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Decoding SFRP2 progenitors in sustaining tooth growth at single-cell resolution

Stem Cell Research & Therapy volume 16, Article number: 58 (2025) Cite this article

Abstract

Background

Single-cell transcriptomics has revolutionized tooth biology by uncovering previously unexplored areas. The mouse is a widely used model for studying human tissues and diseases, including dental pulp tissues. While human and mouse molars share many similarities, mouse incisors differ significantly from human teeth due to their continuous growth throughout their lifespan. The application of findings from mouse teeth to human disease remains insufficiently explored.

Methods

Leveraging multiple single-cell datasets, we constructed a comprehensive dental pulp cell landscape to delineate tissue similarities and species-specific differences between humans and mice.

Results

We identified a distinct cell population, Sfrp2hi fibroblast progenitors, found exclusively in mouse incisors and the developing tooth root of human molars. These cells play a crucial role in sustaining continuous tissue growth. Mechanistically, we found that the transcription factor Twist1, regulated via MAPK phosphorylation, binds to the Sfrp2 promoter and modulates Wnt signaling activation to maintain stem cell identity.

Conclusions

Our study reveals a previously unrecognized subset of dental mesenchymal stem cells critical for tooth growth. This distinct subset, evolutionarily conserved between humans and mice, provides valuable insights into translational approaches for dental tissue regeneration and repair.

Graphical abstract

Introduction

Recent advances in single-cell omics technologies, combined with extensive efforts to integrate these datasets, have resulted in the development of comprehensive cell atlases for a wide range of tissues and organs under both healthy and diseased conditions[1,2,3,4,5], These atlases have significantly advanced our understanding of tissue biology, development, and disease processes[6]. Despite these achievements, the translation of findings from animal models to human applications continues to pose a major challenge in regenerative medicine and biology.

Tooth biology has been profoundly impacted by the advent of single-cell transcriptomics, which enables researchers to explore intricate cellular landscapes previously inaccessible [7]. The mouse model is frequently employed due to its genetic tractability and significant anatomical and physiological similarities with human tissues. Notably, mouse molars resemble human molars, providing a valuable comparative platform. However, key distinctions exist, particularly the continuous growth of mouse incisor, a feature absent in human teeth [8]. This raises critical questions regarding the relevance of findings from mouse models to human dental biology.

Addressing this gap is essential for enhancing the applicability of preclinical research to human therapeutic contexts. Previous studies have established single-cell atlases for mouse and human teeth but have largely focus on the dental epithelial component[9], leaving mesenchymal cells dynamics underexplored. Mesenchymal stem cells within dental pulp (DPSCs) play a crucial role in tissue regeneration, angiogenesis, and immune modulation [10,11,12]. The heterogeneity and functional diversity of these DPSCs offer significant potential for advancing regenerative dentistry [8, 13,14,15].

The Sfrp2 gene encodes Secreted Frizzled-Related Protein 2 (SFRP2), a key modulator of the Wnt signaling pathway [16], which is crucial for tissue development and the maintenance of stem cell identities [17]. SFRP2 is particularly involved in enhancing stem cell self-renewal and regeneration by increasing stem cell survival, inhibiting cell lineage commitment and decreasing stem cell apoptosis [18, 19]. Sfrp2 is found as a key paracrine factor that mediates myocardial survival and repair after ischemic injury, suggesting that protein-based therapies might have advantages over cell-based cardiac therapies for acute myocardial infarction [20].

In this study, we leveraged comprehensive single-cell datasets to map the dental pulp cells in humans and mice, with a particular focus on dental mesenchymal cells. Our goal was to investigate previously unexplored area to uncover species-specific similarities and differences and to identify evolutionally conserved molecular mechanisms underlying dental pulp regeneration. Notably, we discovered distinct Sfrp2-expressing fibroblast progenitors that are exclusively present in the developing human tooth root and continuously growing mouse incisor. This discovery underscores the translation potential of targeting this cell for therapeutic application. Our findings offer new insights into the conserved and divergent aspects of dental biology, providing a foundation for translational research and therapeutic innovations.

Materials and methods

Human sample collection

Human sample collection was approved by the Ethics Review Committee for Human Life Sciences and Medical Research at Jilin University Stomatological Hospital (Approval No. JDKQ20230069), and conducted in accordance with the guidelines of the Declaration of Helsinki. Informed consent was obtained from all participating patients. Tooth samples were collected from the Third Department of Oral and Maxillofacial Surgery at Jilin University Stomatological Hospital between 2023 and 2024. These teeth were extracted as part of orthodontic or other clinical treatments. Both fully developed healthy teeth and undeveloped teeth were obtained for immunofluorescence staining and cell culture experiments.

