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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 increase neuronal differentiation of adult mouse SVZ NSCs.(A) Schematic representation of the experimental design: adult SVZ NSCs were treated with TSA, TSA/AZA or VPA for 5 days and differentiated using a modified protocol developed for the differentiation of mouse ESCs to midbrain DA neurons. (B) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated, TSA-, TSA/AZA- or VPA-treated cultures. Treatment with TSA and VPA increased the number of β-TubIII+ neurons, whereas TSA, TSA/AZA and VPA decreased the number of GFAP+ astrocytes. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01, *** p≤0.001; One-way ANOVA). (C) Immunofluorescence staining for neuron-specific class III β-Tubulin (TUJ1) and the glial-specific marker glial fibrillary acidic protein (GFAP) of untreated, TSA-, TSA/AZA-, and VPA-treated cultures. Nuclei were counterstained with Hoechst. (D) Western blot analysis of differentiated NSC extracts, untreated or differentiated in the presence of TSA, TSA/AZA or VPA. Two independent experiments were performed in triplicate. Error bars indicate SEM. (*p≤0.05, ** p≤0.01; One-way ANOVA). Optical densities of the individual bands were quantified using NIH ImageJ and normalized by the averaged value of GAPDH. (E) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs treated with VPA. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 20 µm (C); 10 µm (E). U, untreated; T, TSA; T/A, TSA/AZA; V, VPA.
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pone-0019926-g001: Chromatin modifying agents increase neuronal differentiation of adult mouse SVZ NSCs.(A) Schematic representation of the experimental design: adult SVZ NSCs were treated with TSA, TSA/AZA or VPA for 5 days and differentiated using a modified protocol developed for the differentiation of mouse ESCs to midbrain DA neurons. (B) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated, TSA-, TSA/AZA- or VPA-treated cultures. Treatment with TSA and VPA increased the number of β-TubIII+ neurons, whereas TSA, TSA/AZA and VPA decreased the number of GFAP+ astrocytes. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01, *** p≤0.001; One-way ANOVA). (C) Immunofluorescence staining for neuron-specific class III β-Tubulin (TUJ1) and the glial-specific marker glial fibrillary acidic protein (GFAP) of untreated, TSA-, TSA/AZA-, and VPA-treated cultures. Nuclei were counterstained with Hoechst. (D) Western blot analysis of differentiated NSC extracts, untreated or differentiated in the presence of TSA, TSA/AZA or VPA. Two independent experiments were performed in triplicate. Error bars indicate SEM. (*p≤0.05, ** p≤0.01; One-way ANOVA). Optical densities of the individual bands were quantified using NIH ImageJ and normalized by the averaged value of GAPDH. (E) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs treated with VPA. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 20 µm (C); 10 µm (E). U, untreated; T, TSA; T/A, TSA/AZA; V, VPA.

Mentions: We first investigated whether epigenetic modifications induced by chromatin modifying agents can increase adult SVZ NSC responsiveness to the patterning signals that regulate midbrain DA neuron specification such as sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). We tested the effects of a variety of chemicals that have been shown to enhance cell dedifferentiation by promoting an ESC-like state: 5-aza-cytidine (AZA), a DNA methyltransferase (DNMT) inhibitor; BIX-01294, a G9a histone methyltransferase inhibitor; valproic acid (VPA) and trichostatin A (TSA), histone deacetylase inhibitors (HDACi) (Table 1) [26]. The non-toxic concentration of each chemical was determined by exposing adult SVZ NSCs to a range of concentrations and determining cell toxicity by Annexin V/Propidium iodide flow-cytometry analysis at 24 and 48 hours after treatment (data not shown). In order to test whether such chromatin modifying agents increase NSC competency to region-specific neuronal differentiation, NSCs were isolated from the SVZ of adult C57/BL6 mice and grown as neurospheres in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). In order to limit the exposure to EGF and FGF-2 that can deregulate the spatial identity and differentiation potential of neural precursors [27], only primary neurospheres were used in these experiments. NSCs were treated with 1 µM AZA, 1 µM BIX-01294, 1 mM VPA, 100 nM TSA, or 500 nM AZA/100 nM TSA (Table 1) for 72 hours. NSCs were then differentiated upon EGF withdrawal, using a modified protocol developed for the differentiation of mouse ESCs to midbrain DA neurons [7], [24] (Fig. 1A). In this protocol, the signaling molecules Shh, FGF-2 and FGF8 induce ventral midbrain DA neuron patterning of neuronal precursors. Final differentiation to mature DA neurons is