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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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DA neurons from Oct4 reprogrammed adult NSCs integrate into the striatum of 6-OHDA lesioned parkinsonian rats and improve behavioural deficits.(A) A low-power overview of an iPSC cell graft 8 weeks after transplantation stained with an antibody against TH (dark brown). (B–C) Higher magnification of a NSC-derived iPSC graft showing TH-positive soma and the reinnervation of the surrounding host striatum by donor-derived neurites. The dashed line indicates the edge of the graft. (D–F) Confocal analysis of NSC-derived iPSC grafts, 8 weeks post-transplantation, showed that most grafts contained midbrain DA neurons. The grafted TH-positive cells (red) were colabeled with antibodies against NeuN (green) (D), Pitx3 (green) (E) and FOXA2 (green) (F). (G–H) DA neurons from NSC-derived iPSCs reversed amphetamine- and apomorphine-induced rotational behaviour upon engraftment into 6-OHDA-lesioned rats. Animals were analysed for amphetamine- and apomorphine-induced rotational behaviour before and 4, 6, and 8 weeks post transplantation. Graphs show mean values±SEM. (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). Scale bars: 200 µm (A–B); 100 µm (C); 20 µm (D–E); 10 µm (F).
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pone-0019926-g007: DA neurons from Oct4 reprogrammed adult NSCs integrate into the striatum of 6-OHDA lesioned parkinsonian rats and improve behavioural deficits.(A) A low-power overview of an iPSC cell graft 8 weeks after transplantation stained with an antibody against TH (dark brown). (B–C) Higher magnification of a NSC-derived iPSC graft showing TH-positive soma and the reinnervation of the surrounding host striatum by donor-derived neurites. The dashed line indicates the edge of the graft. (D–F) Confocal analysis of NSC-derived iPSC grafts, 8 weeks post-transplantation, showed that most grafts contained midbrain DA neurons. The grafted TH-positive cells (red) were colabeled with antibodies against NeuN (green) (D), Pitx3 (green) (E) and FOXA2 (green) (F). (G–H) DA neurons from NSC-derived iPSCs reversed amphetamine- and apomorphine-induced rotational behaviour upon engraftment into 6-OHDA-lesioned rats. Animals were analysed for amphetamine- and apomorphine-induced rotational behaviour before and 4, 6, and 8 weeks post transplantation. Graphs show mean values±SEM. (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). Scale bars: 200 µm (A–B); 100 µm (C); 20 µm (D–E); 10 µm (F).

Mentions: To further analyse the DA fate potential of NSC-derived iPSCs in vivo, we explored the ability of differentiated neurons to survive, integrate and reinnervate the host striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats, a rodent animal model of PD. Adult NSC-derived iPSCs (clone mm3) were differentiated into DA neurons as described above and transplanted into the striatum of 6-OHDA-lesioned rats. Animals received a striatal graft of 2×10∧5 differentiated cells. In order to avoid tumor formation, SSEA1+ cells were eliminated by cell sorting FACS prior to transplantation (Figure S1). Analysis of amphetamine-induced behaviour showed a significant reduction of ipsilateral rotations at 8 weeks after transplantation (Fig. 7G), indicating significant restoration of the 6-OHDA lesion. We found that rats transplanted with differentiated NSC-derived iPSCs showed a significantly reduced number of apomorphine-induced rotations 8 weeks after engraftment, when compared to the non-transplanted rats (Fig. 7H). Eight weeks after surgery, the animal brains were prepared for morphological analysis. TH staining showed that animals displaying improvement in the behavioural assays had grafts containing large numbers of DA neurons (Fig. 7A–D). Importantly, the transplanted neurons expressed the midbrain DA marker Pitx3 and Foxa2 (Fig. 7E–F). TH-immunoreactive fibres were found to extend into the parenchyma of the host striatum (Fig. 7B,C).