Animal work

All animal procedures were conducted in accordance with the guidelines of the Laboratory Animal Ethics Committee at the School of Basic Medical Sciences, Jilin University, China (Approval No. 2023492). C57BL/6 mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. and housed in a specific pathogen-free (SPF) environment. The mice had unrestricted access to food and water and were maintained at a stable temperature of 22 °C with a relative humidity of 55 ± 10%, under a consistent 12-h light–dark cycle. A total of 52 wild-type C57BL/6 mice were used in this study.

Dental pulp tissue was isolated from the mandibular incisors of 7–8-week-old female wild-type C57BL/6 mice for single-cell RNA sequencing experiments. To ensure an adequate number of qualified cells for single-cell RNA sequencing, five mice per group were used, as determined by preliminary testing. For validation experiments, samples were pooled from at least three mice per group. Mice were euthanized using a carbon dioxide (CO₂) system in accordance with ethical guidelines. CO₂ was gradually introduced at a flow rate of 10%-30% of the chamber volume per minute to minimize distress. The process lasted for about 5 min, depending on the body weight of the mice, to ensure complete unconsciousness and cessation of respiration. Death was confirmed by cervical dislocation, in compliance with ethical guidelines. No animal anesthesia was used in this study. The study has been reported in accordance with the ARRIVE guidelines 2.0.

Single-cell suspension preparation

Female wild-type C57BL/6 mice, aged 7–8 weeks, were sacrificed and the mandible tissue were carefully dissected under a stereo microscope. Surrounding soft tissues were meticulously removed using surgical scissors and fine forceps. The dental pulp from the mouse incisors was isolated and washed three times with ice-cold sterile Phosphate Buffered Saline (PBS). The dental pulp tissues were fully extracted and then cut into approximately 3mm segments using ophthalmic scissors. These segments were immediately placed in α-Minimum Essential Medium (α-MEM) containing collagenase and digested for 40 min at 37°C to obtain single cell suspension. Red blood cells were removed using a red blood cell lysis buffer. Finally, the cells were resuspended in α-MEM medium for further analysis.

Single-cell RNA sequencing

The dental pulp single-cell suspension was prepared according to the manufacturer’s instruction for the 10X Chromium platform. Approximately 10,000 cells were captured for barcoding as single-cell gel beads-in-emulsion (GEMs) before being loaded onto the Illumina NovaSeq 6000 system for sequencing. Single-cell cDNA libraries were constructed using the Chromium Single-Cell 5’ Reagent Kits v2, following the manufacturer’s protocols.

The raw sequencing data from the mouse dental pulp were first subjected to quality control, aligned to the mouse reference genome, and processed to generate the cell-gene expression matrix using Cell Ranger version 7.0.0 (10 × Genomics). Subsequent data analysis was performed using the Seurat R Package.

Quality control and analysis

All dental pulp single-cell datasets (Table 1) were imported into Seurat (v5.0.0) for analysis. To ensure unbiased data interpretation, identical quality control criteria were applied to all datasets. Detailed quality control metrics are provided in Table 2. Low-quality sequencing data and potential doublets were excluded based on cell counts, the number of genes detected, and the percentage of mitochondrial gene expression.

Table 1 Sources of human and mouse dental pulp scRNA-seq datasets
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Table 2 Quality Control Parameters for All scRNA-seq Datasets Used
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Data integration and visualization

After quality control, the human and mouse dental pulp single-cell datasets were categorized into four groups: human molar dental pulp, human apical papilla, mouse incisor dental pulp, and mouse molar dental pulp. The human and mouse datasets were integrated separately, with each matrix normalized and variable features identified using the NormalizeData and FindVariableFeatures functions, with dimensions set to 1:30. Principal component analysis (PCA) was performed with the number of principal components set to 50. Batch effects between sequencing data and publicly available databases were mitigated using the RunHarmony function [21]. Subsequently, uniform manifold approximation and projection (UMAP) was applied to map the single-cell data onto a two-dimensional coordinate system, generating UMAP plots. The FindClusters function was used to identify cell clusters with a resolution of 0.6. Differentially expressed gene (DEG) analysis was carried out using the FindAllMarkers function in Seurat (v5.0.0). Furthermore, cell type subgroups within human and mouse dental pulp tissues were identified using SingleR in conjunction with relevant literature [22].

Cell interaction analysis

CellChat was used to investigate potential ligand-receptor interactions in human and mouse molars. The Seurat object was preprocessed using CellChat functions, and potential intercellular communication networks were visualized using the netVisual_circle and netAnalysis_signalingRole_heatmap functions.

