Wnt-ligands are among key morphogens that mediate patterning of the anterior territories of the developing brain in mammals. We qualified the role of Wnt-signals in regional specification and subregional organization of the human telencephalon using human pluripotent stem cells (hPSCs). One step neural conversion of hPSCs using SMAD inhibitors leads to progenitors with a default rostral identity. It provides an ideal biological substrate for investigating the role of Wnt signaling in both anteroposterior and dorso-ventral processes. Challenging hPSCneural derivatives with Wnt-antagonists, alone or combined with sonic hedgehog (Shh), we found that Wntinhibition promote both telencephalic specification and ventral patterning of telencephalic neural precursors in a dose-dependent manner. Using optimal Wnt-antagonist and Shh-agonist signals we produced human ventraltelencephalic precursors, committed to differentiation into striatal projection neurons both in vitro and in vivo after homotypic transplantation in quinolinate-lesioned rats. This study indicates that sequentially organized Wntsignals play a key role in the development of human ventral telencephalic territories from which the striatum arise. In addition, the optimized production of hPSCderived striatal cells described here offers a relevant biological resource for exploring and curing Huntington disease.

Decreased expression of neuronal genes such as brainderived neurotrophic factor (BDNF) is associated with several neurological disorders. One molecular mechanism associated with Huntington disease (HD) is a discrete increase in the nuclear activity of the transcriptional repressor REST/NRSF binding to repressor element-1 (RE1) sequences. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to identify two benzoimidazole-5-carboxamide derivatives that inhibited REST silencing in a RE1-dependent manner. The most potent compound, X5050, targeted REST degradation, but neither REST expression, RNA splicing nor binding to RE1 sequence. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST in wild-type neural cells treated with X5050. This activity was confirmed in neural cells produced from human induced pluripotent stem cells derived from a HD patient. Acute intraventricular delivery of X5050 increased the expressions of BDNF and several other REST-regulated genes in the prefrontal cortex of mice with quinolinate-induced striatal lesions. This study demonstrates that the use of pluripotent stem cell derivatives can represent a crucial step toward the identification of pharmacological compounds with therapeutic potential in neurological affections involving decreased expression of neuronal genes associated to increased REST activity, such as Huntington disease. STEM CELLS

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self-renewal, and differentiation are not fully elucidated. In the present report, we describe a growth-stimulatory activity of liver explant-conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen-activated protein kinase (MAPK) (ERK1/2)-dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c-Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem-like cells that form neurospheres and self-renew. Immunostaining, HGF enzyme-linked immunosorbent assay and MadinDarby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell-conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF-neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin-expressing cells and c-Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF-neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self-renewal.