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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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Reprogrammed adult NSCs can be differentiated into midbrain DA neurons.(A) NSC-derived iPSCs were differentiated into DA neurons according to the five-stage protocol. Neural differentiation was induced by EB formation in ITSFn medium. Subsequently, the neural precursors were expanded in the presence of the growth factors FGF-2, FGF8, and Shh. Terminal differentiation was induced by growth factor withdrawal in the presence of ascorbic acid. (B) Graphs indicate the percentage of cells that stained positive for β-TubIII and TH, relative to nuclear Hoechst staining. Error bars indicate SEM. Three independent experiments were performed in triplicate. (C) Brightfield images showing EB-derived FGF2-responsive neural precursor cells. (D) Brightfield microphotographs of differentiated NSC-derived iPSCs, 7 days after growth factor withdrawal. (E) Immunofluorescence staining of neuronal cultures derived from NSC-derived iPSCs for β-TubIII (green) and TH (red). (F) Immunofluorescence staining for TH (red) and GABA (green), a marker typically expressed in olfactory bulb (OB) glomerular interneurons. Nuclei were counterstained with Hoechst. (G–H) Confocal images of neuronal cultures stained for β-TubIII (green) (G), TH (red) (G–H), En1 (blue) (G) and Pitx3 (green) (H). (I) Quantitative RT-PCR analysis of midbrain transcription factors, DA neurotransmission markers (TH, DAT, Pitx3, En1, Girk2, VMAT, ALDH, Nurr1) and calbindin in differentiated NSC-derived iPSCs. cDNA was isolated from differentiated adult NSC-derived iPSCs, and values were normalized to the level of β-actin. Error bars indicate SEM. Three independent experiments were performed in triplicate. Scale bar: 200 µm (C–D); 50 µm (E–F); 20 µm (G–H). ITSFn, insulin/transferrin/selenium/fibronectin.
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pone-0019926-g006: Reprogrammed adult NSCs can be differentiated into midbrain DA neurons.(A) NSC-derived iPSCs were differentiated into DA neurons according to the five-stage protocol. Neural differentiation was induced by EB formation in ITSFn medium. Subsequently, the neural precursors were expanded in the presence of the growth factors FGF-2, FGF8, and Shh. Terminal differentiation was induced by growth factor withdrawal in the presence of ascorbic acid. (B) Graphs indicate the percentage of cells that stained positive for β-TubIII and TH, relative to nuclear Hoechst staining. Error bars indicate SEM. Three independent experiments were performed in triplicate. (C) Brightfield images showing EB-derived FGF2-responsive neural precursor cells. (D) Brightfield microphotographs of differentiated NSC-derived iPSCs, 7 days after growth factor withdrawal. (E) Immunofluorescence staining of neuronal cultures derived from NSC-derived iPSCs for β-TubIII (green) and TH (red). (F) Immunofluorescence staining for TH (red) and GABA (green), a marker typically expressed in olfactory bulb (OB) glomerular interneurons. Nuclei were counterstained with Hoechst. (G–H) Confocal images of neuronal cultures stained for β-TubIII (green) (G), TH (red) (G–H), En1 (blue) (G) and Pitx3 (green) (H). (I) Quantitative RT-PCR analysis of midbrain transcription factors, DA neurotransmission markers (TH, DAT, Pitx3, En1, Girk2, VMAT, ALDH, Nurr1) and calbindin in differentiated NSC-derived iPSCs. cDNA was isolated from differentiated adult NSC-derived iPSCs, and values were normalized to the level of β-actin. Error bars indicate SEM. Three independent experiments were performed in triplicate. Scale bar: 200 µm (C–D); 50 µm (E–F); 20 µm (G–H). ITSFn, insulin/transferrin/selenium/fibronectin.

Mentions: To test whether NSC-derived iPSCs were able to generate DA neurons in vitro, we differentiated iPSCs according to the mouse ESC five-stage protocol, with some modifications (Fig. 6A) [7], [24]. We induced neural differentiation by EB formation in ITSFn medium (Fig. 6C). Subsequently, the neural precursors were expanded in the presence of the growth factors Shh, FGF-2 and FGF8. Terminal differentiation was induced by growth factor withdrawal in the presence of AA (Fig. 6D). At day 32, we detected a total of 30% β-TubIII+ neurons and a total of 5% TH+/β-TubIII+ neurons (Fig. 6B, E). To assess neuronal regional specification, we double-labeled TH-positive neurons with antibody against Pitx3 and En1, markers typically expressed in midbrain DA neurons (Fig. 6 G–H). Importantly, the vast majority of TH-positive neurons stained positive for these midbrain markers, suggesting their proper midbrain regional specification in vitro (Fig. 6 G–H). We did not find any colocalization between TH and GABA, a marker typically expressed in OB glomerular interneurons (Fig. 6F). qRT-PCR analysis further confirmed expression levels of midbrain DA markers in differentiated cultures: DAT, PITX3, En1, G-protein-activated inwardly rectifying potassium channel subunit (Girk2), vesicular monoamine transporter (VMAT), aldehyde dehydrogenase 2 (ALDH), Calbindin and Nurr1 (Fig. 6I).


