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. 2024 May 22;9(10):e167598. doi: 10.1172/jci.insight.167598

Figure 4. Dex30 hESCs can differentiate to GnRH-expressing neurons.

Figure 4

(A) A representative RNAscope RNA in situ hybridization using probes against Madd (blue) and Gnrh1 (red) in adult (P45) mouse hypothalamus, coronal section. Experiments were repeated >10 times. Arrows indicate examples of double-positive cells. Scale bar: 50 μm. 3v, 3rd ventricle; Hy, hypothalamus. (B) A schematic of the hESC-derived GnRH-expressing neuron differentiation protocol. DM, dorsomorphin; SB, SB431542; FGF8, fibroblast growth factor 8; DAPT, gamma-secretase inhibitor IX. (C) tdTomato fluorescent reporter indicating expression of GNRH1 in WT and dex30 cultures on day 27. Scale bars: 100 μm. (D) The number of tdTomato+ cells on day 27 (n = 6 for WT, n = 12 for dex30). (E) Immunocytochemistry with antibodies against GnRH (green) and neuron-specific class II β-tubulin TuJ1 (red) in WT and dex30 cultures on day 27. Scale bars: 50 μm, region of interest (ROI) scale bar: 20 μm. (F) Secretion of GnRH to culture medium during 48 hours from WT and dex30 GnRH-expressing cells on days 25 and 27 (n = 1–2). NS P ≥ 0.05, analyzed by Student’s t test. CL, clone.