induced by ascorbic acid (AA) in the absence of growth factors. Differentiation medium was supplemented with 1 µM AZA, 1 µM BIX-01294, 1 mM VPA, 100 nM TSA, or 500 nM AZA/100 nM TSA for an additional 48 hours (Fig. 1A). AZA alone and BIX-01294 at effective concentrations were found to be too toxic when added during differentiation, thus were excluded from further studies. Treatment with HDACi (TSA,VPA) induced a significant increase of β-TubIII+ neurons as revealed by immunohistochemistry at day 30 (40.5±0.5%, 55.25±1.6%, 62.5±4% in untreated, TSA and VPA treated cells; p≤0.01 and p≤0.001 compared to untreated respectively) and western blot analysis (Fig. 1B–D). Treatment with TSA, TSA/AZA and VPA decreased the number of GFAP+ astrocytes in differentiated cultures at day 30 (65±1.9%, 37.25±1.8%, 48.34±1.5%, 34±1.9% in untreated, TSA, TSA/AZA, and VPA treated cells; p≤0.001, p≤0.01 and p≤0.001 compared to untreated cells) (Fig. 1B–C). Western blot analysis confirmed the decrease of astrocytic differentiation after treatment with TSA, TSA/AZA and VPA (Figure 1D). We examined the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of DA synthesis, and other midbrain DA neuronal markers [engrailed-1 (En1), paired-like homeodomain transcription factor 3 (Pitx3), dopamine transporter (DAT), and nuclear receptor related 1 (Nurr1)] by immunocytochemistry and RT-PCR at day 16, 26 and 30. Some TH+ neurons were transiently detected at day 16 in HDACi treated cultures (data not shown). However, after the completion of the differentiation protocol (day 30), differentiated neurons did not express any midbrain DA marker. We found that differentiated neurons almost exclusively expressed GAD67, a marker for GABAergic interneurons (Fig. 1E).


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 increase neuronal differentiation of adult mouse SVZ NSCs.(A) Schematic representation of the experimental design: adult SVZ NSCs were treated with TSA, TSA/AZA or VPA for 5 days and differentiated using a modified protocol developed for the differentiation of mouse ESCs to midbrain DA neurons. (B) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated, TSA-, TSA/AZA- or VPA-treated cultures. Treatment with TSA and VPA increased the number of β-TubIII+ neurons, whereas TSA, TSA/AZA and VPA decreased the number of GFAP+ astrocytes. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01, *** p≤0.001; One-way ANOVA). (C) Immunofluorescence staining for neuron-specific class III β-Tubulin (TUJ1) and the glial-specific marker glial fibrillary acidic protein (GFAP) of untreated, TSA-, TSA/AZA-, and VPA-treated cultures. Nuclei were counterstained with Hoechst. (D) Western blot analysis of differentiated NSC extracts, untreated or differentiated in the presence of TSA, TSA/AZA or VPA. Two independent experiments were performed in triplicate. Error bars indicate SEM. (*p≤0.05, ** p≤0.01; One-way ANOVA). Optical densities of the individual bands were quantified using NIH ImageJ and normalized by the averaged value of GAPDH. (E) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs treated with VPA. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 20 µm (C); 10 µm (E). U, untreated; T, TSA; T/A, TSA/AZA; V, VPA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104995&req=5

pone-0019926-g001: Chromatin modifying agents increase neuronal differentiation of adult mouse SVZ NSCs.(A) Schematic representation of the experimental design: adult SVZ NSCs were treated with TSA, TSA/AZA or VPA for 5 days and differentiated using a modified protocol developed for the differentiation of mouse ESCs to midbrain DA neurons. (B) Quantification of β-TubIII+ neurons and GFAP+ astrocytes in untreated, TSA-, TSA/AZA- or VPA-treated cultures. Treatment with TSA and VPA increased the number of β-TubIII+ neurons, whereas TSA, TSA/AZA and VPA decreased the number of GFAP+ astrocytes. Error bars indicate SEM. Three independent experiments were performed in triplicate (** p≤0.01, *** p≤0.001; One-way ANOVA). (C) Immunofluorescence staining for neuron-specific class III β-Tubulin (TUJ1) and the glial-specific marker glial fibrillary acidic protein (GFAP) of untreated, TSA-, TSA/AZA-, and VPA-treated cultures. Nuclei were counterstained with Hoechst. (D) Western blot analysis of differentiated NSC extracts, untreated or differentiated in the presence of TSA, TSA/AZA or VPA. Two independent experiments were performed in triplicate. Error bars indicate SEM. (*p≤0.05, ** p≤0.01; One-way ANOVA). Optical densities of the individual bands were quantified using NIH ImageJ and normalized by the averaged value of GAPDH. (E) Immunofluorescence staining for β-TubIII (green) and GAD67 (red) of differentiated adult SVZ NSCs treated with VPA. The majority of β-TubIII+ neurons co-expressed GAD67. Scale bars: 20 µm (C); 10 µm (E). U, untreated; T, TSA; T/A, TSA/AZA; V, VPA.
