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Oct4-induced reprogramming is required for adult brain neural stem cell differentiation into midbrain dopaminergic neurons.

Deleidi M, Cooper O, Hargus G, Levy A, Isacson O - PLoS ONE (2011)

Bottom Line: Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation.Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons.Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroregeneration Research, Harvard Medical School/McLean Hospital, Belmont, Massachusetts, United States of America.

ABSTRACT
Neural stem cells (NSCs) lose their competency to generate region-specific neuronal populations at an early stage during embryonic brain development. Here we investigated whether epigenetic modifications can reverse the regional restriction of mouse adult brain subventricular zone (SVZ) NSCs. Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation. Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons. DA neurons derived from Oct4-reprogrammed NSCs improved behavioural motor deficits in a rat model of Parkinson's disease (PD) upon intrastriatal transplantation. Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.

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Chromatin modifying agents induce the expression of pluripotency-associated genes and promote partial demethylation of Oct4 promoter in adult SVZ NSCs.(A) SVZ adult NSCs were treated with VPA, TSA, or TSA/AZA and mRNA level of Nanog, Sox2, Oct4 and Klf4 was analysed by qRT-PCR. Values were normalized to the level of β-actin and expressed as relative increase over vehicle-treated cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01; # p≤0.05; One-way ANOVA). (B) The DNA methylation profile of 12 CpG sites located in the Oct4 proximal promoter from −469 to the ATG start codon in mouse ESCs, untreated SVZ NSCs and NSCs treated with AZA, TSA, VPA or AZA/TSA is shown. The methylated and non-methylated CpG positions are presented as black and white circles, respectively. Ratios indicate average of methylated to unmethylated sites at the 12 CpG sites±SEM.
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pone-0019926-g002: Chromatin modifying agents induce the expression of pluripotency-associated genes and promote partial demethylation of Oct4 promoter in adult SVZ NSCs.(A) SVZ adult NSCs were treated with VPA, TSA, or TSA/AZA and mRNA level of Nanog, Sox2, Oct4 and Klf4 was analysed by qRT-PCR. Values were normalized to the level of β-actin and expressed as relative increase over vehicle-treated cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01; # p≤0.05; One-way ANOVA). (B) The DNA methylation profile of 12 CpG sites located in the Oct4 proximal promoter from −469 to the ATG start codon in mouse ESCs, untreated SVZ NSCs and NSCs treated with AZA, TSA, VPA or AZA/TSA is shown. The methylated and non-methylated CpG positions are presented as black and white circles, respectively. Ratios indicate average of methylated to unmethylated sites at the 12 CpG sites±SEM.

Mentions: We then analysed the expression of pluripotency-associated genes such as Nanog, Sox2, Oct4, and Klf4 in adult SVZ NSCs at 24 and 48 hours after treatment with VPA, TSA or TSA/AZA. We found that TSA alone or TSA/AZA significantly induced the expression of Oct4 and Klf4 at 24 and 48 hours (Fig. 2A). We investigated the DNA methylation profile of Oct4 promoter. Oct-4 is the master regulator of stem cell pluripotency and differentiation [28] and the methylation of its promoter drives the conversion from primitive NSCs (pNSCs) to definitive NSCs (dNSCs) [29], thus limiting their competency to undergo region-specific neuronal differentiation. The methylation status of 12 CpG sites in the Oct4 promoter region (between 470 to the ATG start codon) was assessed by bisulfite sequencing in mouse ESCs, untreated adult SVZ NSCs and adult SVZ NSCs treated with AZA, VPA, TSA or AZA/TSA for 48 hours (Fig. 2B). CpG sites within Oct4 promoter were mostly unmethylated in mouse ESCs (methylation rate: 2.075±1.3%). Conversely, untreated NSCs and AZA-treated NSCs were highly methylated (methylation rate: 90±3.9% and 89.9±4.9%, respectively). Upon treatment with VPA, TSA or AZA/TSA we found more unmethylated CpG sites (methylation rate: 80±6.7%, 64±3.4%, 60±4.1%, respectively) (Fig. 2B). These data indicate that the chromatin-modifying agents (TSA, VPA and TSA/AZA) induced de novo expression of pluripotency genes and partial demethylation of Oct4 promoter in adult SVZ NSCs. However, these changes were not sufficient to reverse NSC fate restriction and induce the competency to patterning by midbrain developmental cues.


Oct4-induced reprogramming is required for adult brain neural stem cell differentiation into midbrain dopaminergic neurons.

