Limits...
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.

Show MeSH

Related in: MedlinePlus

NPC-derived iPSCs can be differentiated into three germ layers in vitro.NSC-derived iPSCs were differentiated into three germ layers by embryoid body (EB) differentiation. (A) In vitro EB formation. (B–D) EBs expressed markers of the three germ layers including α-fetoprotein (AFP) (endoderm), brachyury (mesoderm), and β-TubIII (ectoderm). Scale bars: 25 µm (A–C); 50 µm (D).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3104995&req=5

pone-0019926-g005: NPC-derived iPSCs can be differentiated into three germ layers in vitro.NSC-derived iPSCs were differentiated into three germ layers by embryoid body (EB) differentiation. (A) In vitro EB formation. (B–D) EBs expressed markers of the three germ layers including α-fetoprotein (AFP) (endoderm), brachyury (mesoderm), and β-TubIII (ectoderm). Scale bars: 25 µm (A–C); 50 µm (D).

Mentions: We overexpressed Oct-4 in rTA-Oct4 SVZ NSC cultures by DOX and 48 hours later NSCs were plated on mouse embryonic fibroblasts (MEF) in ESC medium. In order to improve the efficiency of reprogramming, ESC medium was supplemented with 0.5 mM VPA for 7–10 days [31] (Fig. 4A). After 5–6 weeks, we were able to select mouse ESC-like colonies [from now on termed as NSCs-derived induced pluripotent stem cells (iPSCs)]. We characterized 2 NSC-derived iPSC clones (clones mm3 and mm4) (Fig. 4B). The estimated reprogramming efficiency of NSCs was 0.001%. We observed no colonies when VPA was omitted. NSC-derived iPSCs expressed alkaline phosphates (AP), Nanog and SSEA-1, and were morphologically indistinguishable from mouse ESCs (Fig. 4 B–E). As expected, the Oct4 promoter region of NSC-derived iPSCs was found to be hypomethylated (Fig. 4F). Interestingly, the Oct4 promoter region of NSCs derived from iPSCs was hypomethylated compared to adult SVZ NSCs (Fig. 4F and 2B). These iPSC colonies could be expanded up to 25 passages. We then examined the ability of NSC-derived iPSCs to differentiate into the three germ layers by embryoid body (EB) differentiation. EBs derived from NSC-iPSCs expressed markers of the three germ layers including α-fetoprotein (AFP) (endoderm), brachyury (mesoderm), and β-TubIII (ectoderm) as determined by immunocytochemical analysis (Fig. 5 A–D).


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)

NPC-derived iPSCs can be differentiated into three germ layers in vitro.NSC-derived iPSCs were differentiated into three germ layers by embryoid body (EB) differentiation. (A) In vitro EB formation. (B–D) EBs expressed markers of the three germ layers including α-fetoprotein (AFP) (endoderm), brachyury (mesoderm), and β-TubIII (ectoderm). Scale bars: 25 µm (A–C); 50 µm (D).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019926-g005: NPC-derived iPSCs can be differentiated into three germ layers in vitro.NSC-derived iPSCs were differentiated into three germ layers by embryoid body (EB) differentiation. (A) In vitro EB formation. (B–D) EBs expressed markers of the three germ layers including α-fetoprotein (AFP) (endoderm), brachyury (mesoderm), and β-TubIII (ectoderm). Scale bars: 25 µm (A–C); 50 µm (D).
Mentions: We overexpressed Oct-4 in rTA-Oct4 SVZ NSC cultures by DOX and 48 hours later NSCs were plated on mouse embryonic fibroblasts (MEF) in ESC medium. In order to improve the efficiency of reprogramming, ESC medium was supplemented with 0.5 mM VPA for 7–10 days [31] (Fig. 4A). After 5–6 weeks, we were able to select mouse ESC-like colonies [from now on termed as NSCs-derived induced pluripotent stem cells (iPSCs)]. We characterized 2 NSC-derived iPSC clones (clones mm3 and mm4) (Fig. 4B). The estimated reprogramming efficiency of NSCs was 0.001%. We observed no colonies when VPA was omitted. NSC-derived iPSCs expressed alkaline phosphates (AP), Nanog and SSEA-1, and were morphologically indistinguishable from mouse ESCs (Fig. 4 B–E). As expected, the Oct4 promoter region of NSC-derived iPSCs was found to be hypomethylated (Fig. 4F). Interestingly, the Oct4 promoter region of NSCs derived from iPSCs was hypomethylated compared to adult SVZ NSCs (Fig. 4F and 2B). These iPSC colonies could be expanded up to 25 passages. We then examined the ability of NSC-derived iPSCs to differentiate into the three germ layers by embryoid body (EB) differentiation. EBs derived from NSC-iPSCs expressed markers of the three germ layers including α-fetoprotein (AFP) (endoderm), brachyury (mesoderm), and β-TubIII (ectoderm) as determined by immunocytochemical analysis (Fig. 5 A–D).

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