Trajectory analysis

Pseudotime trajectory analysis of dental pulp stem cells and dental pulp fibroblasts was performed using Monocle2 and Monocle3 [23,24,25]. To generate cell trajectories, the Seurat object was preprocessed with Monocle2 functions, with a gene mean expression cutoff of ≥ 0.1 for temporal ordering. Dimensionality reduction was performed using DDRTree, and the trajectories were visualized with the plot_cell_trajectory function. Additionally, Monocle3 was employed to infer pseudotime differentiation trajectories, visualized using the plot_cells function.

CytoTRACE analysis

CytoTRACE2 package was used to estimate the transcriptional diversity of each dental pulp cell in terms of differentiation or stemness status. Each cell was assigned a CytoTRACE score based on its differentiation potential, with higher score indicating greater stemness and less differentiated characteristics[26].

Multigene co-expression analysis

Multigene co-expression analysis was conducted using Seurat’s FeaturePlot function with the parameter `blend = TRUE`. Two selected gene pairs of interest were visualized, with overlapping color regions representing areas of co-expression between the two genes.

Immunofluorescence and imaging

Human molars, apical papilla tissues, and mouse mandibles were collected and fixed overnight in 4% PFA. Decalcification was carried out using 10% EDTA solution for 3–6 weeks. The samples were dehydrated through graded ethanol and xylene treatments and then embedded in paraffin. Tissue samples were sectioned into 4 μm slices using a Lecia microtome. The paraffin sections were deparaffinized, followed by antigen retrieval, blocking, permeabilization, and overnight incubation with primary antibodies at 4°C. Fluorescently labeled secondary antibodies (FITC) were applied for 1 h, followed by counterstaining with DAPI and mounting. Images were captured using a Nikon confocal microscope (AX R, Nikon).

Quantitative real-time PCR

RNA was extracted from human dental pulp organoids using RNA-easy Isolation Reagent (Vazyme, China) following the manufacturer’s protocol. Briefly, the organoids were lysed at 4°C, and the lysate was centrifuged to collect the supernatant. RNA was precipitated with isopropanol, washed with 75% ethanol, resuspended in RNase-free water, and stored at − 80°C.

Primers were designed using NCBI Primer-BLAST. cDNA synthesis was performed with Hifair III 1st Strand cDNA Synthesis SuperMix (Yeasen, China). Quantitative PCR was conducted on a Light Cycler 480 (Roche, Switzerland) using Hieff qPCR SYBR Green Master Mix (Yeasen, China). Each reaction mix contained 10 μL of SYBR Green Master Mix, 0.4 μL of each primer (10 μM), 2.5 μL of cDNA, and 6.7 μL of ddH2O. The PCR program included an initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Melting curves analysis was performed to confirm amplification specificity, and all samples were tested in triplicate. Relative gene expression levels were calculated using the 2 − ΔΔCt method with β-actin as the reference gene. Statistical analysis was carried out using Student’s t-test, with P < 0.05 considered significant. Primer sequences are listed below:

TWIST1 Forward: AATTCAAAGAAACAGGGCGTG.

TWIST1 Reverse: AATGCAGAGGTGTGAGGATG.

SFRP2 Forward: TCCTTAGCAACTTTTCCCCTC.

SFRP2 Reverse: TGGTAAGGCTCTGGTGATTTG.

Dental pulp organoid culture

Freshly extracted undeveloped human teeth were collected from healthy individuals aged 12 to 20 years. The dental apical papilla tissues were carefully dissected from the root and thoroughly rinsed with PBS. The tissue was then minced into approximately 1 mm3 fragments using sterile scissors and subjected to enzymatic digestion in α-MEM basal medium containing 3 mg/ml type I collagenase (Sigma) at 37°C for 25 min to obtain a single-cell suspension. The resulting single cells were resuspended in α-MEM basal medium supplemented with 20% fetal bovine serum (FBS), 1% Glutamax (ThermoFisher), and 1% penicillin/streptomycin solution. The cells were seeded into 60 mm culture dishes and incubated at 37°C in a humidified atmosphere with 5% CO2, with medium changes every two days. After one week, the cells reached approximately 90% confluence and were passaged into 6-well plates (P1). Upon reaching 75%-80% confluence, the cells were further passaged at a 1:2 ratio to obtain passage 2 (P2) cells. P2 cells were seeded into 96-well ultra-low attachment plates (Corning, 7007) at a density of 5,000 cells per well, where they were cultured until forming spheroids.

In Vitro application of MAPK inhibitor

A 1 mM stock solution of the MAPK pathway inhibitor SB 203580 (MCE) was prepared in DMSO (absin) and sonicated to ensure complete solubility. Once the dental pulp organoids had stably formed and reached an approximately diameter of 200 µm, the MAPK inhibitor was added to the experimental group at a final concentration of 30 µM in each well of the 96-well ultra-low attachment plates. To serve as a vehicle control, an equivalent volume of DMSO was added to the control group organoids. The organoids were then cultured for an additional 24 h before harvested for subsequent qPCR analysis.

