br Introduction Proper function of

Introduction Proper function of the cerebral cortex requires the coordinated activity of two distinct neuronal populations: excitatory projection neurons and inhibitory GABAergic interneurons (cINs). In both mice and humans, roughly half of all cINs originate within the medial ganglionic eminence (MGE) of the subcortical telencephalon and can be separated into two non-overlapping categories defined by their expression of either parvalbumin (Pv) or somatostatin (Sst) (Kepecs and Fishell, 2014; Kubota and Kawaguchi, 1994). While Sst interneurons primarily target the dendrites of their synaptic partners, Pv interneurons mainly target the cell body, proximal dendrites, or the axon initial segment of pyramidal neurons (Rudy et al., 2011). Interneuron dysfunction is implicated in major neurological and psychiatric diseases including autism, schizophrenia, and epilepsy (Marin, 2012). Due to their remarkable capacity to migrate, survive, and integrate into cortical circuitry after transplantation, cINs are attractive candidates for use in cell-based therapies of disorders of cortical inhibition, such as epilepsy (Southwell et al., 2014; Tyson and Anderson, 2014). Although progress has been made in generating enriched populations of interneuron subgroups from pluripotent stem PalMitoyl Tripeptide-1 (Harmacek et al., 2014; Tyson et al., 2015), protocols to efficiently generate highly enriched samples of Pv interneurons are lacking. We recently demonstrated that Pv interneurons originate primarily from divisions of intermediate progenitors in the subventricular zone (SVZ) of the MGE (Petros et al., 2015). This finding is consistent with a previous study that loss of cyclin D2 (Ccnd2), which is expressed in intermediate progenitors throughout the telencephalon, results in reduced numbers of Pv interneurons without affecting the Sst-expressing subgroup (Glickstein et al., 2007). Loss of Nr2f1, which results in increased expression of Ccnd2 in the dorsal region of the MGE where most Sst interneurons normally originate (Inan et al., 2012), also results in supernumerary production of Pv interneurons (Lodato et al., 2011). Together, these findings suggest that enhancement of intermediate progenitor-like divisions should enhance the production of Pv interneurons from stem cell differentiation. The atypical protein kinase C (aPKC)-CREB-binding protein (CBP) signaling pathway regulates the differentiation of interneurons from ventral forebrain neural progenitors (Tsui et al., 2014). Activation of aPKC results in the phosphorylation of CREB, thereby promoting neural differentiation (Wang et al., 2010). In addition, aPKC is an integral component of the aPKC/Par complex that regulates cell polarity and the localization of cell-fate determinants (Vorhagen and Niessen, 2014) including the Notch inhibitor Numb (Klezovitch et al., 2004). Since aPKC inhibition enhances intermediate neurogenesis in the neocortex (Wang et al., 2012), we examined whether aPKC inhibition during directed differentiations of embryonic stem cells (ESCs) into cortical interneurons will bias progenitors to undergo SVZ-like divisions. We find that a PKC pseudosubstrate peptide inhibitor (aPKCi), applied to our “MGE” protocol (Tyson et al., 2015), significantly increases the fraction of these progenitors that express Ccnd2. Moreover, treatment of stem cell differentiations with aPKCi greatly enriches for the generation of Pv-expressing interneurons at the expense of those expressing Sst. Also consistent with our studies in vivo (Petros et al., 2015), Notch signaling inhibition promotes the generation of Sst subtype fate. Taken together, our system provides a platform for further study of cortical interneuron genesis, fate determination, and their use in the development of cell-based therapies.
Results
Discussion Our previous study showed that manipulations of sonic hedgehog (Shh) exposure and time in culture differentially enrich for Pv- versus Sst-fated mESC-derived cINs (Tyson et al., 2015). While early-born cells exposed to higher levels of Shh produced a ∼6.4:1 ratio of Sst to Pv, increased duration in culture combined with lower levels of Shh generated a ∼2.6:1 ratio of Pv to Sst. Another study using the forced expression of transcription factors in a gain-of-function approach found that Lmo3 expression after the expression of Nkx2.1 and Dlx2 was able to achieve a 2.7:1 ratio of Pv to Sst (Harmacek et al., 2014). In this study, we used aPKC inhibition to achieve a ∼5.8:1 ratio of Pv to Sst. This, to our knowledge, is the best enrichment for Pv-expressing subtypes that has been obtained from mESCs to date.