Characteristics of endoplasmic reticulum stress changes during the differentiation of adipose-derived stromal cells...

Adipose-derived stromal cells (ADSC) show promise for neuronal differentiation, but their utility is limited by late-stage cell death, which may be driven by endoplasmic reticulum stress (ERS). To investigate this mechanism, we employed an integrated approach combining immunocytochemistry, western blotting, single-cell RNA sequencing (scRNA-Seq), and transmission electron microscopy (TEM) to systematically profile ERS-related gene expression, dynamic changes of key proteins, and ultrastructural evolution of the ER during neuronal induction. Our results demonstrate that ERS pathways are activated throughout the differentiation process. In early stages, the endoplasmic reticulum (ER) chaperone GRP78 initially increased but markedly declined at 6 h and 8 h. Key UPR sensors IRE1α, XBP1s, PERK, and ATF6 peaked in undifferentiated ADSC and Pre-induction (Prei-1d) cells, then gradually decreased as differentiation progressed. In contrast, pro-apoptotic markers CHOP and Caspase-3 were continuously upregulated in later phases, accompanied by ultrastructural hallmarks of ER dilation, disrupted mitochondrial cristae, and cytoplasmic vacuolization. These findings indicate that ERS initially activates the unfolded protein response to maintain ER homeostasis and support differentiation, whereas sustained ERS at later stages shifts toward CHOP/Caspase-3-dependent apoptosis, leading to cellular injury. This study provides a theoretical basis for optimizing neuronal differentiation protocols through time-dependent modulation of ERS pathways.