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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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Short-term overexpression of Oct4 increases proliferation, self-renewal and neuronal differentiation of adult SVZ NSCs.(A) Adult SVZ NSCs were isolated from doxycycline (DOX)-inducible Oct4 transgenic mice (rTA-Oct4) and grown as neurospheres in growth medium with EGF/FGF-2. Oct4 was induced by 2 µg/mL DOX. Fluorescence images of rTA-Oct4 NSCs cultured for 48 hours, with or without DOX, and stained with an antibody to Oct4. Percentages of Oct4+ cells are indicated. (B) DOX-dependent induction of Oct-4 protein in adult SVZ NSCs from rTA-Oct4 transgenic mice determined by Western blot analysis. Mouse embryonic stem cells (mESC) were used as control. GAPDH was used as loading control. (C) Growth curves of DOX-induced and -uninduced adult SVZ NSCs. Time points represent average values from triplicate measurements and their standard deviations (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). (D) Clonal analysis of SVZ adult NSCs after Oct4 induction. NSCs were grown as neurospheres and treated with DOX or left untreated. After dissociation, single cells were replated and the total number of neurospheres with a diameter >100 µm was assessed and expressed as % over plated cells (** p≤0.01, t-test). Error bars indicate SEM. (E) Schematic representation of the experimental design: SVZ adult NSCs were isolated from rTA-Oct4 transgenic mice and grown as neurospheres in growth medium with EGF/FGF-2, with or without DOX. Forty-eight hours after Oct4 induction, neurospheres were dissociated and replated in differentiation medium with Shh, FGF8, FGF-2, with or without DOX. DOX was withdrawn after 48 hours. Cells were finally differentiated with ascorbic acid (AA). (F–I) Immunofluorescence staining for β-TubIII (green, F–G) and GFAP (green, H–I) of adult SVZ NSCs differentiated with or without Oct4 induction. Nuclei were counterstained with Hoechst. (J) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated and DOX-treated neuronal cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate. (*** p≤0.001; t-test). (K) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs upon Oct4 induction. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 50 µm (F–I); 10 µm (K).
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pone-0019926-g003: Short-term overexpression of Oct4 increases proliferation, self-renewal and neuronal differentiation of adult SVZ NSCs.(A) Adult SVZ NSCs were isolated from doxycycline (DOX)-inducible Oct4 transgenic mice (rTA-Oct4) and grown as neurospheres in growth medium with EGF/FGF-2. Oct4 was induced by 2 µg/mL DOX. Fluorescence images of rTA-Oct4 NSCs cultured for 48 hours, with or without DOX, and stained with an antibody to Oct4. Percentages of Oct4+ cells are indicated. (B) DOX-dependent induction of Oct-4 protein in adult SVZ NSCs from rTA-Oct4 transgenic mice determined by Western blot analysis. Mouse embryonic stem cells (mESC) were used as control. GAPDH was used as loading control. (C) Growth curves of DOX-induced and -uninduced adult SVZ NSCs. Time points represent average values from triplicate measurements and their standard deviations (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). (D) Clonal analysis of SVZ adult NSCs after Oct4 induction. NSCs were grown as neurospheres and treated with DOX or left untreated. After dissociation, single cells were replated and the total number of neurospheres with a diameter >100 µm was assessed and expressed as % over plated cells (** p≤0.01, t-test). Error bars indicate SEM. (E) Schematic representation of the experimental design: SVZ adult NSCs were isolated from rTA-Oct4 transgenic mice and grown as neurospheres in growth medium with EGF/FGF-2, with or without DOX. Forty-eight hours after Oct4 induction, neurospheres were dissociated and replated in differentiation medium with Shh, FGF8, FGF-2, with or without DOX. DOX was withdrawn after 48 hours. Cells were finally differentiated with ascorbic acid (AA). (F–I) Immunofluorescence staining for β-TubIII (green, F–G) and GFAP (green, H–I) of adult SVZ NSCs differentiated with or without Oct4 induction. Nuclei were counterstained with Hoechst. (J) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated and DOX-treated neuronal cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate. (*** p≤0.001; t-test). (K) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs upon Oct4 induction. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 50 µm (F–I); 10 µm (K).

Mentions: We then examined whether overexpression of Oct-4 can alter the region-specific differentiation potential of adult SVZ NSCs. We established neurosphere cultures from the lateral ventricular walls of rTA/Oct-4 mice [30]. These mice express rtTA-M2, an optimized form of reverse tetracycline-controlled transactivator (rtTA) protein directed to multiple tissues by the Gt(ROSA)26Sor promoter. Following doxycycline (DOX) administration, high Oct-4 expression is induced in several tissues [30]. In vivo, Oct-4 expression was not originally detected in the brain probably due to the low penetration of DOX through the blood brain barrier (BBB) [30]. Therefore, we first tested Oct-4 expression in adult SVZ NSC cultures derived from rTA/Oct-4 mice. We found that 25-35% of cells expressed Oct-4 as revealed by immunocytochemistry and western-blot analysis 48 hours after DOX induction (Fig. 3A–B). NSCs that were not exposed to DOX did not show any detectable Oct-4 expression (Fig. 3A–B). We then determined the effect of Oct-4 induction on adult SVZ NSC proliferation and self-renewal. rTA/Oct-4 NSCs were grown as neurospheres in the presence of EGF and FGF-2, with or without DOX, and propagated for up to 8 passages. Growth curves revealed increased long-term proliferation rate in DOX-treated NSCs (Fig. 3C). NSC self-renewal was assessed by the neurosphere-colony forming assay. We found more neurosphere-forming cells in DOX-treated cultures (Fig. 3D).