Results

Single-cell landscapes reveal conserved cellular profiles in human and mouse molars

Human and mouse molars share several similarities in morphogenesis and physiology, making mice a valuable model for studying human dental biology. To scrutinize cellular concordance and diversity, we established a comprehensive single-cell landscape by integrating twelve publicly available scRNA-seq datasets, comprising ten from human molars and two from mouse molars (Fig. 1A; Table 1). In total, 45,383 cells were analyzed. After quality control, batch effect removal, and dimensionality reduction, we annotated 41,039 of pulp cells into five major cell types based on canonical gene expression markers: epithelial (KRT14, KRT5), fibroblasts (PDGFRB, COL6A1, COL6A2), immune cells (PTPRC), endothelial (CD34, PECAM1, PLVAP) and neuro glia (PLP1, SOX10) (Fig. 1B, 1C and supplementary Fig. 1A). These cells were further divided into nine clusters using SingleR package and top marker gene expression (Fig. 1D and 1E and supplementary Fig. 1B). Both cell types and clusters were similarly distributed across human and mouse samples. Cell proportion analysis revealed a comparable composition of all cell clusters, with some notable differences: pulp cells were more abundant in human molars, whereas macrophages were enriched in mouse molars (Fig. 1F). This discrepancy likely reflects inherent species-specific. Despite this, a strong alignment of shared gene expression between humans and mice across was observed across all cell clusters, as illustrated in the dot plot (Fig. 1G). To assess overall cell cluster correlation between species, we conducted Pearson’s correlation coefficient analysis. The results showed a positive correlation for most of cell clusters, indicating a general comparability between human and mouse (Fig. 1H). Using CellChat analysis, we compared intercellular communication strength in terms of interaction number and weight. T-cells, Macrophages, endothelial cells, pulp cells and neuro glial cells exhibited strong reciprocal correlations between humans and mice. In contrast, B cells epithelial cells and dental follicle cells displayed weaker correlation, indicating substantial divergence in gene expression despite general similarities in cell type composition (Fig. 1I).

Fig. 1
figure 1

Single-Cell Atlas of Human and Mouse Molar Pulp Cells. A Overview of data collection and quality control processes. B, C UMAP visualization of scRNA-seq data, highlighting the five major cell types common to both human and mouse molars. D, E Further subdivision of all cells into nine distinct clusters based on their top differentially expressed genes (DEGs) in both species. F Cell proportion analysis illustrating the percentage of different cell clusters within the entire pulp tissue. G Dot plot displaying the genes commonly shared in each cluster between humans and mice. H Pearson correlation analysis demonstrating a strong correlation between corresponding cell clusters across the two species. I CellChat analysis revealing similar cell–cell interaction patterns in humans and mice

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Overall, our analysis indicated a high degree of conservation in cell types, clusters, marker gene expression and cell–cell interactions between human and mouse molar. These findings suggest that studies on mouse molar provide a valid translation perspective for human teeth.

A distinct Sfrp2hi fibroblast population exists exclusively in continuously growing mouse incisors

Unlike human teeth and mouse molar, which do not exhibit continuous growth, mouse incisors demonstrate sustained growth, suggesting the presence of unique regulatory mechanisms for tissue homeostasis and regeneration. To investigate this phenotype difference, we analyzed two single-cell datasets from mouse incisor and two from molar dental pulp (Fig. 2A). After rigorous quality control and batch effect correction, cells were classified into five main cell types based on canonical marker genes: (Krt5, Krt14, Krt17), fibroblasts (Col1a1, Col1a2, Tnc), immune cells (Ptprc), endothelial (Pecam1, Plvap) and neuro glia (Plp1, Mbp) (Fig. 2B, 2C and supplementary Fig. 2A). These classifications were consistent across mouse incisors and molars and aligned with human dental tissue, as showed by UMAP analysis of commonly expressed marker genes. Using the SingleR package, we further subdivided the cells into eleven distinct clusters, revealing a unique fibroblast cluster (FIB_1) present only in the mouse incisor (Fig. 2D and 2E). Proportion analysis confirmed the absence of this FIB_1 cluster in molar dental pulp, as highlighted with a thick dashed line in red color in Fig. 2F. Examination of gene expression across clusters identified Sfrp2 as the dominant gene in the FIB_1 cluster, leading us to classify these cells as Sfrp2hi fibroblasts (Fig. 2G). UMAP analysis further highlighted the exclusive expression of Sfrp2 in the FIB_1 cluster within the mouse incisor, contrasting with its absence in molar pulp cells (Fig. 2H).

