NSCs acquire a regional identity already at the formation of the neural plate during embryogenesis 
. Therefore, fetal and adult NSCs have passed crucial checkpoints that restrict their developmental capacity to generate region-specific neuronal subtypes.
Epigenetic modifications (i.e. histone code and DNA methylation) play a critical role in regulating adult NSC differentiation and fate determination 
. Here we investigated whether epigenetic remodeling induced by chromatin-modifying agents can reverse adult SVZ NSC developmental restriction and enable their differentiation into region-specific neuronal subtypes such as midbrain DA neurons. Chromatin remodeling factors can improve the induction of an ESC-like state and have been extensively used to enhance dedifferentiation of somatic cells to the pluripotent state 
. We showed that chromatin-modifying agents (TSA and TSA in combination with AZA) reactivate specific pluripotency-associated genes such as Oct4 and Klf-4 in adult SVZ NSCs. These agents induced partial demethylation of Oct4
promoter, thus promoting a dedifferentiation toward a primitive neural stage. We observed an increased neuronal differentiation, paralleled by a decreased number of astrocytes in treated NSC cultures. However, these modifications were not sufficient to reverse the resistance of NSCs to the patterning signals that regulate midbrain DA development, since the majority of differentiated neurons showed a GABAergic phenotype. Previous reports have shown the derivation of iPSCs from NSCs 
. Specifically, the overexpression of Oct-4 alone induces the reprogramming of NSCs obtained from mouse whole brain and fetal human telencephalon NSCs 
. In the current work we show for the first time that Oct4 overexpression in association with the chromatin-modifying agent VPA induced adult SVZ NSC reprogramming into iPSCs. Such Oct4-reprogrammed NSCs were then successfully differentiated into midbrain DA neurons. Importantly, we show for the first time that single-factor reprogrammed adult SVZ NSCs generated functional midbrain DA neurons that successfully improved the motor behavioural deficits in a rodent model of PD. Such functional motor behavioural recovery in this model is further significant evidence for the correct subtype of DA neurons generated from NSC-derived iPSCs.
With respect to functional cell therapy for the motor symptoms of PD, it is critical to obtain DA neurons that have the molecular properties of midbrain DA neurons 
. Both substantia nigra (SN-A9) and ventral tegmental area (VTA-A10) DA neurons contribute to widespread and dense axonal arborization 
, and SN-A9 DA neurons are responsible for appropriate striatal reinnervation and behavioural motor recovery in rodent models of PD 
. However, no studies have reported successful in vitro differentiation of adult SVZ NSCs into midbrain DA neurons. When grown as neurospheres and differentiated according to the most dedicated differentiation culture protocols, fetal and adult NSCs only generate a small number of TH+
. Furthermore, such differentiated TH+
neurons do not express markers of midbrain DA neurons 
, which is the required cell type for grafting and appropriate striatal reinnervation in PD. In vivo, mobilization of SVZ endogenous precursor cells has been envisaged as a promising alternative to cell transplantation for the treatment of neurodegenerative diseases including PD 
. Therefore, different approaches have been tested to promote the differentiation of adult SVZ NSCs to midbrain DA neurons and endogenous NPC proliferation in order to achieve tissue repair and functional recovery in several animal models of neurodegenerative diseases 
. However, there is no evidence that such strategies promote the generation of functional midbrain DA neurons that integrate into the injured or naïve nigrostriatal DA system 
Adult NSCs are resistant to the signaling molecules (Shh, FGF8) that control the development of midbrain and hindbrain 
. The precise mechanism by which such restriction is controlled and maintained in adult SVZ NSCs is still poorly understood. During mouse development, the earliest NSCs can be isolated starting at embryonic day 5.5 (E5.5) 
. These pNSCs retain ESC characteristics such as high Oct-4 expression and in vitro LIF-dependence 
. pNSCs are highly responsive to regionalization cues, allowing the efficient generation of region-specific neuronal subtypes. LIF-dependent NSCs with similar antigenic and functional properties can also be isolated from E5.5–7.5 mouse embryos 
. Between embryonic day 7.5 (E7.5) and E8.5, germ cell nuclear factor (GNFC)-mediated Oct4
promoter methylation drives the conversion from pNSCs to dNSCs 
. In the developing neuroectoderm and NSC cultures, such transition from pNSCs to dNSCs restricts the potential of dNSCs to form non-neural cell types and their capacity to generate region-specific neuron populations.
Interestingly, we found that Oct-4 overexpression, without reprogramming to an ESC state, increased NSC self-renewal and long-term proliferation in the presence of EGF and FGF-2. In addition, we showed that Oct4 overexpression increases neuronal differentiation of NSC cultures in the presence of Shh, FGF-2 and FGF8. However, our data indicate that such short-term Oct-4 overexpression alone does not confer competence for adult SVZ NSC midbrain regionalization. In contrast, long-term overexpression (30–40 days) of Oct-4 in adult SVZ NSCs grown on MEFs in ESC medium with LIF induced complete dedifferentiation to a pluripotent state. Only after Oct-4 induced reprogramming to a pluripotent state were adult SVZ NSCs successfully patterned to midbrain DA neurons.
Conclusion and summary
In summary, we show that epigenetic modifications are not sufficient to reverse the resistance of SVZ NSCs to the patterning signals that regulate midbrain regional specification. Such modifications do not promote the dedifferentiation of adult SVZ dNSCs toward a primitive neural stage. The current work describes for the first time the reprogramming of adult SVZ NSCs by means of Oct4 overexpression and provides the first evidence that functional midbrain DA neurons can be derived from Oct4-reprogrammed adult SVZ NSCs. These results indicate that the complex and precise regulatory processes responsible for the regional specification of adult NSCs are irreversible, and further de-differentiation steps are required to regain the competency to generate region-specific neuronal phenotypes. These findings have major fundamental scientific and practical implications for regenerative neuroscience.