Deleidi M, Cooper O, Hargus G, Levy A, Isacson O - PLoS ONE (2011)

Chromatin modifying agents induce the expression of pluripotency-associated genes and promote partial demethylation of Oct4 promoter in adult SVZ NSCs.(A) SVZ adult NSCs were treated with VPA, TSA, or TSA/AZA and mRNA level of Nanog, Sox2, Oct4 and Klf4 was analysed by qRT-PCR. Values were normalized to the level of β-actin and expressed as relative increase over vehicle-treated cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01; # p≤0.05; One-way ANOVA). (B) The DNA methylation profile of 12 CpG sites located in the Oct4 proximal promoter from −469 to the ATG start codon in mouse ESCs, untreated SVZ NSCs and NSCs treated with AZA, TSA, VPA or AZA/TSA is shown. The methylated and non-methylated CpG positions are presented as black and white circles, respectively. Ratios indicate average of methylated to unmethylated sites at the 12 CpG sites±SEM.
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Related In: Results  -  Collection

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pone-0019926-g002: Chromatin modifying agents induce the expression of pluripotency-associated genes and promote partial demethylation of Oct4 promoter in adult SVZ NSCs.(A) SVZ adult NSCs were treated with VPA, TSA, or TSA/AZA and mRNA level of Nanog, Sox2, Oct4 and Klf4 was analysed by qRT-PCR. Values were normalized to the level of β-actin and expressed as relative increase over vehicle-treated cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01; # p≤0.05; One-way ANOVA). (B) The DNA methylation profile of 12 CpG sites located in the Oct4 proximal promoter from −469 to the ATG start codon in mouse ESCs, untreated SVZ NSCs and NSCs treated with AZA, TSA, VPA or AZA/TSA is shown. The methylated and non-methylated CpG positions are presented as black and white circles, respectively. Ratios indicate average of methylated to unmethylated sites at the 12 CpG sites±SEM.
Mentions: We then analysed the expression of pluripotency-associated genes such as Nanog, Sox2, Oct4, and Klf4 in adult SVZ NSCs at 24 and 48 hours after treatment with VPA, TSA or TSA/AZA. We found that TSA alone or TSA/AZA significantly induced the expression of Oct4 and Klf4 at 24 and 48 hours (Fig. 2A). We investigated the DNA methylation profile of Oct4 promoter. Oct-4 is the master regulator of stem cell pluripotency and differentiation [28] and the methylation of its promoter drives the conversion from primitive NSCs (pNSCs) to definitive NSCs (dNSCs) [29], thus limiting their competency to undergo region-specific neuronal differentiation. The methylation status of 12 CpG sites in the Oct4 promoter region (between 470 to the ATG start codon) was assessed by bisulfite sequencing in mouse ESCs, untreated adult SVZ NSCs and adult SVZ NSCs treated with AZA, VPA, TSA or AZA/TSA for 48 hours (Fig. 2B). CpG sites within Oct4 promoter were mostly unmethylated in mouse ESCs (methylation rate: 2.075±1.3%). Conversely, untreated NSCs and AZA-treated NSCs were highly methylated (methylation rate: 90±3.9% and 89.9±4.9%, respectively). Upon treatment with VPA, TSA or AZA/TSA we found more unmethylated CpG sites (methylation rate: 80±6.7%, 64±3.4%, 60±4.1%, respectively) (Fig. 2B). These data indicate that the chromatin-modifying agents (TSA, VPA and TSA/AZA) induced de novo expression of pluripotency genes and partial demethylation of Oct4 promoter in adult SVZ NSCs. However, these changes were not sufficient to reverse NSC fate restriction and induce the competency to patterning by midbrain developmental cues.

Bottom Line: Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation.Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons.Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.

View Article: PubMed Central - PubMed

Affiliation: Center for Neuroregeneration Research, Harvard Medical School/McLean Hospital, Belmont, Massachusetts, United States of America.

ABSTRACT
Neural stem cells (NSCs) lose their competency to generate region-specific neuronal populations at an early stage during embryonic brain development. Here we investigated whether epigenetic modifications can reverse the regional restriction of mouse adult brain subventricular zone (SVZ) NSCs. Using a variety of chemicals that interfere with DNA methylation and histone acetylation, we showed that such epigenetic modifications increased neuronal differentiation but did not enable specific regional patterning, such as midbrain dopaminergic (DA) neuron generation. Only after Oct-4 overexpression did adult NSCs acquire a pluripotent state that allowed differentiation into midbrain DA neurons. DA neurons derived from Oct4-reprogrammed NSCs improved behavioural motor deficits in a rat model of Parkinson's disease (PD) upon intrastriatal transplantation. Here we report for the first time the successful differentiation of SVZ adult NSCs into functional region-specific midbrain DA neurons, by means of Oct-4 induced pluripotency.

Show MeSH
Related in: MedlinePlus