OSNs and GNRHNs arise from hPSC-derived olfactory placode. (A) qRT-PCR results at day 30 for immature neuron (NESTIN), OSN (OMP), and GNRHN (GNRH1). Values are normalized to 18S and compared with vehicle-treated group (n = 5, mean ± SEM). (B) Immunocytochemistry analysis comparing protein expression on day 30 of differentiation for OMP (green) and NESTIN (red). Scale bars, 100 μM. (C) Quantification of OMP, NESTIN, and OMP+NESTIN-positive cells. Values are normalized to DAPI and compared with vehicle-treated group (n = 5, mean ± SEM). (D) Immunocytochemistry analysis comparing protein expression on day 30 of differentiation for GNRH1 (green) and NESTIN (red). Scale bars, 100 μM. (E) Quantification of GNRH1, NESTIN, and GNRH1+NESTIN-positive cells. Values are normalized to DAPI and compared with vehicle-treated group (n = 5, mean ± SEM). P-values (*P < 0.05, **P < 0.01, ***P < 0.001). GNRH1, gonadotropin-releasing hormone 1; GNRHNs. gonadotropin-releasing hormone neuron; hPSC, human pluripotent stem cell; OMP, olfactory marker protein; OSNs, olfactory sensory neurons.