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)

Short-term overexpression of Oct4 increases proliferation, self-renewal and neuronal differentiation of adult SVZ NSCs.(A) Adult SVZ NSCs were isolated from doxycycline (DOX)-inducible Oct4 transgenic mice (rTA-Oct4) and grown as neurospheres in growth medium with EGF/FGF-2. Oct4 was induced by 2 µg/mL DOX. Fluorescence images of rTA-Oct4 NSCs cultured for 48 hours, with or without DOX, and stained with an antibody to Oct4. Percentages of Oct4+ cells are indicated. (B) DOX-dependent induction of Oct-4 protein in adult SVZ NSCs from rTA-Oct4 transgenic mice determined by Western blot analysis. Mouse embryonic stem cells (mESC) were used as control. GAPDH was used as loading control. (C) Growth curves of DOX-induced and -uninduced adult SVZ NSCs. Time points represent average values from triplicate measurements and their standard deviations (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). (D) Clonal analysis of SVZ adult NSCs after Oct4 induction. NSCs were grown as neurospheres and treated with DOX or left untreated. After dissociation, single cells were replated and the total number of neurospheres with a diameter >100 µm was assessed and expressed as % over plated cells (** p≤0.01, t-test). Error bars indicate SEM. (E) Schematic representation of the experimental design: SVZ adult NSCs were isolated from rTA-Oct4 transgenic mice and grown as neurospheres in growth medium with EGF/FGF-2, with or without DOX. Forty-eight hours after Oct4 induction, neurospheres were dissociated and replated in differentiation medium with Shh, FGF8, FGF-2, with or without DOX. DOX was withdrawn after 48 hours. Cells were finally differentiated with ascorbic acid (AA). (F–I) Immunofluorescence staining for β-TubIII (green, F–G) and GFAP (green, H–I) of adult SVZ NSCs differentiated with or without Oct4 induction. Nuclei were counterstained with Hoechst. (J) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated and DOX-treated neuronal cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate. (*** p≤0.001; t-test). (K) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs upon Oct4 induction. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 50 µm (F–I); 10 µm (K).
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getmorefigures.php?uid=PMC3104995&req=5

pone-0019926-g003: Short-term overexpression of Oct4 increases proliferation, self-renewal and neuronal differentiation of adult SVZ NSCs.(A) Adult SVZ NSCs were isolated from doxycycline (DOX)-inducible Oct4 transgenic mice (rTA-Oct4) and grown as neurospheres in growth medium with EGF/FGF-2. Oct4 was induced by 2 µg/mL DOX. Fluorescence images of rTA-Oct4 NSCs cultured for 48 hours, with or without DOX, and stained with an antibody to Oct4. Percentages of Oct4+ cells are indicated. (B) DOX-dependent induction of Oct-4 protein in adult SVZ NSCs from rTA-Oct4 transgenic mice determined by Western blot analysis. Mouse embryonic stem cells (mESC) were used as control. GAPDH was used as loading control. (C) Growth curves of DOX-induced and -uninduced adult SVZ NSCs. Time points represent average values from triplicate measurements and their standard deviations (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). (D) Clonal analysis of SVZ adult NSCs after Oct4 induction. NSCs were grown as neurospheres and treated with DOX or left untreated. After dissociation, single cells were replated and the total number of neurospheres with a diameter >100 µm was assessed and expressed as % over plated cells (** p≤0.01, t-test). Error bars indicate SEM. (E) Schematic representation of the experimental design: SVZ adult NSCs were isolated from rTA-Oct4 transgenic mice and grown as neurospheres in growth medium with EGF/FGF-2, with or without DOX. Forty-eight hours after Oct4 induction, neurospheres were dissociated and replated in differentiation medium with Shh, FGF8, FGF-2, with or without DOX. DOX was withdrawn after 48 hours. Cells were finally differentiated with ascorbic acid (AA). (F–I) Immunofluorescence staining for β-TubIII (green, F–G) and GFAP (green, H–I) of adult SVZ NSCs differentiated with or without Oct4 induction. Nuclei were counterstained with Hoechst. (J) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated and DOX-treated neuronal cultures. Error bars indicate SEM. Three independent experiments were performed in triplicate. (*** p≤0.001; t-test). (K) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs upon Oct4 induction. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 50 µm (F–I); 10 µm (K).
Mentions: We then examined whether overexpression of Oct-4 can alter the region-specific differentiation potential of adult SVZ NSCs. We established neurosphere cultures from the lateral ventricular walls of rTA/Oct-4 mice [30]. These mice express rtTA-M2, an optimized form of reverse tetracycline-controlled transactivator (rtTA) protein directed to multiple tissues by the Gt(ROSA)26Sor promoter. Following doxycycline (DOX) administration, high Oct-4 expression is induced in several tissues [30]. In vivo, Oct-4 expression was not originally detected in the brain probably due to the low penetration of DOX through the blood brain barrier (BBB) [30]. Therefore, we first tested Oct-4 expression in adult SVZ NSC cultures derived from rTA/Oct-4 mice. We found that 25-35% of cells expressed Oct-4 as revealed by immunocytochemistry and western-blot analysis 48 hours after DOX induction (Fig. 3A–B). NSCs that were not exposed to DOX did not show any detectable Oct-4 expression (Fig. 3A–B). We then determined the effect of Oct-4 induction on adult SVZ NSC proliferation and self-renewal. rTA/Oct-4 NSCs were grown as neurospheres in the presence of EGF and FGF-2, with or without DOX, and propagated for up to 8 passages. Growth curves revealed increased long-term proliferation rate in DOX-treated NSCs (Fig. 3C). NSC self-renewal was assessed by the neurosphere-colony forming assay. We found more neurosphere-forming cells in DOX-treated cultures (Fig. 3D).

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