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)

DA neurons from Oct4 reprogrammed adult NSCs integrate into the striatum of 6-OHDA lesioned parkinsonian rats and improve behavioural deficits.(A) A low-power overview of an iPSC cell graft 8 weeks after transplantation stained with an antibody against TH (dark brown). (B–C) Higher magnification of a NSC-derived iPSC graft showing TH-positive soma and the reinnervation of the surrounding host striatum by donor-derived neurites. The dashed line indicates the edge of the graft. (D–F) Confocal analysis of NSC-derived iPSC grafts, 8 weeks post-transplantation, showed that most grafts contained midbrain DA neurons. The grafted TH-positive cells (red) were colabeled with antibodies against NeuN (green) (D), Pitx3 (green) (E) and FOXA2 (green) (F). (G–H) DA neurons from NSC-derived iPSCs reversed amphetamine- and apomorphine-induced rotational behaviour upon engraftment into 6-OHDA-lesioned rats. Animals were analysed for amphetamine- and apomorphine-induced rotational behaviour before and 4, 6, and 8 weeks post transplantation. Graphs show mean values±SEM. (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). Scale bars: 200 µm (A–B); 100 µm (C); 20 µm (D–E); 10 µm (F).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104995&req=5

pone-0019926-g007: DA neurons from Oct4 reprogrammed adult NSCs integrate into the striatum of 6-OHDA lesioned parkinsonian rats and improve behavioural deficits.(A) A low-power overview of an iPSC cell graft 8 weeks after transplantation stained with an antibody against TH (dark brown). (B–C) Higher magnification of a NSC-derived iPSC graft showing TH-positive soma and the reinnervation of the surrounding host striatum by donor-derived neurites. The dashed line indicates the edge of the graft. (D–F) Confocal analysis of NSC-derived iPSC grafts, 8 weeks post-transplantation, showed that most grafts contained midbrain DA neurons. The grafted TH-positive cells (red) were colabeled with antibodies against NeuN (green) (D), Pitx3 (green) (E) and FOXA2 (green) (F). (G–H) DA neurons from NSC-derived iPSCs reversed amphetamine- and apomorphine-induced rotational behaviour upon engraftment into 6-OHDA-lesioned rats. Animals were analysed for amphetamine- and apomorphine-induced rotational behaviour before and 4, 6, and 8 weeks post transplantation. Graphs show mean values±SEM. (* p≤0.05, Two-way ANOVA with post hoc analysis by Bonferroni test). Scale bars: 200 µm (A–B); 100 µm (C); 20 µm (D–E); 10 µm (F).
Mentions: To further analyse the DA fate potential of NSC-derived iPSCs in vivo, we explored the ability of differentiated neurons to survive, integrate and reinnervate the host striatum of 6-hydroxydopamine (6-OHDA)-lesioned rats, a rodent animal model of PD. Adult NSC-derived iPSCs (clone mm3) were differentiated into DA neurons as described above and transplanted into the striatum of 6-OHDA-lesioned rats. Animals received a striatal graft of 2×10∧5 differentiated cells. In order to avoid tumor formation, SSEA1+ cells were eliminated by cell sorting FACS prior to transplantation (Figure S1). Analysis of amphetamine-induced behaviour showed a significant reduction of ipsilateral rotations at 8 weeks after transplantation (Fig. 7G), indicating significant restoration of the 6-OHDA lesion. We found that rats transplanted with differentiated NSC-derived iPSCs showed a significantly reduced number of apomorphine-induced rotations 8 weeks after engraftment, when compared to the non-transplanted rats (Fig. 7H). Eight weeks after surgery, the animal brains were prepared for morphological analysis. TH staining showed that animals displaying improvement in the behavioural assays had grafts containing large numbers of DA neurons (Fig. 7A–D). Importantly, the transplanted neurons expressed the midbrain DA marker Pitx3 and Foxa2 (Fig. 7E–F). TH-immunoreactive fibres were found to extend into the parenchyma of the host striatum (Fig. 7B,C).

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