Mentions: We first investigated whether epigenetic modifications induced by chromatin modifying agents can increase adult SVZ NSC responsiveness to the patterning signals that regulate midbrain DA neuron specification such as sonic hedgehog (Shh) and fibroblast growth factor 8 (FGF8). We tested the effects of a variety of chemicals that have been shown to enhance cell dedifferentiation by promoting an ESC-like state: 5-aza-cytidine (AZA), a DNA methyltransferase (DNMT) inhibitor; BIX-01294, a G9a histone methyltransferase inhibitor; valproic acid (VPA) and trichostatin A (TSA), histone deacetylase inhibitors (HDACi) (Table 1) [26]. The non-toxic concentration of each chemical was determined by exposing adult SVZ NSCs to a range of concentrations and determining cell toxicity by Annexin V/Propidium iodide flow-cytometry analysis at 24 and 48 hours after treatment (data not shown). In order to test whether such chromatin modifying agents increase NSC competency to region-specific neuronal differentiation, NSCs were isolated from the SVZ of adult C57/BL6 mice and grown as neurospheres in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). In order to limit the exposure to EGF and FGF-2 that can deregulate the spatial identity and differentiation potential of neural precursors [27], only primary neurospheres were used in these experiments. NSCs were treated with 1 µM AZA, 1 µM BIX-01294, 1 mM VPA, 100 nM TSA, or 500 nM AZA/100 nM TSA (Table 1) for 72 hours. NSCs were then differentiated upon EGF withdrawal, using a modified protocol developed for the differentiation of mouse ESCs to midbrain DA neurons [7], [24] (Fig. 1A). In this protocol, the signaling molecules Shh, FGF-2 and FGF8 induce ventral midbrain DA neuron patterning of neuronal precursors. Final differentiation to mature DA neurons is induced by ascorbic acid (AA) in the absence of growth factors. Differentiation medium was supplemented with 1 µM AZA, 1 µM BIX-01294, 1 mM VPA, 100 nM TSA, or 500 nM AZA/100 nM TSA for an additional 48 hours (Fig. 1A). AZA alone and BIX-01294 at effective concentrations were found to be too toxic when added during differentiation, thus were excluded from further studies. Treatment with HDACi (TSA,VPA) induced a significant increase of β-TubIII+ neurons as revealed by immunohistochemistry at day 30 (40.5±0.5%, 55.25±1.6%, 62.5±4% in untreated, TSA and VPA treated cells; p≤0.01 and p≤0.001 compared to untreated respectively) and western blot analysis (Fig. 1B–D). Treatment with TSA, TSA/AZA and VPA decreased the number of GFAP+ astrocytes in differentiated cultures at day 30 (65±1.9%, 37.25±1.8%, 48.34±1.5%, 34±1.9% in untreated, TSA, TSA/AZA, and VPA treated cells; p≤0.001, p≤0.01 and p≤0.001 compared to untreated cells) (Fig. 1B–C). Western blot analysis confirmed the decrease of astrocytic differentiation after treatment with TSA, TSA/AZA and VPA (Figure 1D). We examined the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of DA synthesis, and other midbrain DA neuronal markers [engrailed-1 (En1), paired-like homeodomain transcription factor 3 (Pitx3), dopamine transporter (DAT), and nuclear receptor related 1 (Nurr1)] by immunocytochemistry and RT-PCR at day 16, 26 and 30. Some TH+ neurons were transiently detected at day 16 in HDACi treated cultures (data not shown). However, after the completion of the differentiation protocol (day 30), differentiated neurons did not express any midbrain DA marker. We found that differentiated neurons almost exclusively expressed GAD67, a marker for GABAergic interneurons (Fig. 1E).

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