Investigations into the basal neural-like properties of dental pulp stem cells reveal they possess a functional type 2 muscarinic receptor which regulates quiescence.

BACKGROUND: Dental pulp stem cells (DPSCs) are a population of mesenchyme-derived cells residing within the dental pulp known for their multipotent differentiation potential and neural-like properties. While a functional cholinergic system has been described in various mesenchymal stem cell (MSC) populations, muscarinic receptor mediated cholinergic signalling remains unexplored in DPSCs. METHODS: The expression of neurotransmitter-associated genes was investigated using a targeted array panel and immunocytochemistry. Functionality of acetylcholine receptors (AChRs) was confirmed using receptor-specific agonists and antagonists. The effects of type 2 muscarinic receptor (m2AChR) signalling on DPSCs viability and proliferation were evaluated using an LDH release assay, CCK-8 assay and annexin V/PI staining. The effect of m2AChR signalling on the cell cycle was determined by flow cytometry and gene expression profiling, and the downstream effects on DPSCs osteogenic differentiation and migration were determined using an osteogenic differentiation assay and a wound healing assay. Finally, the effect of m2AChR signalling on the transcriptome was determined by RNAseq and the role of the MAPK/ERK pathway in mediating m2AChR signalling determined using an in-cell ELISA. RESULTS: Analysis of the neurotransmitter profile of DPSCs revealed they have cholinoceptive properties and pharmacological investigations confirmed they express a functional m2AChR. Activation of the m2AChR led to a reversible reduction in DPSCs proliferation, without compromising cell viability or pluripotency. Flow cytometric analysis and gene expression profiling confirmed that activation of the m2AChR caused cell cycle arrest at the G2/M phase which coincided with upregulated expression of CDKN1A (P21), a canonical marker of quiescence. Activation of the DPSC m2AChR also impaired their migration and osteogenic differentiation capabilities. RNAseq analysis revealed differentially expressed genes involved in regulating the cell cycle and MAPK/ERK signalling. Furthermore, analysis of ERK1/2 phosphorylation suggested that the MAPK/ERK pathway may play a role in m2AChR mediated regulation of quiescence. CONCLUSIONS: DPSCs exhibit cholinoceptive properties, and activation of the m2AChR engages the MAPK/ERK pathway and is associated with a reversible cell-cycle arrest consistent with a quiescent-like state, without affecting viability or pluripotency. These data support the m2AChR as a putative target for manipulating DPSC behaviour and transient quiescence in future regenerative applications.