Endoplasmic Reticulum Stress Inhibits the Neuronal Differentiation of Human Stem Cells From Apical Papillae by Attenuating the Activity of ERK-IRE1α Axis In Vitro.

Aim: Stem cells from apical papillae (SCAPs) are promising seed cells for angiogenesis, neurogenesis and dental pulp regeneration, which are contingent upon endoplasmic reticulum (ER) homeostasis. Due to the narrow anatomical structure of the root canal system and slow ingrowth of vasculatures, the presence of hypoxia and nutrient-deficient microenvironment within the sterilised root canal space may induce ER stress in the transplanted cells and affect their differentiation into neural lineages. This study aimed to investigate the role of ER stress in the neuronal differentiation of human SCAPs and its underlying mechanisms.

Methodology: Thapsigargin (TG) was employed to induce ER stress in SCAPs. ER Ca²⁺ level was quantified by Mag-Fluo 4 AM. Quantitative real-time PCR (qRT-PCR) and western blot were conducted to detect ER stress markers. SCAPs, with or without ER stress, were guided towards neuronal differentiation. We measured the expression of neuronal markers and the activation of the extracellular signal-regulated kinase (ERK1/2) and the unfolded protein response (UPR) signalling. Immunofluorescence staining was applied to observe SCAP-derived neuron-like cells. The kinetic Ca²⁺ influx of SCAP-derived neuron-like cells was monitored using a fluorescence microscope. SCH772984 and MKC8866 were used to selectively inhibit ERK1/2 and inositol-requiring enzyme 1α (IRE1α) activation, respectively. Statistical analyses were conducted using the GraphPad Prism 10 software.

Results: After TG stimulation, ER Ca²⁺ levels in all TG-treated SCAP groups were markedly reduced, the ER stress markers were significantly upregulated and UPR activation was found. Following neuronal induction, ER stress induced by 20 nM TG did not inhibit SCAP neuronal differentiation. However, ER stress induced by 40 or 80 nM TG significantly inhibited neuronal marker expression, neurite outgrowth and Ca²⁺ influx in SCAP-derived neuron-like cells. The phosphorylated ERK1/2 decreased during neuronal differentiation, along with the reduction of phosphorylated-IRE1α (p-IRE1α). Inhibition of ERK1/2 activation led to neuronal marker protein reduction, neurite outgrowth restraint and p-IRE1α decrease. Selective inhibition of IRE1α activity suppressed NeuN expression and neurite outgrowth.

Conclusion: Severe ER stress inhibits the neuronal differentiation of SCAPs via decreasing ERK1/2 and IRE1α activity, whereas ER stress at an appropriate level is essential for the neuronal differentiation of SCAPs.