Soft hydrogels supported high viability of DS-NPC spheroids and promoted neuronal differentiation of DS-NPC spheroids. (A) Representative fluorescent images of living cells (green) and dead cells (red) of DS-NPC spheroids cultured in hydrogel-free medium or laden in hydrogel scaffolds in NDM after 28-day culture. Scale bars= 300 μm. (B) Representative immunofluorescent staining for NeuN (red), βIII-tubulin (green) and nuclei (blue) of DS-NPC spheroids cultured in hydrogel-free medium or laden in hydrogel scaffolds in NDM after 28-day culture. Scale bars= 300 μm, (C) qPCR analysis of DLG4, Synapsin1, TUBB3, and NeuN of DS-NPC spheroids cultured in hydrogel-free medium or laden in hydrogel scaffolds in NDM after 28-day culture. Relative gene expression is presented as normalized to 18S and expressed relative to DS-NPC spheroids cultured in hydrogel-free medium (n=3; bars that do not share letters are significantly different from each other (p<0.05); **p<0.01).