Fig. 2
figure 2

Distinct Sfrp2-Expressing Fibroblasts Identified in Mouse Incisor Whereas Was Absent in Mouse Molar. A Overview of data collection and quality control. B, C UMAP visualization displaying the five major cell types in both mouse incisor and molar. D, E SingleR analysis identified eleven distinct clusters based on their top differentially expressed genes (DEGs) in both groups, with the FIB_1 cluster absent in the mouse molar sample. F Proportion analysis showing the percentage of each cell cluster. G Dot plot highlighting the top marker gene in each cluster, with Sfrp2 as the top marker gene in the FIB_1 cluster. (H) UMAP visualization showing the expression of Sfrp2 in the FIB_1 cluster, which is absent in mouse molar clusters. (I) Immunofluorescence staining demonstrating specific expression of the SFRP2 gene near the cervical loop of the mouse incisor, with no expression observed in mouse molar tissues

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Immunofluorescence staining on tissue sections from mouse incisors and molars confirmed the primary localization of SFRP2-expressing cells in the labial and lingual cervical loops of the mouse incisors, with no corresponding presence in molars (Fig. 2I). Notably, these regions have been previously identified stem cell niches in the mouse incisors [27, 28]. Our findings reveal significant cellular heterogeneity between the dental pulp of mouse incisors and molars, characterized by the presence of a distinct population of SFRP2-expressing fibroblasts in the stem cell niche of mouse incisors. Given that SFRP2 function acts as a Wnt signaling pathway inhibitor, it plays a significant role in maintaining stem cell stemness[20]. The high expression of SFRP2 in the cervical loop region suggests its potential role in maintaining dental pulp stem cell stemness, thereby supporting the continuous growth of mouse incisors, a feature absent in mouse molars.

Developing teeth in humans contain sfrp2+ fibroblasts unlike mature teeth

In humans, mature teeth do not continue to grow. However, developing teeth contain a unique population of dental stem cells known as stem cells from the root apical papilla (SCAP). These cells are located at the apical end of growing tooth roots to sustain root development prior to tooth eruption into the oral cavity [29]. To investigate the presence and characteristics of this specific subset of pulp cells in humans and their relevance to Sfrp2 expression, similar to those in mouse incisors, we conducted a comparative analysis of ten single cell datasets from adult molar and two from developing teeth (Fig. 3A). A total of 53,048 high-quality cells were included in the UMAP analysis. All cells were classified into five major cell types, consistent with previous classifications (Fig. 1B, 1C, Fig. 2B, 2C and Fig. 3B, 3C). We further subdivided these cells into eleven cell clusters across human molar and apical papilla tissues (Fig. 3D and 3E) and identified Sfrp2 expression specifically in one subset of pulp fibroblasts (FIB_1) (Fig. 3F). Notably, Sfrp2-expressing cell were absent in mature human molar dental pulp (Fig. 3F), paralleling the lack of these cells in non-growing mouse molars. Immunofluorescence staining on tissue sections of human molar and apical papilla further validated these findings (Fig. 3G), showing high expression levels of SFRP2 in the apical papilla of developing tooth root, whereas no SFRP2 expression was detected in mature molar pulp. To assess the differential potential of SFRP2-expressing cells, we performed the Cytotrace analysis, a computational framework developed by Stanford University that evaluates cellular differentiation potential in single-cell RNA-seq data using a gene count signature (GCS), without requiring prior knowledge. Our results revealed that Sfrp2-expressing pulp fibroblasts exhibited higher developmental potential compared to other dental pulp cells in human apical papilla (Fig. 3H).

Fig. 3
figure 3

SFRP2hi Fibroblasts Present Exclusively in Human Developing Teeth, Not in Mature Teeth. A Overview of data collection and quality control processes. B, C UMAP visualization of the five major cell types identified in both human developing and mature tooth samples. D, E Cells were grouped into twelve distinct clusters based on their top differentially expressed genes (DEGs), with the FIB_1 cluster being uniquely present in the human apical papilla, representative of developing teeth. F UMAP highlighting SFRP2 expression, showing its presence in the human apical papilla and absence in the mature molar sample. G Immunofluorescence confirming SFRP2 gene expression in the dental pulp and apical papilla of human molars, which is absent in mature molar tissues. H Cytotrace2 analysis revealing the differentiation potential across various cell types in human molar dental pulp and apical papilla, with the SFRP2-expressing FIB_1 cells showing the highest differentiation potential

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These findings suggest that a subset of dental pulp mesenchymal cells with high SFRP2 expression represents a unique cell population exclusively present in developing human tooth root, similar to those in continuously growing mouse incisor. This indicates a conserved cell population that sustains tissue growth in both human and mouse teeth [30].

