Introduction: Human embryonic stem cells (hESCs) are susceptible to dissociation-induced stress, activating the unfolded protein response (UPR) and triggering apoptosis. BiP (GRP78), a key ER chaperone, regulates UPR signaling and maintains ER homeostasis. However, the specific role of BiP in dissociation-induced stress in hESCs is unclear. This study investigates how BiP knockout (BiP-KO) alters UPR pathways and apoptosis, and evaluates TUDCA’s potential to improve cell survival.
Methods: BiP was knocked out in hESC line (H9) using CRISPR/Cas9 technology. BiP-KO and wild-type (WT) hESCs were dissociated and analyzed over a 24-hour time course. UPR markers (EIF2α, ATF4, ATF6, ERN1, spliced XBP1, total XBP1) were quantified using qRT-PCR. Apoptosis markers (CHOP, cleaved caspase-3) were assessed by Western blotting and immunofluorescence. TUDCA treatment was used to assess protective effects on ER stress and apoptosis.
Results: BiP-KO cells exhibited significant alterations in UPR signaling. EIF2α levels increased over time and were significantly higher in BiP-KO cells, indicating enhanced UPR activation. In contrast, ATF4 and ATF6 levels decreased over time and were significantly lower in BiP-KO cells, while WT cells showed an increase, reflecting adaptive UPR activation. Spliced XBP1 levels increased over time and were significantly higher in BiP-KO cells, while total XBP1 levels decreased and were lower in BiP-KO cells, indicating altered XBP1 dynamics. No significant difference in ERN1 expression was observed between BiP-KO and WT cells. BiP-KO cells demonstrated increased apoptosis, with elevated cleaved caspase-3 and CHOP levels. TUDCA treatment partially alleviated ER stress and reduced apoptosis markers in both BiP-KO and WT cells.
Conclusion: BiP is crucial for maintaining ER homeostasis and UPR signaling in hESCs under dissociation-induced stress. Its absence causes dysregulated UPR pathways, increased EIF2α, spliced XBP1 activation, and heightened apoptosis. While ERN1 expression remains unchanged, elevated spliced XBP1 suggests BiP influences ERN1 activity indirectly. TUDCA shows promise in reducing ER stress and apoptosis, supporting its potential as a therapeutic approach to enhance hESC viability and address ER stress-related diseases.
Funding Source: Shahnaz Babaei was supported by a Cumming School of Medicine Postdoctoral Fellowship.
