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  • Although previous work showed that SOX is sufficient to conv

    2018-10-20

    Although previous work showed that SOX2 is sufficient to convert fibroblasts into NES+ multipotent NSCs under a unique culture condition (Ring et al., 2012), our genetic approach using Nes-CreER;Rosa-YFP mice failed to detect a stable NSC state during the in vivo reprogramming process. This difference highlights the critical importance of the microenvironment in cell fate reprogramming and suggests that additional factors are required to enable SOX2 to convert glial cells into multipotent stem cells in the adult CNS. Our initial in vivo screens for reprogramming factors included the four factors for induced pluripotent stem cells (Niu et al., 2013); however, this combination of factors has been subsequently Triptolide shown to induce tumorigenesis in vivo (Abad et al., 2013; Ohnishi et al., 2014) and raised serious concerns about the therapeutic value of in vivo induced pluripotent stem cells. In contrast, we have not observed any histological signs of tumorigenesis in brains or spinal cords with ectopic SOX2 expression when examined up to 1 year post virus injection. This is consistent with a recent study showing that multiple key transcription factors are required to initiate Triptolide tumors in vivo (Suvà et al., 2014). Adult neurogenesis is a multistep process beginning with multipotent NSCs that yield transient amplifying neural progenitors and then mature neurons (Gage, 2000; Hsieh, 2012; Kriegstein and Alvarez-Buylla, 2009; Lie et al., 2004). Our immunohistochemistry and genetic fate-mapping experiments demonstrate that SOX2-driven in vivo reprogramming passes through an ASCL1+ intermediate progenitor stage. These progenitors give rise to DCX+ neuroblasts, which can further proliferate and expand the neuronal lineage (Niu et al., 2013). This sequential and multistage amplification process is consistent with what has been observed in the adult neurogenic niches, suggesting that the reprogrammed glial cells have adopted the behavior of endogenous progenitors (Kim et al., 2011b; Lugert et al., 2012). ASCL1 serves as a molecular marker of neural progenitors and also controls their activity, including proliferation and differentiation (Andersen et al., 2014; Battiste et al., 2007; Jessberger et al., 2008; Kim et al., 2007, 2008). Consistently, our conditional deletion of Ascl1 in astrocytes shows that it is critically important for robust in vivo reprogramming. Nonetheless, ASCL1 cannot replace SOX2 in the induction of neurogenesis from adult astrocytes, as DCX+ iANBs are rarely detected in striatal regions with ectopic ASCL1 during a series of time course analyses. This result agrees with a recent in vivo study (Heinrich et al., 2014) and suggests that additional factors are also required in combination with ASCL1 to mediate SOX2 function in cell fate reprograming in the adult brain. In contrast, ASCL1 alone is able to transdifferentiate early postnatal astroglia (Heinrich et al., 2010, 2012), fibroblasts (Chanda et al., 2014), and embryonic stem cells (Chanda et al., 2014) into functional neurons in culture. This discrepancy between results from in vitro and in vivo studies once again highlights the importance of the cellular milieu in cell fate specification, determination, and reprogramming. A survey of markers for neuronal subtypes reveals that a larger population of SOX2-induced neurons expresses calretinin, a calcium-binding protein in GABAergic interneurons (Tepper et al., 2010). Interestingly, calretinin was shown to be transiently co-expressed with DCX during the early stage of adult hippocampal neurogenesis and subsequently downregulated once newly born neurons become mature 6 weeks later (Brandt et al., 2003). In contrast, calretinin is rarely detectable in iANBs but continuously expressed once they develop into mature neurons by 8 wpi, suggesting the stable generation of this neuronal subtype. The preferential reprogramming of striatal astrocytes by SOX2 to calretinin+ neurons was unexpected but is most likely due to signaling cues present in the adult striatum or intrinsic factors residing in the striatal astrocytes. It will be interesting in the future to tease out these instructive factors for the generation of specific neuron subtypes.