Sfrp2hi fibroblasts represent an evolutionarily conserved stem cell population in humans and mice

To further investigate the cell identity of Sfrp2+ fibroblasts in both humans and mice, we subdivided the pulp fibroblasts in human apical papilla into four clusters based on SFRP2 expression levels: SFRP2hi, SFRP2low and SFRP2-negative fibroblast1 and fibroblast2 (Fig. 4A and supplementary Fig. 3A). Similarly, pulp fibroblasts in mouse incisor were categorized into six clusters, including one cluster with high Sfrp2 expression and five clusters with lower or no expression of Sfrp2 (Fig. 4B and supplementary Fig. 3B). Cell trajectory analysis of human apical papilla and mouse incisor fibroblasts revealed that subsets with high SFRP2 expression occupy the earliest stages of dental pulp cell development (Fig. 4C and 4D). These SFRP2hi fibroblasts exhibit potential to differentiate into various cell types within the dental pulp tissue. GSEA analysis showed that the SFRP2hi fibroblast subsets in both human and mouse dental pulp are closely associated with the regulation of cell differentiation processes (Fig. 4E). Pseudotime analysis demonstrated that a gradual decreased in SFRP2/Sfrp2 expression levels along the differentiation trajectory (Fig. 4F), indicating the differentiation ability of this cell population. Additionally, Gene enrichment analysis of human and mouse Sfrp2hi fibroblasts revealed shared stem cell characteristics and functions including terms such as “system development”, “multicellular organism development”, “extracellular matrix” and “protein binding” (Fig. 4G and 4H). These findings underscore the stem cell properties of this conserved fibroblast population.

Fig. 4
figure 4

SFRP2hi Fibroblasts Represent a Conserved Stem Cell Population Across Humans and Mice. A, B Re-clustering of dental pulp fibroblasts identified four subsets in human apical papilla and six subsets in mouse incisors. C, D Cell trajectory analysis demonstrated that SFRP2hi cells are positioned at the initiation point of differentiation in both human and mouse samples, suggesting their stem cell potential. E, F Gene Set Enrichment Analysis (GSEA) indicated that SFRP2 expression positively regulates cell differentiation, with SFRP2/Sfrp2 expression decreasing along the differentiation pathway. G, H Gene enrichment analysis revealed similar functions for the SFRP2/Sfrp2 cell clusters in both humans and mice

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Overall, our findings suggest that SFRP2hi fibroblasts represent an evolutionarily conserved stem cell population essential for dental growth and development in both humans and mice.

Twist1 regulates Sfrp2 expression via MAPK pathway to maintain stem cell identity

Sfrp2 functions as an inhibitor of the Wnt signaling pathway [31, 32], playing a crucial role in maintaining stem cell stemness. To elucidate the mechanism underlying the high levels of Sfrp2 in this unique group of dental pulp mesenchymal stem cells, we used the ChIP-Atlas database to predict transcription factors binding to the Sfrp2 promoter [33]. Our analysis revealed significant Twist1 binding peaks at the Sfrp2 gene promoter, indicating that Twist1 is a potential candidate for enhancing Sfrp2 transcription (Fig. 5A). Twist1 is a key transcription factor involved in fundamental biological processes, including embryonic development [34], stem cell maintenance[35, 36] and cellular responses to environmental signals [37]. Multigene co-expression analysis of single-cell data from human molar apical papilla and mouse incisor dental pulp revealed significant co-expression of Twist1 and Sfrp2 in DPSCs (Fig. 5B).

Fig. 5
figure 5

Twist1 Regulates Sfrp2 Expression Through the MAPK Pathway. A ChIP-Atlas enrichment analysis shows that the TWIST1 transcription factor binds to the promoter region of the SFRP2 gene in both human and mouse models. B Co-expression of TWIST1 and SFRP2 is observed in single-cell data from human and mouse dental pulp. C A dual luciferase assay confirms TWIST1 binding to the SFRP2 gene promoter. D PhosphoSite predictions identify phosphorylation sites on the TWIST1 transcription factor protein. E Specifically, PhosphoSite predictions indicate that phosphorylation of the S68 site on TWIST1 is linked to proteins associated with P38A. F Kinase gene enrichment analysis for TWIST1’s S68 phosphorylation site reveals a strong connection to the MAPK pathway. G Immunofluorescence staining of SFRP2 and P-P38 in human apical papilla and mouse incisor tissue sections shows co-localized positive expression in both tissues. H Treatment with a MAPK inhibitor in dental pulp organoids culture system for 48 h results in decreased levels of TWIST1 and SFRP2, as shown by qPCR analysis

