FGF8-FGFR1 signaling regulates human GnRH neuron differentiation in a time- and dose-dependent manner

Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of gonadotropin-releasing hormone-expressing neurons (GnRH neurons). We hypothesized that FGF8 regulates differentiation of human GnRH neurons in a time-and dose-dependent manner via FGFR1. To investigate this further, human pluripotent stem cells were differentiated during 10 days of dual-SMAD inhibition into neural progenitor cells, followed either by treatment with FGF8 at different concentrations (25 ng/ml, 50 ng/ml or 100 ng/ml) for 10 days or by treatment with 100 ng/ml FGF8 for different durations (2, 4, 6 or 10 days); cells were then matured through DAPT-induced inhibition of Notch signaling for 5 days into GnRH neurons. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with gene expression of GNRH1 (PPeer reviewe

HiHo-AID2: boosting homozygous knock-in efficiency enables robust generation of human auxin-inducible degron cells

Recent developments in auxin-inducible degron (AID) technology have increased its popularity for chemogenetic control of proteolysis. However, generation of human AID cell lines is challenging, especially in human embryonic stem cells (hESCs). Here, we develop HiHo-AID2, a streamlined procedure for rapid, one-step generation of human cancer and hESC lines with high homozygous degron-tagging efficiency based on an optimized AID2 system and homology-directed repair enhancers. We demonstrate its application for rapid and inducible functional inactivation of twelve endogenous target proteins in five cell lines, including targets with diverse expression levels and functions in hESCs and cells differentiated from hESCs

Deciphering the Transcriptional Landscape of Human Pluripotent Stem Cell-Derived GnRH Neurons : The Role of Wnt Signaling in Patterning the Neural Fate

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons lay the foundation for human development and reproduction; however, the critical cell populations and the entangled mechanisms underlying the development of human GnRH neurons remain poorly understood. Here, by using our established human pluripotent stem cell-derived GnRH neuron model, we decoded the cellular heterogeneity and differentiation trajectories at the single-cell level. We found that a glutamatergic neuron population, which generated together with GnRH neurons, showed similar transcriptomic properties with olfactory sensory neuron and provided the migratory path for GnRH neurons. Through trajectory analysis, we identified a specific gene module activated along the GnRH neuron differentiation lineage, and we examined one of the transcription factors, DLX5, expression in human fetal GnRH neurons. Furthermore, we found that Wnt inhibition could increase DLX5 expression and improve the GnRH neuron differentiation efficiency through promoting neurogenesis and switching the differentiation fates of neural progenitors into glutamatergic neurons/GnRH neurons. Our research comprehensively reveals the dynamic cell population transition and gene regulatory network during GnRH neuron differentiation.Peer reviewe