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

Abstract

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.

Keywords: Acetylcholine; Cholinergic receptors; Dental pulp stem cells; Muscarinic type 2 receptor; Neurotransmitters.

Fig. 1

Analysis of the dental pulp stem cell neurotransmitter profile reveals expression of muscarinic receptors. A Mean gene expression values for the 43/92 neurotransmitter genes determined to be expressed in DPSCs calculated using the e-ΔCT method. The data are derived from six biological replicates (n = 6). B Immunocytochemical analysis of m2AChR, m3AChR and m5AChR expression by DPSCs. (i) Expression of the m2AChR protein in DPSCs, (ii) Merged images showing DPSCs nuclei stained with DAPI (blue) and actin filaments with phalloidin (green), (iii) Merged images of (i) and (ii), and (iv) Negative control with no primary antibody showing no non-specific binding. (v) Expression of the m3AChR protein in DPSCs, (vi) Merged images showing DPSCs nuclei stained with DAPI (blue) and actin filaments with phalloidin (green), (vii) Merged images of (v) and (vi), and (viii) Negative control with no primary antibody showing no non-specific binding. (ix) Expression of the m5AChR protein in DPSCs, (x) Merged images showing DPSCs nuclei stained with DAPI (blue) and actin filaments with phalloidin (green), (xi) Merged images of (ix) and (x), and (xii) Negative control with no primary antibody showing no non-specific binding. All images show 2D projections of confocal stacks and are representative of three independent experiments (n = 3). Scale bars = 100 μm

Fig. 2

Pharmacological induction and inhibition of dental pulp stem cell proliferation confirm the expression of a functional m2AChR. The effect of the m2AChR agonists A McN-A 343 (McN, 0.1–100 µM) and B Arecaidine propargyl ester hydrobromide (APE, 0.1–100 µM) on DPSC proliferation determined using an MTT assay. The effects on DPSC proliferation after 2 h pretreatment with 100 µM Pirenzepine (PZ) and 0.1 µM Methoctramine (MC), alone or in combination, followed by 72 h treatment with C 100 µM McN and D 100 µM APE. Comparison made to untreated group (CTRL); negative controls are cells without serum (No FBS). E Time course of DPSC proliferation after stimulation with 100 µM APE. Ctrl denotes DPSCs cultured in media alone, 100 µM denotes DPSCs cultured in media containing 100 µM APE and recovery denotes DPSCs cultured in media containing 100 µM APE for 72 h and then replacing with culture media alone. F CCK-8 assessment of live cells over a 120 h period. m2AChR agonist depicts viable cells after stimulation with 100 µM APE. Serum starved cells (No FBS) acted as a negative control and cells in media alone containing serum (Ctrl) acted as a positive control. Data are presented as mean ± SEM from duplicate wells across three independent experiments. Statistical analysis was performed using one-way ANOVA; Dunnett’s multiple comparisons test was used for panels A–D, and Tukey’s post hoc test was used for panels E and F. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Full dose–response and recovery data for APE are shown in Supplementary Fig. 3B, and antagonist-alone controls are provided in Supplementary Fig. 3A

