Scientific Research (B); and a Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science (JSPS) to YO. This work was also supported by a Grant-in-Aid for Scientific Research in Innovative Areas (Foundation of Synapse Neurocircuit Pathology to YO; a Brain Protein Aging and Dementia Control to YO and HO) from the JSPS and the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); the Project for the Realization of Regenerative Medicine and Support for the Core Institutes for iPS cellresearch from the MEXT; a grant from the Program for Intractable Disease Research utilizing disease-specific iPS Cells from the Japan Science and Technology Agency (JST) and AMED to HO; and a Grant-in-Aid for JSPS Fellows to DS.
Publisher Copyright:
© 2015 Shimojo et al.
PY - 2015/12/1
Y1 - 2015/12/1
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.
AB - 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.
KW - Human embryonic stem cells
KW - Human induced pluripotent stem cells
KW - Lentiviral reporter
KW - Long-term culture of motor neurons
KW - Motor neurons
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U2 - 10.1186/s13041-015-0172-4
DO - 10.1186/s13041-015-0172-4
M3 - Article
C2 - 26626025
AN - SCOPUS:84956938336
SN - 1756-6606
VL - 8
JO - Molecular Brain
JF - Molecular Brain
IS - 1
M1 - 79
ER -