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

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 - Mol Brain (2015)

Bottom Line: 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.Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.

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.

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Derivation of motor neurons from hiPSCs. a Immunocytochemical analysis of motor neurons derived from 201B7, TIGE-9 and YFE-16 for HB9, ISL-1, and βIII-Tubulin after 2 weeks of monolayer differentiation and ChAT after 4 weeks of monolayer differentiation. Scale bar, 100 μm. b Quantitative analysis of the number of the cells positive for motor neuron markers in cultures derived from each hiPSC clone. n = 3, mean ± SEM. c Time-course analysis of the expression of HB9, ISL-1, and ChAT during monolayer differentiation of hiPSC-derived motor neurons via quantitative RT-PCR. n = 3, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation of hiPSC-derived motor neurons. B7, 201B7. T9, TIGE-9. Y16, YFE-16. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. Protein expression levels are normalized to β-Actin. n = 3, mean ± SEM. *, p < 0.05, (Student’s t test)
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Fig4: Derivation of motor neurons from hiPSCs. a Immunocytochemical analysis of motor neurons derived from 201B7, TIGE-9 and YFE-16 for HB9, ISL-1, and βIII-Tubulin after 2 weeks of monolayer differentiation and ChAT after 4 weeks of monolayer differentiation. Scale bar, 100 μm. b Quantitative analysis of the number of the cells positive for motor neuron markers in cultures derived from each hiPSC clone. n = 3, mean ± SEM. c Time-course analysis of the expression of HB9, ISL-1, and ChAT during monolayer differentiation of hiPSC-derived motor neurons via quantitative RT-PCR. n = 3, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation of hiPSC-derived motor neurons. B7, 201B7. T9, TIGE-9. Y16, YFE-16. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. Protein expression levels are normalized to β-Actin. n = 3, mean ± SEM. *, p < 0.05, (Student’s t test)

Mentions: We further examined whether this motor neuron differentiation system is applicable to hiPSCs. Three hiPSC clones (201B7, TIGE-9, and YFE-16) were differentiated into motor neurons following the same protocol (Fig. 4). 201B7 was kindly provided by Dr. Yamanaka [1], and TIGE-9 and YFE-16 were established in our laboratory from fibroblasts from adult males (36 and 24 years of age, respectively) by introducing OCT4, SOX2, KLF4, L-MYC, LIN28, and shTP53 via episomal vectors; the last two clones were subjected to analysis of pluripotent marker expression via immunocytochemistry and quantitative RT-PCR, silencing of episomal transgenes, karyotype analysis, and teratoma formation capacity testing (Additional file 1: Figure S1). All of these clones efficiently differentiated into HB9+ and ISL-1+ motor neurons that expressed the mature motor neuron marker ChAT by 4 weeks of monolayer differentiation (Fig. 4a). The proportions of HB9+ and ISL-1+ cells 1 week after monolayer differentiation of dissociated EBs derived from the three hiPSC clones were similar to those obtained from KhES1 hESCs, at approximately 40-50 % (Fig. 4b). We also examined the time course of motor neuron marker expression via quantitative RT-PCR and western blotting and confirmed similar expression profiles in all of the hiPSC clones to those observed in KhES1 cells (Fig. 4c-e). These results suggest that our differentiation protocol is applicable to hiPSCs, thereby minimizing clonal variation among the hPSC clones.Fig. 4


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

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 - Mol Brain (2015)

Derivation of motor neurons from hiPSCs. a Immunocytochemical analysis of motor neurons derived from 201B7, TIGE-9 and YFE-16 for HB9, ISL-1, and βIII-Tubulin after 2 weeks of monolayer differentiation and ChAT after 4 weeks of monolayer differentiation. Scale bar, 100 μm. b Quantitative analysis of the number of the cells positive for motor neuron markers in cultures derived from each hiPSC clone. n = 3, mean ± SEM. c Time-course analysis of the expression of HB9, ISL-1, and ChAT during monolayer differentiation of hiPSC-derived motor neurons via quantitative RT-PCR. n = 3, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation of hiPSC-derived motor neurons. B7, 201B7. T9, TIGE-9. Y16, YFE-16. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. Protein expression levels are normalized to β-Actin. n = 3, mean ± SEM. *, p < 0.05, (Student’s t test)
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Fig4: Derivation of motor neurons from hiPSCs. a Immunocytochemical analysis of motor neurons derived from 201B7, TIGE-9 and YFE-16 for HB9, ISL-1, and βIII-Tubulin after 2 weeks of monolayer differentiation and ChAT after 4 weeks of monolayer differentiation. Scale bar, 100 μm. b Quantitative analysis of the number of the cells positive for motor neuron markers in cultures derived from each hiPSC clone. n = 3, mean ± SEM. c Time-course analysis of the expression of HB9, ISL-1, and ChAT during monolayer differentiation of hiPSC-derived motor neurons via quantitative RT-PCR. n = 3, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation of hiPSC-derived motor neurons. B7, 201B7. T9, TIGE-9. Y16, YFE-16. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. Protein expression levels are normalized to β-Actin. n = 3, mean ± SEM. *, p < 0.05, (Student’s t test)
Mentions: We further examined whether this motor neuron differentiation system is applicable to hiPSCs. Three hiPSC clones (201B7, TIGE-9, and YFE-16) were differentiated into motor neurons following the same protocol (Fig. 4). 201B7 was kindly provided by Dr. Yamanaka [1], and TIGE-9 and YFE-16 were established in our laboratory from fibroblasts from adult males (36 and 24 years of age, respectively) by introducing OCT4, SOX2, KLF4, L-MYC, LIN28, and shTP53 via episomal vectors; the last two clones were subjected to analysis of pluripotent marker expression via immunocytochemistry and quantitative RT-PCR, silencing of episomal transgenes, karyotype analysis, and teratoma formation capacity testing (Additional file 1: Figure S1). All of these clones efficiently differentiated into HB9+ and ISL-1+ motor neurons that expressed the mature motor neuron marker ChAT by 4 weeks of monolayer differentiation (Fig. 4a). The proportions of HB9+ and ISL-1+ cells 1 week after monolayer differentiation of dissociated EBs derived from the three hiPSC clones were similar to those obtained from KhES1 hESCs, at approximately 40-50 % (Fig. 4b). We also examined the time course of motor neuron marker expression via quantitative RT-PCR and western blotting and confirmed similar expression profiles in all of the hiPSC clones to those observed in KhES1 cells (Fig. 4c-e). These results suggest that our differentiation protocol is applicable to hiPSCs, thereby minimizing clonal variation among the hPSC clones.Fig. 4

Bottom Line: 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.Our motor neuron differentiation system and lentivirus-based reporter system for motor neurons facilitate the analysis of disease-specific hiPSCs for motor neuron diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Aichi Medical University School of Medicine, Aichi, 480-1195, Japan.

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.

Show MeSH
Related in: MedlinePlus