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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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Long-term culture of hESC-derived motor neurons. a Immunocytochemical analysis of HB9, ISL-1, βIII-Tubulin, and ChAT after 1, 2, or 4 weeks of monolayer differentiation. Scale bar, 100 μm, and 20 μm (inset). b Quantification of the number of the cells positive for each marker. n = 4, mean ± SEM. *, p < 0.05 (Student’s t test). c Time-course analysis of the expression of PAX6, SOX1, NGN2, OLIG2, NKX2.2, HB9, ISL-1, and ChAT during monolayer differentiation via quantitative RT-PCR. n = 4, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. The protein expression levels are normalized to β-Actin. n = 4, mean ± SEM. *, p < 0.05, (Student’s t test)
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Fig2: Long-term culture of hESC-derived motor neurons. a Immunocytochemical analysis of HB9, ISL-1, βIII-Tubulin, and ChAT after 1, 2, or 4 weeks of monolayer differentiation. Scale bar, 100 μm, and 20 μm (inset). b Quantification of the number of the cells positive for each marker. n = 4, mean ± SEM. *, p < 0.05 (Student’s t test). c Time-course analysis of the expression of PAX6, SOX1, NGN2, OLIG2, NKX2.2, HB9, ISL-1, and ChAT during monolayer differentiation via quantitative RT-PCR. n = 4, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. The protein expression levels are normalized to β-Actin. n = 4, mean ± SEM. *, p < 0.05, (Student’s t test)

Mentions: To investigate the effects of the long-term culture of hESC-derived motor neurons, we examined temporal alterations of the expression of several motor neuron markers during 4 weeks of monolayer differentiation in MNM. Based on immunocytochemical analysis, the proportion of HB9+ and ISL-1+ cells did not change greatly and consistently comprised approximately 50–60 % of the total cells (Fig. 2a, b). By contrast, the proportion of cells positive for ChAT, a marker of mature motor neurons, was 54.6 ± 4.5 % at the end of one week and increased to 68.1 ± 4.1 % by 4 weeks of monolayer differentiation. Moreover, after 4 weeks of monolayer differentiation, the somas of the motor neurons had grown larger and were stained more clearly for ChAT compared with those observed after 1 week of monolayer differentiation (Fig. 2a, insets). Quantitative RT-PCR analysis revealed that the expression of genes known to be expressed in neural progenitor cells (PAX6, SOX1, NGN2, OLIG2, and NKX2.2) decreased during 4 weeks of monolayer differentiation, whereas markers of motor neurons (HB9, ISL-1, and ChAT) were increased during the first 2 weeks of differentiation, followed by a gradual decrease during the subsequent 2 weeks (Fig. 2c). To address the discrepancy in the time course of motor neuron marker expression between the immunocytochemistry and quantitative RT-PCR analyses, we also performed a western blot analysis (Fig. 2d, e). We found that the expression of the ChAT protein significantly increased during 4 weeks of differentiation, whereas the HB9 protein level was not significantly altered during this period (Fig. 2e). The ISL-1 protein was significantly decreased during monolayer differentiation as observed in the quantitative RT-PCR analysis (Fig. 2c). These results suggest that the expression of HB9 and ChAT might be regulated post-transcriptionally during the culture of hESC-derived motor neurons and that 4 weeks of monolayer differentiation facilitates the maturation of hESC-derived motor neurons.Fig. 2


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)

Long-term culture of hESC-derived motor neurons. a Immunocytochemical analysis of HB9, ISL-1, βIII-Tubulin, and ChAT after 1, 2, or 4 weeks of monolayer differentiation. Scale bar, 100 μm, and 20 μm (inset). b Quantification of the number of the cells positive for each marker. n = 4, mean ± SEM. *, p < 0.05 (Student’s t test). c Time-course analysis of the expression of PAX6, SOX1, NGN2, OLIG2, NKX2.2, HB9, ISL-1, and ChAT during monolayer differentiation via quantitative RT-PCR. n = 4, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. The protein expression levels are normalized to β-Actin. n = 4, mean ± SEM. *, p < 0.05, (Student’s t test)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666063&req=5

Fig2: Long-term culture of hESC-derived motor neurons. a Immunocytochemical analysis of HB9, ISL-1, βIII-Tubulin, and ChAT after 1, 2, or 4 weeks of monolayer differentiation. Scale bar, 100 μm, and 20 μm (inset). b Quantification of the number of the cells positive for each marker. n = 4, mean ± SEM. *, p < 0.05 (Student’s t test). c Time-course analysis of the expression of PAX6, SOX1, NGN2, OLIG2, NKX2.2, HB9, ISL-1, and ChAT during monolayer differentiation via quantitative RT-PCR. n = 4, mean ± SEM. d Western blot analysis of the expression of the HB9, ISL-1, and ChAT proteins during monolayer differentiation. e Quantitative analysis of the expression of the HB9, ISL-1, and ChAT proteins through densitometry using ImageJ. The protein expression levels are normalized to β-Actin. n = 4, mean ± SEM. *, p < 0.05, (Student’s t test)
Mentions: To investigate the effects of the long-term culture of hESC-derived motor neurons, we examined temporal alterations of the expression of several motor neuron markers during 4 weeks of monolayer differentiation in MNM. Based on immunocytochemical analysis, the proportion of HB9+ and ISL-1+ cells did not change greatly and consistently comprised approximately 50–60 % of the total cells (Fig. 2a, b). By contrast, the proportion of cells positive for ChAT, a marker of mature motor neurons, was 54.6 ± 4.5 % at the end of one week and increased to 68.1 ± 4.1 % by 4 weeks of monolayer differentiation. Moreover, after 4 weeks of monolayer differentiation, the somas of the motor neurons had grown larger and were stained more clearly for ChAT compared with those observed after 1 week of monolayer differentiation (Fig. 2a, insets). Quantitative RT-PCR analysis revealed that the expression of genes known to be expressed in neural progenitor cells (PAX6, SOX1, NGN2, OLIG2, and NKX2.2) decreased during 4 weeks of monolayer differentiation, whereas markers of motor neurons (HB9, ISL-1, and ChAT) were increased during the first 2 weeks of differentiation, followed by a gradual decrease during the subsequent 2 weeks (Fig. 2c). To address the discrepancy in the time course of motor neuron marker expression between the immunocytochemistry and quantitative RT-PCR analyses, we also performed a western blot analysis (Fig. 2d, e). We found that the expression of the ChAT protein significantly increased during 4 weeks of differentiation, whereas the HB9 protein level was not significantly altered during this period (Fig. 2e). The ISL-1 protein was significantly decreased during monolayer differentiation as observed in the quantitative RT-PCR analysis (Fig. 2c). These results suggest that the expression of HB9 and ChAT might be regulated post-transcriptionally during the culture of hESC-derived motor neurons and that 4 weeks of monolayer differentiation facilitates the maturation of hESC-derived motor neurons.Fig. 2

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