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Further analysis using the JASPAR database predicted specific Twist1 binding sequences in the Sfrp2 gene promoter region. This prediction was validated through dual-luciferase assays, confirming that Twist1 binds directly to the Sfrp2 promoter site [38], and enhances its transcription, thereby maintaining high Sfrp2 levels in DPSCs (Fig. 5C). For Twist1 to function as a transcription factor, it must undergo phosphorylation to enter the nucleus and bind DNA[39]. Studies have shown that phosphorylation of Twist1 by Akt and MAPKs at specific serine residues is essential for its stability, nuclear localization, and DNA binding activity [40]. Using PhosphoSitePlus, we identified ser68 as the primary phosphorylation site of Twist1 (Fig. 5D). Kinase prediction analysis suggested that P38 is the likely kinase responsible for this modification (Fig. 5E). GO analysis of Sfrp2+ cells revealed enrichment of "phosphorylation" in the biological process category, while "MAPK pathway" and "TP53 regulation" were prominent in the pathway analysis. These findings suggest that MAPK and TP53 pathway influence Twist1 activity and subsequently regulates Sfrp2 expression. This is aligns with previous studies demonstrating that p38, a critical component of the MAPK pathway, regulates TP53 activity [41], emphasizing a coordinated regulatory mechanism. Immunofluorescence staining confirmed the specific co-expression of SFRP2 and phosphorylated p38 in both human apical papilla and mouse incisor tissue sections (Fig. 5G). In an ex vivo dental pulp organoid model, inhibition of MAPK pathway led to a significant reduction in both Twist1 and Sfrp2 expression (Fig. 5H), underscoring the importance of MAPK signaling in regulating the fate of Sfrp2+ DPSCs.

Our findings demonstrated that Twist1 binds to the Sfrp2 promoter region and regulates Sfrp2 expression via the MAPK pathway to support stem cell identity. This regulatory mechanism highlights the critical role of MAPK-mediated Twist1 phosphorylation in maintaining stem cell function and underscores it importance in dental tissue homeostasis and regeneration.

Discussion

Our integrative analysis of single-cell datasets from human and mouse dental pulp highlights both conserved and species-specific cellular characteristics. Notably, we identified, for the first time, a distinct subset of Sfrp2-expressing fibroblasts uniquely present in mouse incisor and developing human tooth root. This cell population plays a pivotal role in sustaining tissue growth and regeneration, underscoring its evolutionary significance.

Recent studies support our findings, showing that SFRP2+ DPSCs enhances dentin regeneration in a rabbit model [42]. Similarly, overexpression of SFRP2 in stem cells from the apical papilla (SCAPs) has been demonstrated to enhance periodontal tissue regeneration in a miniature pig model [43]. These findings align with our single cell data from humans and mice, emphasizing the role of SFRP2hi progenitors as an evolutionarily conserved cell population essential for mammalian dental tissue development and homeostasis.

The Wnt pathway has been identified as playing a dual role in stem cell regulation. While activation of Wnt signaling promotes stem cell self-renewal and proliferation, its inhibition is necessary to maintain stem cell quiescence under homeostatic conditions [44, 45]. Given that Sfrp2, a Wnt pathway inhibitor, is highly expressed in dental pulp progenitors, our findings suggest that SFRP2hi fibroblasts mediate a delicate balance between quiescence and activation. This balance allows effective tissue regeneration while preserving the stem cell pool. These insights provide a nuanced understanding of Wnt signaling in stem cell regulation and highlights its context-dependent functionality.

Mechanistically, we demonstrated that Twist1 regulates Sfrp2 expression via MAPK signaling, thereby maintaining stem cell identities. Pathway Enrichment analysis identified additional roles for TP53 activity regulation, suggesting that the interplay between MAPK and TP53 dynamics is crucial for cellular responses, consistent with prior studies [46]. This robust regulatory framework appears to preserve genomic integrity in SFRP2hi Fibroblasts [47].

While most cell clusters showed a positive correlation between human and mouse molar datasets in our investigation, indicating general comparability, certain cluster, such as B cells, epithelial cells, and dental follicle cell exhibited poor correlation. These discrepancies highlight the limitations of directly extrapolating findings from mouse models to human tooth biology. Such species-specific differences underscore the importance of validating key findings in human systems.

Conclusions

This study provides a comprehensive single-cell analysis of dental pulp stem cell populations in humans and mice, revealing significant species-specific differences and conserved features. The identification of Sfrp2hi fibroblast progenitors, exclusive to mouse incisors and developing human tooth roots, underscores their pivotal role in sustaining tissue growth. Mechanistic insights into how Twist1, modulated through MAPK phosphorylation, actives Wnt signaling through the Sfrp2 promoter deepen our understanding of dental pulp regeneration at the molecular level. These discoveries hold substantial implications for regenerative dentistry and translational research.

