Fig. 2

Differentiation of hiPSCs into neurons and analysis of neuronal markers in HS and sAD neurons. A Schematic representation of the Ngn2-mediated differentiation process used to convert hiPSCs from HS and sAD donors into neurons. Neurons were induced using doxycycline (Doxy) and selected with puromycin (Puro). B Diagram of the lentiviral constructs used in the differentiation process, including Ngn2, EGFP, and Puromycin resistance genes. C Timeline of neuronal differentiation showing key steps from hiPSC plating (DIV0), doxycycline induction (DIV1), puromycin selection (DIV3), and final analysis at DIV30. D Immunofluorescence staining of neuronal markers in HS and sAD-derived neurons at DIV30. DAPI (blue) stains nuclei, EGFP (green) marks neurons, and NEUN (red) marks mature neurons. Merged images confirm co-localization of neuronal markers. Scale bars: 50 µm. E Immunofluorescence staining of the neuronal marker MAP2 (red) in HS and sAD-derived neurons, with DAPI and EGFP labeling. F Quantification of MAP2 and NEUN median fluorescence intensity (in arbitrary units, a.u.) in HS and sAD neurons, showing no significant difference (n.s.) between groups (n = 5/group). G Representative Western blot images of MAP2 and NEUN in HS and sAD neurons, with GAPDH as the loading control. H Densitometric analysis of MAP2 and NEUN expression in HS and sAD neurons showing no significant differences in fold change(n = 5/group). Each dot represents the average of three independent experiments. Data are presented as mean ± SEM. n.s. p > 0.05, assessed by Mann Whitney Rank Sum test