In accordance with recently published data our approach invo

In accordance with recently published data, our approach involves use of target-specific neurogenic factors for direct conversion toward a neural fate. Findings of other groups have provided strong evidence of reprogramming of mouse and human fibroblasts to functional neurons following forced buy something online of a single transcription factor or a combination of transcription factors (Lujan et al., 2012; Ring et al., 2012; Vierbuchen et al., 2010). This conversion is either direct driving fibroblasts to post-mitotic neurons (Vierbuchen et al., 2010) or first drives them toward an NPC state (Han et al., 2012; Lujan et al., 2012; Ring et al., 2012). Here, we have used a reprogramming protocol that primes naive MEFs toward acquisition of NESTIN+ neuroepithelial cell identity. By forced expression of CEND1 and NEUROG2, these primed fibroblasts have acquired a neuronal precursor fate, and upon addition of bFGF and BDNF they differentiated to subtype-specific functional neurons capable of firing action potentials. NEUROG2 has been previously shown to possess reprogramming action, directly converting human fibroblasts to cholinergic neurons when overexpressed together with the transcription factor SOX11 (Liu et al., 2013) and dopaminergic neurons upon its overexpression together with MASH1, SOX2, NURR1, and PITX3 (Liu et al., 2012). However, as recently demonstrated, NEUROG2 alone is not capable of inducing neuronal reprogramming of MEFs unless downstream effectors are also co-expressed (Chanda et al., 2013). In support of this, our findings demonstrate that NEUROG2 alone possesses a poor neuronal reprogramming action in MEFs, unless co-expressed together with CEND1. The generation of neuronal precursors from fibroblasts with subsequent differentiation to neurons observed in our system exhibits the advantage that induced NPCs can self-renew and expand for many passages, an event facilitating applications where large cell numbers are needed, such as high-throughput drug screening or cell transplantation. CEND1 is associated with the dynamics of neuron-generating divisions of embryonic and postnatal NPCs (Katsimpardi et al., 2008; Politis et al., 2007) and as shown here of primary astrocytes in vitro. Our previous data also indicate that CEND1 participates in bHLH proneural genes pathway(s) and is activated by bHLH factors, such as NEUROG1/2 and MASH1 in neuronal precursors (Katsimpardi et al., 2008; Papadodima et al., 2005; Politis et al., 2007). Here we show that CEND1 can also enhance the endogenous expression levels of NEUROG2 in reprogramming astrocytes, indicating the existence of a cross-activating feedback loop and, most importantly, that CEND1 expression is a key downstream player in NEUROG2-driven astrocytic reprogramming. NEUROG2 protein has a short half-life in NPCs—less than 30 min (Ali et al., 2011)—while its expression is dynamically regulated at both the mRNA and protein level by a number of factors. NEUROG2 mRNA stabilization promotes neuronal differentiation (Knuckles et al., 2012), while NEUROG2 protein phosphorylation by Cyclin-dependent kinases diminishes it (Hindley et al., 2012). In particular, it has been demonstrated that the cell-cycle machinery negatively regulates neurogenesis by diminishing NEUROG2 protein stability and binding to the E box of its downstream effectors (Hindley et al., 2012). Our findings demonstrate that CEND1 also affects endogenous NEUROG2 expression and/or stability and that its presence is required for NEUROG2 to exert its neurogenic action. Of relevance, since proneural genes are readily downregulated in differentiated neurons, their ability to sustain neuronal differentiation not only during development but also during reprogramming relies on activation of downstream genes participating in cellular differentiation networks, one of which is CEND1 (Katsimpardi et al., 2008; Politis et al., 2007). Therefore, sustained expression of CEND1 in differentiated neurons above a certain threshold level (Katsimpardi et al., 2008) is crucial for astrocytic reprogramming to differentiated neurons by NEUROG2. This observation is in line with the findings of Chanda et al. (2013), who show that NEUROG2 cannot induce efficient neurogenic reprogramming of MEFs unless the neuronal differentiation mediator MYTL1 is co-expressed. Last, while NEUROG2 is a proneural factor linked to dorsal cell identity of cortical neurons, CEND1 has a wider neuronal distribution being highly enriched in GABAergic striatal neurons and motor neurons of the spinal cord. Indeed, our previous in vivo CEND1-overexpresssion studies in the early chick neural tube (Politis et al., 2007) indicated that CEND1 can give rise to both ventral and dorsal neuronal identities. Thus, unlike NEUROG2, CEND1 seems to exhibit a general—not cell-type-specific—potential in conferring neuronal identity, a fact that explains the different neuronal phenotypes present upon CEND1 or NEUROG2 overexpression.