Our results show that anterior NPCs

Our results show that anterior NPCs can give rise to GnRH-expressing adenosine receptor antagonist when cultured with FGF8, which has been shown to promote progenitor cell survival in the anterior neural structures (Forni et al., 2013; Kawauchi et al., 2005). In mouse anterior neural ridge, FGF8 has been shown to positively regulate Foxg1 expression (Shimamura and Rubenstein, 1997), and mouse embryos that carry hypomorphic and conditional Fgf8 mutations display reduced telencephalons, which is suggested to result from a decreased Foxg1 expression (Storm et al., 2006). In our model, FOXG1 expression persisted following FGF8 treatment, and importantly, FOXG1 was also expressed in migrating GnRH neurons of a human fetus. The final step in our protocol comprised Notch inhibition, which induces neuronal maturation from progenitor cells (Borghese et al., 2010; Imayoshi et al., 2010; Noisa et al., 2014; Yoon and Gaiano, 2005). As expected, the blocking of Notch in our FGF8-treated cells after d20 decelerated cell proliferation, increased the expression of neuron-specific markers TUJ1 and MAP2, and induced GNRH1 expression, with up to 15% of the cells being immunopositive for GnRH. This efficiency is high, given that humans only have few thousand GnRH neurons. Two recent publications have reported hPSC differentiation to hypothalamic-like neuroendocrine cells, such as POMC-, αMSH-, and AGRP-secreting neurons (Merkle et al., 2015; Wang et al., 2015). In both approaches, dual SMAD inhibition in combination with SHH activation led to ventral forebrain progenitor stage, as suggested by marker expression patterns including high NKX2.1 and low FOXG1 expression. It is noteworthy that these two protocols did not report an induction of GNRH1 expression, which highlights the known differences in the embryonic origin of these cells. In contrast, our protocol produced GnRH-expressing cells with long cellular processes consistent with previously described morphology of GnRH neurons (Herde et al., 2013). The GABAA receptor agonist muscimol suppressed the migration of GnRH-expressing cells, in agreement with the studies performed in embryonic mouse GnRH neurons (Casoni et al., 2012). In the majority of GnRH-expressing cells the GnRH staining appeared in punctate structures, indicating GnRH prepropeptide processing to mature decapeptide and packaging into secretory vesicles. The cultures indeed robustly secreted GnRH into culture medium. Future studies are required to further characterize the neuroendocrine properties of these GnRH-expressing cells. Our protocol was reproducible in hiPSCs, which implicates that it can be employed to model diseases that affect GnRH neuron specification, such as hypogonadotropic hypogonadism due to congenital GnRH deficiency.
Experimental Procedures See also Supplemental Experimental Procedures.
Acknowledgments We thank the Biomedicum Stem Cell Center and the Biomedicum Imaging Unit for advice and assistance. We thank Professor Erik Hrabovszky for the generous gift of GnRH antibody, Professor Timo Otonkoski for valuable comments, Mr. Samuel Malone for the human fetus collection and processing of the samples for immunohistochemical procedures, and M.A. Annika Tarkkanen for linguistic guidance. This work was supported by the Academy of Finland, Foundation for Pediatric Research, Sigrid Juselius Foundation, Novo Nordisk Foundation, Emil Aaltonen Foundation, University of Helsinki, Helsinki University Central Hospital, and Agence Nationale de la Recherche, ANR, France (ANR-14-CE12-0015-01 RoSes and GnRH).
Introduction The liver bud in the mouse embryo is formed from the foregut endoderm at around embryonic day 9.5 (E9.5) by the migration into the septum transversum of the fetal liver progenitors, hepatoblasts expressing the hepatic markers α-fetoprotein (AFP), and albumin (ALB). Hepatoblasts proliferate considerably to form the fetal liver mass and finally differentiate in midgestation into either hepatocytes or cholangiocytes based on their proximity to portal veins (Gordillo et al., 2015). In mammals, the fetal liver is also the major site of hematopoiesis (Golub and Cumano, 2013). Murine liver hematopoiesis is initiated at E10 with the colonization of the fetal liver by hematopoietic progenitors migrating from the yolk sac and the region of the aorta-gonad-mesonephros. The fetal liver hematopoietic activity decreases around E15 and disappears shortly after birth.