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)

Reprogrammed adult NSCs can be differentiated into midbrain DA neurons.(A) NSC-derived iPSCs were differentiated into DA neurons according to the five-stage protocol. Neural differentiation was induced by EB formation in ITSFn medium. Subsequently, the neural precursors were expanded in the presence of the growth factors FGF-2, FGF8, and Shh. Terminal differentiation was induced by growth factor withdrawal in the presence of ascorbic acid. (B) Graphs indicate the percentage of cells that stained positive for β-TubIII and TH, relative to nuclear Hoechst staining. Error bars indicate SEM. Three independent experiments were performed in triplicate. (C) Brightfield images showing EB-derived FGF2-responsive neural precursor cells. (D) Brightfield microphotographs of differentiated NSC-derived iPSCs, 7 days after growth factor withdrawal. (E) Immunofluorescence staining of neuronal cultures derived from NSC-derived iPSCs for β-TubIII (green) and TH (red). (F) Immunofluorescence staining for TH (red) and GABA (green), a marker typically expressed in olfactory bulb (OB) glomerular interneurons. Nuclei were counterstained with Hoechst. (G–H) Confocal images of neuronal cultures stained for β-TubIII (green) (G), TH (red) (G–H), En1 (blue) (G) and Pitx3 (green) (H). (I) Quantitative RT-PCR analysis of midbrain transcription factors, DA neurotransmission markers (TH, DAT, Pitx3, En1, Girk2, VMAT, ALDH, Nurr1) and calbindin in differentiated NSC-derived iPSCs. cDNA was isolated from differentiated adult NSC-derived iPSCs, and values were normalized to the level of β-actin. Error bars indicate SEM. Three independent experiments were performed in triplicate. Scale bar: 200 µm (C–D); 50 µm (E–F); 20 µm (G–H). ITSFn, insulin/transferrin/selenium/fibronectin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104995&req=5

pone-0019926-g006: Reprogrammed adult NSCs can be differentiated into midbrain DA neurons.(A) NSC-derived iPSCs were differentiated into DA neurons according to the five-stage protocol. Neural differentiation was induced by EB formation in ITSFn medium. Subsequently, the neural precursors were expanded in the presence of the growth factors FGF-2, FGF8, and Shh. Terminal differentiation was induced by growth factor withdrawal in the presence of ascorbic acid. (B) Graphs indicate the percentage of cells that stained positive for β-TubIII and TH, relative to nuclear Hoechst staining. Error bars indicate SEM. Three independent experiments were performed in triplicate. (C) Brightfield images showing EB-derived FGF2-responsive neural precursor cells. (D) Brightfield microphotographs of differentiated NSC-derived iPSCs, 7 days after growth factor withdrawal. (E) Immunofluorescence staining of neuronal cultures derived from NSC-derived iPSCs for β-TubIII (green) and TH (red). (F) Immunofluorescence staining for TH (red) and GABA (green), a marker typically expressed in olfactory bulb (OB) glomerular interneurons. Nuclei were counterstained with Hoechst. (G–H) Confocal images of neuronal cultures stained for β-TubIII (green) (G), TH (red) (G–H), En1 (blue) (G) and Pitx3 (green) (H). (I) Quantitative RT-PCR analysis of midbrain transcription factors, DA neurotransmission markers (TH, DAT, Pitx3, En1, Girk2, VMAT, ALDH, Nurr1) and calbindin in differentiated NSC-derived iPSCs. cDNA was isolated from differentiated adult NSC-derived iPSCs, and values were normalized to the level of β-actin. Error bars indicate SEM. Three independent experiments were performed in triplicate. Scale bar: 200 µm (C–D); 50 µm (E–F); 20 µm (G–H). ITSFn, insulin/transferrin/selenium/fibronectin.
Mentions: To test whether NSC-derived iPSCs were able to generate DA neurons in vitro, we differentiated iPSCs according to the mouse ESC five-stage protocol, with some modifications (Fig. 6A) [7], [24]. We induced neural differentiation by EB formation in ITSFn medium (Fig. 6C). Subsequently, the neural precursors were expanded in the presence of the growth factors Shh, FGF-2 and FGF8. Terminal differentiation was induced by growth factor withdrawal in the presence of AA (Fig. 6D). At day 32, we detected a total of 30% β-TubIII+ neurons and a total of 5% TH+/β-TubIII+ neurons (Fig. 6B, E). To assess neuronal regional specification, we double-labeled TH-positive neurons with antibody against Pitx3 and En1, markers typically expressed in midbrain DA neurons (Fig. 6 G–H). Importantly, the vast majority of TH-positive neurons stained positive for these midbrain markers, suggesting their proper midbrain regional specification in vitro (Fig. 6 G–H). We did not find any colocalization between TH and GABA, a marker typically expressed in OB glomerular interneurons (Fig. 6F). qRT-PCR analysis further confirmed expression levels of midbrain DA markers in differentiated cultures: DAT, PITX3, En1, G-protein-activated inwardly rectifying potassium channel subunit (Girk2), vesicular monoamine transporter (VMAT), aldehyde dehydrogenase 2 (ALDH), Calbindin and Nurr1 (Fig. 6I).

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