Fig. 3

Activation of the m2AChR arrests dental pulp stem cell proliferation at the G2/M phase. A Cell cycle analysis of DPSCs after 72 h of stimulation with the M2 agonist (100 µM APE) using bivariate analysis of BrdU incorporation (ordinate) and propidium iodide (PI) labelled DNA content (abscissa). (i) Example bivariate plot of DPSCs cultured in media alone (ctrl) and gating strategy. (ii) Example bivariate plot of DPSCs cultured in media containing 100 µM APE (M2 agonist) and gating strategy. (iii) Graphical representation of cell cycle analysis. B Analysis of expression of genes involved in proliferation and the cell cycle following stimulation with the M2 agonist (100 µM APE) for 72 h. (i) cyclin-A2 (CCNA2), (ii) cyclin-B1 (CCNB1), (iii) cyclin-D1 (CCND1), (iv) cyclin-D2 (CCND2), (v) cyclin-E2 (CCNE2), (vi) cell division cycle 14 A (CDC14A), (vii) M phase inducer phosphatase 1 (CDC25A), (viii) cyclin dependent kinase inhibitor 1 A (CDKN1A), (ix) glycogen synthase kinase 3 beta (GSK3B) and (x) proliferating cell nuclear antigen (PCNA). Cells cultured in media (undifferentiated) alone acted as a control (ctrl). M2 agonist = cells cultured in media containing 100 µM APE. Data are presented as mean ± SEM from duplicate wells across three independent experiments. Statistical analysis was performed using unpaired two-tailed t-tests for all comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Activation of the dental pulp stem cell m2AChR inhibits their migration and osteogenic differentiation. A Determination of the effect of m2AChR activation on DPSC migration using a scratch assay. (i) DPSCs grown on glass coverslips in control media, exposed to a scratch and stained for actin (green) after 8 h. (ii) DPSCs grown on glass coverslips in media containing 100 µM APE, exposed to a scratch and stained for actin (green) after 8 h. (iii) DPSCs grown on glass coverslips in media containing 0.01 µM MC (m2AChR antagonist) and 100 µM APE, exposed to a scratch and stained for actin (green) after 8 h. Images are representative of duplicate coverslips from three independent experiments. (iv) Quantification of DPSC migration measured by determining the difference in the width of the gap (as shown by yellow arrow) at 0 h and 8 h. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. Data are presented as mean ± SEM (n = 3). ns: not significant, ****p < 0.0001. Scale bars = 1200 μm. APE = Arecaidine propargyl ester, MC = Methoctramine. B Determination of the effect of m2AChR activation on DPSC osteogenic differentiation. DPSCs grown on glass coverslips for 4 weeks in normal media and stained with Alizarin red. (ii) DPSCs grown on glass coverslips for 4 weeks in osteogenic differentiation media and stained with Alizarin red. (iii) DPSCs grown on glass coverslips for 4 weeks in osteogenic differentiation media containing 100 µM APE and stained with Alizarin red. (iv) DPSCs grown on glass coverslips for 4 weeks in normal media and stained with von Kossa. (v) DPSCs grown on glass coverslips for 4 weeks in osteogenic differentiation media and stained with von Kossa. (vi) DPSCs grown on glass coverslips for 4 weeks in osteogenic differentiation media containing 100 µM APE and stained with von Kossa. Images are representative of duplicate coverslips from three independent experiments. (vii) Alizarin Red stain quantification showing significantly less mineralisation compared to cells that had undergone osteogenic differentiation. Statistical analysis was performed using one-way ANOVA with Kruskal–Wallis post hoc test. Data are presented as mean ± SEM (n = 3). *p < 0.05, ****p < 0.0001

Fig. 5

Overview of differential gene expression by dental pulp stem cells after activation of the m2AChR. Volcano plots for differentially expressed genes (DEGs) of DPSCs after activation of the m2AChR for 4 h (A) and 24 h (B) compared to untreated controls. Scatter points represent genes. The x-axis represents the log2 fold change of m2AChR activated vs. untreated cells. The y-axis represents the -log 10 of the padj-value, in which 1.3 is equal to a padj of < 0.05. Heatmaps of the top 50 significantly (DEGs) of DPSCs after activation of the m2AChR for 4 h (C) and 24 h (D) compared to untreated controls. The genes are clustered based on the normalised log2 fold change in gene expression, where the red and green colour scale at the right of the heatmap represents higher and lower relative expression levels, respectively. Each row represents one gene, and each column represents a single sample of the experimental groups. The gene symbols are shown on the right side of the rows. The data is derived from four independent experiments. Statistical analysis was performed using DESeq2 with adjusted p-values calculated by the Benjamini–Hochberg method; genes with padj ≤ 0.05 were considered significantly differentially expressed

Fig. 6

Overview of the biological pathway and protein-protein interaction analysis to determine pathways involved in the m2AChR signalling in dental pulp stem cells. KEGG enrichment analysis of significantly enriched KEGG pathways. (A) KEGG enrichment analysis highlighting the number of significantly upregulated and downregulated KEGG pathways after activation of the DPSC m2AChR for 4 and 24 h. (B) Protein-protein interaction analysis representing the significant protein coding DEGs after activation of the DPSC m2AChR for 4 and 24 h. (C) Interactions between highlighted genes involved in cell cycle (red band), cell migration (purple band), and genes involved in the MAPK cascades (green band)

Fig. 7

m2AChR signalling in dental pulp stem cells is mediated by the MAPK/ERK signalling pathway. A Analysis of expression of genes involved in the MAPK/ERK signalling pathway following stimulation of DPSCs with the M2 agonist (100 µM APE) for 72 h. (i) ERK1 (MAPK3), (ii) ERK2 (MAPK1) and (iii) Proliferating cell nuclear antigen (PCNA), Cells cultured in media alone acted as a control (ctrl). M2 agonist = cells cultured in media containing 100 µM APE. Statistical analysis was performed using unpaired two-tailed t-tests. B Time course of phosphorylation of ERK 1/2 after activation of the dental pulp stem cell m2AChR determined using an in-cell ELISA. Cells cultured in media alone acted as a control (ctrl). M2 agonist = cells cultured in media containing 100 µM APE. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean ± SEM from duplicate wells across three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001