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  • Tibbe McGinnis posted an update 3 months, 1 week ago

    The environment in the postnatal cerebral cortical may not be permissive for all lineage-reprogrammed astroglial hiv protease inhibitors to differentiate into iNs. In fact, after transplantation in the neurogenic subventricular zone, we observed that both cerebellar and cortical astroglia nucleofected with ASCL1 could migrate throughout the RMS and differentiate in the OB as GCL- and PGL-like interneurons. This suggests that the milieu in the postnatal SVZ is not only more permissive to lineage-reprogrammed astroglia iNs, but also plays instructive roles in the phenotype of the iNs. Interestingly, however, despite this instructive role of environment, ASCL1 lineage-reprogrammed cortical and cerebellar astroglia iNs generated GCL- and PGL-like interneurons at different ratios, suggesting that the origin of the astroglial cell still play some role in fate determination. A few iNs were also detected in the OB after transplantation of cortical astroglia nucleofected with NEUROG2. One possible explanation for this difference could be the distinct roles played by ASCL1 and NEUROG2 in the postnatal SVZ. While the former is required for generation of most OB interneurons, especially granule cells (Parras et al., 2004), NEUROG2 contributes to the generation of a very small proportion of juxtaglomerular neurons (Winpenny et al., 2011).Of note, we observed GFP+ cells with astrocytic morphologies in the RMS and OB of all animals transplanted with both cortical and cerebellar astroglia at postnatal stages, regardless of the plasmid used for transfection (control-, Neurog2-, or Ascl1-DsRed). Most of these cells did not express dsRed (non-transfected cell) and some did (transfected but not reprogrammed). This observation suggests that transplanted astrocytic cells can also respond to migration cues in the SVZ-RMS-OB system and integrate in the OB. Accordingly it has been recently shown that astrocytes are constantly added to the OB after generation in the SVZ (Sohn et al., 2015).Experimental ProceduresAuthor ContributionsAcknowledgmentsThis work was supported by CNPq (MRC 473254/2013-1 and 466959/2014-1) and CAPES fellowships to M.C. and D.M. We thank Dr. Benedikt Berninger for providing the plasmids encoding for proneural factors and for helpful discussion of the manuscript. We also thank Dr. Cecilia Hedin Pereira, Dr. Cláudio Queiroz, Dra. Katarina Leão, and Dr. Ricardo Reis for valuable discussion of the work, and Rebecca Diniz, Ana Cristina, and Giovanna Andrade for technical assistance.IntroductionNeural stem cells/progenitor cells (NSCs) promise great hope for various neurological diseases. Researchers have demonstrated that NSCs are able to migrate and differentiate into adult rat brain and spinal cord (Flax et al., 1998; Snyder and Teng, 2012; Tabar et al., 2005). The human brain, however, poses a particular challenge for migrating NSCs or neuroblasts due to our larger brain size and the distances that cells must travel. Endogenous neuroblasts reside in the subventricular zone (SVZ) and hippocampus, deep in the brain. Neuroblasts from these niches have to migrate long distances to reach lesions in the cortex or other extra-hippocampal regions. Another hurdle is that transplanted NSCs have very poor motility due to suppression of migration by NSCs to each other and their progenies (Ladewig et al., 2014). Expanding the limits of migration of stem cells in brain is therefore a key step in stem cell therapies for the human brain (Trounson and McDonald, 2015). We aim to develop techniques that can mobilize and guide stem cells in the brain in vivo, which has not yet been achieved.We chose the rostral migration stream (RMS) to develop our stimulation technique because this is one of the most active migratory paths in the brain, and its cellular and molecular mechanisms are well understood (Anton et al., 2004; Curtis et al., 2007; Mobley and McCarty, 2011; Sanai et al., 2011; Staquicini et al., 2009). Newly born neuroblasts and transplanted hNSCs placed at the SVZ normally migrate directionally downstream to the olfactory bulb (OB), guided by various cues, including multiple chemical gradients and flow of cerebral spinal fluid (Flax et al., 1998; Sawamoto et al., 2006; Snyder and Teng, 2012; Tabar et al., 2005; Wu et al., 1999). This model allows us to test whether our technique is able to guide hNSCs to travel upstream toward the lateral ventricle (LV) region on the ipsilateral side, against endogenous directional cues.

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