Identification of Smmhc-expressing mesenchymal cells in orofacial bone at single-cell resolution
Yi Fan, Yali Wei, Zhuoxuan Wu, Qin Huang, Chen Cui, Zucen Li, Ruoshi Xu, Quan Yuan, Chenchen Zhou
TL;DR
This study identifies a new type of stem cell in mouse orofacial bone that is crucial for bone development and tissue balance.
Contribution
The discovery of Smmhc-expressing mesenchymal stem/stromal cells as a novel and functionally essential subset for craniofacial bone homeostasis.
Findings
Smmhc+ MSCs are multipotent and give rise to osteoblasts, osteocytes, periodontal ligament cells, and dental pulp cells.
Ablation of Smmhc+ MSCs impairs orofacial bone development and disrupts tissue homeostasis.
Smmhc+ MSCs regulate the balance between osteogenesis and bone resorption in the orofacial skeletal niche.
Abstract
Craniofacial bone regeneration remains a major clinical challenge, yet the identity of orofacial mesenchymal stem/stromal cells (OMSCs) has not been fully elucidated. Here, we performed single-cell RNA sequencing (scRNA-seq) on mouse orofacial bone and identified multiple stromal cell clusters. Cell-cell communication mapping and trajectory inference uncovered the heterogeneity of OMSCs and functional divergence among subpopulations. We identified a previously unrecognized population, Smmhc-expressing mesenchymal stem/stromal cells (MSCs), at the earliest stage of the progenitor lineage trajectory. In vivo lineage tracing demonstrated that Smmhc+ MSCs are multipotent, giving rise to osteoblasts, osteocytes, periodontal ligament (PDL) cells, and dental pulp cells. Targeted ablation of Smmhc+ MSCs using SmmhcCreER;iDTR mouse model led to impaired orofacial bone development and disrupted…
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Figure 7- —https://doi.org/10.13039/501100001809National Natural Science Foundation of China (National Science Foundation of China)
- —Natural Science Foundation of Sichuan Province(2024NSFSC0545) Youth Innovation Project of Sichuan Province (Q23007) Research Funding from West China School/Hospital of Stomatology, Sichuan University
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Taxonomy
TopicsSingle-cell and spatial transcriptomics · Mesenchymal stem cell research · Cancer Cells and Metastasis
Introduction
Orofacial bone regeneration holds significant clinical importance due to the unique anatomical and developmental characteristics of craniofacial skeletal tissues. Standard surgical reconstruction of orofacial defects typically relies on autologous or allogeneic bone grafts harvested from distant anatomical sites, procedures often associated with donor-site morbidity, limited tissue availability, and unpredictable clinical outcomes.^1,2^ These challenges underscore the need for alternative regenerative strategies. Stem cell-based therapies offer a promising avenue, particularly with the advent of orofacial mesenchymal stem/stromal cells (OMSCs), which are readily accessible from the oral cavity and possess inherently high proliferative potential.^3,4^ Unlike long bones, which are mesoderm-derived, orofacial bones originate from neural crest-derived mesenchyme, suggesting that OMSCs represent a more suitable cell source for craniofacial repair than long bone skeletal stem cells (SSCs).^5,6^ OMSCs present higher proliferation and osteogenic differentiation capacity compared to SSCs.^7^ Moreover, OMSCs are intrinsically adapted to the craniofacial microenvironment, making them a more effective cell source for site-specific orofacial bone regeneration.^8^
A critical aspect of successful bone regeneration is the recognition of stem cell heterogeneity within craniofacial microenvironment. Multiple OMSCs subsets have been identified in orofacial regions, such as Gli1^+^, Axin2^+^, Prrx1^+^, PTHrP^+^, Lepr^+^, αSMA^+^, Ifitm5^+^, Ctsk^+^ and Fat4^+^ cells, each exhibiting distinct expression patterns and differentiation potentials across specific anatomical sites.^5,9–14^ Current knowledge of their heterogeneity, lineage hierarchies, and functional dynamics remains limited. This gap largely arises from the fact that single-cell RNA sequencing (scRNA-seq) approaches primarily capture all bone marrow-derived cells, with OMSCs comprising less than 10% of the total cell population, leaving their transcriptomic and functional profiles largely underexplored.^15,16^
Here we enriched the OMSCs by cell sorting and identified stromal subclusters. ScRNA-seq analysis revealed multiple stromal clusters in the orofacial bone marrow, including early mesenchymal progenitors (EMP), later mesenchymal progenitors (LMP), MSC_osteogenic lineage cell (MSC_OLC), pre_osteoblast, osteoblast, endothelial cell, Cxcl12_abundant reticular cell (CAR cell), MSC_chondrocyte, MSC_endothelial, and MSC_neurological cell. We also characterized a previously unrecognized subpopulation of OMSCs, termed smooth muscle myosin heavy chain (Smmhc)-expressing mesenchymal progenitors. This cluster represents an early progenitor state in the OMSCs differentiation hierarchy and contributes to osteogenic lineage cells. Lineage tracing experiment using Smmhc^CreER^;Rosa26^Ai14^ mouse model revealed that these progenitors can differentiate into osteoblasts, osteocytes, periodontal ligament (PDL) cells and dental pulp cells in vivo. Depletion of Smmhc^+^ OMSCs using Smmhc^CreER^;iDTR strategy resulted in significant reduction in bone formation and bone resorption, leading to impaired orofacial bone development and disrupted bone homeostasis. These findings highlighted the critical role of OMSCs heterogeneity in craniofacial biology and underscored the potential of targeting specific OMSCs subsets, such as Smmhc^+^ MSCs, to design precise, site-specific regenerative therapies.