Although the specific markers we identified and validated from single cell transcriptomic analysis are located in typical regions of DPSC niches [8, 13,14,15, 48], further studies using genetic lineage tracing and gene deletion techniques are need to confirm the stem cell identity and functional role of Sfrp2-expressing fibroblasts. Such efforts will refine our understanding of their regenerative potential and enhance their therapeutic application.

Data availability

Our single cell data from mouse samples have been uploaded to the Gene Expression Omnibus (GEO) database under accession number GSE (275119). Additionally, publicly available datasets used in this study were obtained from the GEO database, including samples from human molars, human apical papillae, mouse incisors, and mouse molars [7, 49, 50]. Detailed information about these datasets is provided in Table 1.

Abbreviations

DPSC:

Dental pulp stem cell

PCA:

Principal component analysis

UMAP:

Uniform manifold approximation and projection

DEG:

Differentially expressed gene

PFA:

Paraformaldehyde

EDTA:

Ethylenediaminetetraacetic acid

DMSO:

Dimethyl sulfoxide

SCAP:

Stem cells from the apical papilla

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Acknowledgements

We acknowledge all members of the Zhengwen An Laboratory for their support and insightful discussions. We also thank Prof. Peng Chen from the Department of Genetics, College of Basic Medical Sciences, Jilin University, for his expert consultation on the initial setup of our single-cell data analysis, and Prof. Qilin Liu from the Third Department of Oral and Maxillofacial Surgery at Jilin University Stomatological Hospital for providing the freshly extracted tooth samples. The authors declare that they have not use AI-generated work in this manuscript.

Funding

This work was supported by: National Key Research and Development Program of China (2022YFC2504200), National Natural Science Foundation of China (82270960), Science & Technology Development Talent Project of Jilin Financial Department (JCSZ2021893-35), The Startup Fund from Jilin University and School & Hospital of Stomatology, Jilin University to AZ.

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Authors and Affiliations

  1. Department of Oral Biology, School and Hospital of Stomatology, Jilin University, Changchun, China

    Tianyuan Zhao, Qing Zhong, Zewen Sun, Xiaoyi Yu, Tianmeng Sun & Zhengwen An

  2. Key Laboratory of Tooth Development and Bone Remodeling of Jilin Province, School and Hospital of Stomatology, Jilin University, Changchun, China

    Tianyuan Zhao, Qing Zhong, Zewen Sun, Xiaoyi Yu, Tianmeng Sun & Zhengwen An

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  1. Tianyuan Zhao
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  2. Qing Zhong
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Contributions

Tianyuan Zhao: Data collection, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft; Qing Zhong: Investigation, Methodology, Validation; Zewen Sun: Investigation, Formal analysis; Xiaoyi Yu: Investigation, Formal analysis; Tianmeng Sun: Investigation, Methodology; Zhengwen An: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing. All authors gave their final approval and agreed to be accountable for all aspects of work.

Corresponding author

Correspondence to Zhengwen An.

Ethics declarations

Ethical approval and consent to participate

Human sample collection was approved by the Ethics Review Committee for Human Life Sciences and Medical Research at Jilin University Stomatological Hospital (Project title: Investigation of the Molecular Mechanisms Underlying the Interactions Between Dental Pulp Cells and Immune Microenvironment; Approval number: JDKQ20230069; Date of approval: 11/29/2023). This study was conducted in accordance with the guidelines of the Declaration of Helsinki. Informed consent was obtained from all participating patients.

All animal procedures were conducted in compliance with the guidelines of the Laboratory Animal Ethics Committee at the School of Basic Medical Sciences, Jilin University, China (Project title: Single-cell transcriptomics reveals conserved immunoregulatory mechanisms in human and mouse dental pulp tissues; Approval number: 2023492; Date of approval: 11/01/2023).

The acquisition of human SCAPs for this study was conducted with informed consent from the patients. Written informed consent was obtained from the patients or their guardians for participation and the use of samples.

Consent for publication

All the authors approved this manuscript to be published in the Journal Stem Cell Research & Therapy.

Competing interests

The authors declare no potential conflicts of interest concerning the research, authorship, or publication of this article.

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Zhao, T., Zhong, Q., Sun, Z. et al. Decoding SFRP2 progenitors in sustaining tooth growth at single-cell resolution. Stem Cell Res Ther 16, 58 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04190-z

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Stem Cell Research & Therapy

ISSN: 1757-6512