Characterising Expression of Muscarinic Acetylcholine Receptors in Human Dental Pulp Stem Cells: An In-Vitro Study

Abstract

Dental pulp stem cells (DPSCs) are a promising subgroup of mesenchymal stem cells (MSCs) that have the potential for regenerative applications. They have been reported to express a regenerative response for tooth structure by producing reparative dentine. One of the sources of DPSCs is the pulp of an extracted permanent tooth, which provides a convenient collection method. In addition, there are other groups of MSCs which share similar characteristics among them. Recent studies identified Acetylcholine (ACh) in some of the MSCs and the two major categories of Acetylcholine receptors (AChRs), muscarinic AChRs (m-AChRs) and nicotinic (n-AChRs). Although ACh is a neurotransmitter, there is evidence that it is produced by and influences non-neuronal cells. The involvement of ACh in the non-neuronal cells is called the non-neuronal cholinergic system (NNCS), which includes ACh-synthesizing enzymes, transporters, receptors, and degrading enzymes. Because DPSCs share similar characteristics with other MSCs, it is worth investigating the expression of m-AChRs in DPSCs. Also, to look into the role of m-AChRs in the regenerative function of DPSCs. There is potential for using DPSCs outside the tooth in regenerative applications, such as musculoskeletal regenerative medicine. The first part of the project started with m-AChRs by identifying messenger ribonucleic acid (mRNA) and protein expression. Secondly, to observe the influence of the most prominent m-AChRs in DPSCs, we attempted to generate a knock-out (KO) mutant in DPSCS using the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) genome-editing system. The results showed that m-AChRs are identified on a genetic level through gene expression of mRNA in DPSCs and expressed the following genes CHRM2, CHRM3, and CHRM5. They are functional and can identify the following proteins: M2, M3, and M5. Then, we attempted to generate a CHRM2 KO mutant DPSCs and could only produce a low percentage of the mutant population of DPSCs, as we only managed to generate 13% of CHRM2 KO mutant DPSCs. Although the protocol proved its effectiveness on different types of cells, but it did not work with DPSCs in this experiment. Therefore, the protocol steps can be revisited for further investigation and optimisation to generate a sufficient population of CHRM2 KO mutant DPSCs to allow the exploration of the role of m-AChRs in DPSCs so it can be used in clinical applications in regenerative medicine.