Figure 2.

Generation of human orexin neuron by treatment with ManNAc derivatives. (A-C) Activities of 13 ManNAc derivatives for HCRT gene expression in neural cells derived from hiPSCs. hiPSCs were cultured in SDIA+BMP4 medium for 20 d (Fig. 1A). ManNAc derivatives were added on day 7, and HCRT mRNA expression was evaluated by RT-PCR (A) or RT-qPCR (B). Values were normalized to ACTB expression. Means ± SD (n = 3). The broken red line indicates the activity of ManNAc for comparison. IF assay to confirm OREXIN-A expression in the hiONs induced by ManNAc and ManNFAc (C). Right panel indicates the high magnification images in ManNAc- and ManNFAc-treated cells. Scale bars, 100 μm (left panel) and 10 μm (right panel). (D) Efficiencies of neural cells treated with ManNAc, ManNFAc, or the inhibitors BADGP and EX-527 (see also Fig. 1F). Percentages of OREXIN-A positive area were determined by counting the OREXIN-A positive areas, and normalized by the DAPI-positive areas in 20 random images.