Results
Characterization of orofacial bone marrow single-cell atlas
Although scRNA-seq has been applied to profile orofacial bone marrow cells,^15,16^ due to the low frequency of the bone marrow stromal stem/progenitor cells, a comprehensive mapping of the cellular populations of non-hematopoietic progenitors has not yet been fully characterized. We explored the cellular composition of the bone marrow stroma by scRNA-seq profiling of the non-hematopoietic cell containing Cd45^-^*/*Ter119^-^ population via magnetic-activated cell sorting (MACS) (Fig. 1a).^17^ After viability assessment, 3 568 high-quality cells were captured using the 10x Genomics Chromium Single Cell platform, with rigorous quality control thresholds: >500 UMIs/cell, <25% mitochondrial reads. The transcriptomes of individual cells were analyzed, and dimensionality reduction was performed via t-distributed stochastic neighbor embedding (t-SNE) implemented in Seurat v4.1.0. Unsupervised clustering identified 13 transcriptionally distinct populations (Fig. 1b). Non-hematopoietic cells including bone marrow stromal cells (93.47%) as well as non-stromal cells, such as endothelial cells (Flt1) (1.65%), Krt14-enriched epithelial cell (1.12%) and Granulocyte (S100a9) (3.76%) could be identified. There are 10 stromal progenitor populations expressed well-established stromal markers and novel markers, including EMP (Gsn) (3.56%), LMP (Aspn) (10.37%), CAR cell (Cxcl12) (2.38%), MSC_OLC (Col1a1) (46.80%), MSC_chondrocyte (Acan) (0.9%), MSC_endothelial (Plvap) (7.15%), MSC_neurological cell (Mpz) (1.57%), Pre_osteoblast (Ptn) (9.09%), Osteoblast (Bglap) (6.59%), and Smmhc^+^_MSC (Smmhc) (5.07%) (Fig. 1b, c, e). The dot plot of signature genes in all subpopulations supported the clustering result (Fig. 1d). We identified and compared the highly enriched genes within each defined cluster, revealing that these cell populations possess distinct transcriptional signatures (Fig. 1e, f). Interestingly, we did not observe significant adipogenic transcriptional markers (Pparg, Plin1) in all clusters, implying OMSCs had less of an adipocytic signature than known SSCs (Fig. 1e).^18^Fig. 1. Decoding niche-specific cell states in orofacial bone via scRNA-seq integration. a Schematic diagram of the experimental design. b t-Stochastic neighbor embedding (t-SNE) representation of aligned gene expression data in single cells extracted from mandibular bone marrow showing 13 distinct clusters. c Relative abundance of 13 cell populations composing orofacial bone. d Expression of gene markers in distinct cell types. e Gene expression patterns projected onto t-SNE plots of marker genes. f Volcano plot of marker gene differences among cell populations
MSC_OLC, Smmhc+_MSC, LMP and osteolineage cells act as signal hub to cooperate bone formation
To uncover the interactions among cell populations, we employed CellChat analysis to identify a wide range of the varied and unique communications among stromal subclusters. The differential counts and intensities of ligand-receptor pair interactions among the principal cell types are depicted. Quantitative analysis revealed that pre_osteoblast, LMP, CAR cell, Smmhc^+^_MSC, EMP and osteoblast exhibited significantly enriched ligand-receptor interaction networks. Among these, pre_osteoblasts and LMP displayed the highest number of putative interactions (Fig. 2a, b). Furthermore, interaction weight/strength analysis demonstrated that MSC_OLC were engaged in highest interactions with pre_osteoblast, LMP, osteoblast, and Smmhc^+^_MSC (Fig. 2c, d). It was notable that nearly all cell clusters were found to emit robust communication signals towards both pre_osteoblast and LMP (Fig. 2d). Subsequently, we explored the strength of the incoming and outgoing interactions over the subclusters. Interestingly, MSC_OLC had the highest outgoing signaling contributions (indicating peak activity as signal senders) among all cell types within the bone microenvironment. Moreover, pre_osteoblast, LMP, Smmhc^+^_MSC, and osteoblast also showed prominent signaling input intensity (reflecting maximal concentration as signal receivers) (Fig. 2e). These observations indicated that MSC_OLC, LMP, Smmhc^+^_MSC and osteolineage cells may serve as central communication hubs in coordinating osteogenic differentiation and the maintenance of the orofacial bone microenvironment.Fig. 2. Construction of intercellular communication networks and pseudotime of subclusters. a Circular diagram illustrating the interaction quantity. b Heatmap depicting the differential number of intercellular interactions. c Circular diagram illustrating the interaction weight/strength. d Network diagram of each cell population interacting strength with other cell populations. e Scatter chart visualizing the strength of outgoing and incoming interactions. f Sangki diagram of cell incoming and outgoing communication patterns. g Trajectory order of populations by pseudotime value. h Distribution of single cells on the developmental tree by clusters
Through systematic analysis of ligand-receptor pair interactions, we further inferred potential signaling pathways implicated in intercellular communication. Their interactions were notably enriched in core molecular pathways regulating osteogenic differentiation, including Collagen, Ptn, Fn1, Spp1, Periostin, and Bsp. This pattern suggested that activation of osteogenic differentiation-associated pathways within the orofacial bone marrow stromal cell communication network constituted a critical mechanism for maintaining bone tissue homeostasis (Fig. 2f).
To understand the differentiation dynamics of stromal subsets and infer the directionality of individual clusters during differentiation, pseudotime trajectory analysis was then performed. Psedudotime analysis predicted that the OMSCs cluster has two main developmental directions (Fig. 2g, h) with the first originating from EMP, CAR cell, and Smmhc^+^_MSC and moving towards the MSC_OLC cluster, and the second main differentiation path was directed towards pre_osteoblast and osteoblast clusters.
Smmhc+ MSCs are distinct in orofacial bone marrow
ScRNA-seq analysis identified a distinct subpopulation of stromal cells in orofacial bone, termed Smmhc^+^_MSC (Fig. 3a). Trajectory mapping positioned these cells at the onset of OMSCs differentiation, and cell-cell communication analysis revealed their central role within the stromal cell network (Fig. 2e, g, h). This previously uncharacterized subpopulation was thus selected for further functional investigation. CellChat-based intercellular communication analysis revealed that Smmhc^+^_MSC established significant functional connectivity with LMP, MSC_OLC, MSC_endothelial, pre_osteoblast, and osteoblast (Fig. 2d). Subsequent ligand-receptor pair interrogation demonstrated Smmhc^+^_MSC primarily regulate pre_osteoblast through Pdgfa ligand signaling via Pdgfa and Pdgfb receptors. Moreover, modulation of LMP occurs predominantly via Gdf15 -Tgfbr2 mediation, while osteoblasts and MSC_OLC are co-regulated by synergistic Pdgfa-Pdgfrb and Fgf1-Fgfr1 pathways (Fig. 3b, c). Crucially, Smmhc^+^_MSC predominantly receive signals originating from MSC_OLC, including collagen isoforms Col1a1/Col1a2 and osteogenic regulators Ptn/Mdk (Fig. 3c). This bidirectional communication pattern supports the critical role of Smmhc^+^_MSC as orchestrators within the osteogenic differentiation network.Fig. 3. Smmhc^+^_MSC is a distinct stromal cluster in orofacial bone marrow. a Violin plots showing the expression of Smmhc in each cluster. b Dot plot of ligand-receptor pair for Smmhc^+^_MSC with other cell populations. c String diagram of Smmhc^+^_MSC sending and receiving receptor-ligand pair. d Dot plot of gene expression in Smmhc^+^_MSC. e Stacking histogram of Smmhc^+^_MSC and other stem cell clusters. f Gene Ontology (GO) enrichment analysis of the biological functions of Smmhc^+^_MSC. g t-SNE representation an unsupervised clustering of single cells in the Smmhc^+^_MSC cluster. h Heatmap showing the 10 most upregulated genes in each subpopulations. i Relative abundance of two subpopulations in Smmhc^+^_MSCs. j, k Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the biological functions of two subpopulations. l Trajectory diagram of pseudotime analysis based on UMAP. The black line shows the structure of the graph. Numbers with white circles represent the root. Numbers with gray circles indicate different endings. Numbers with black circles represent branching points. m The expression of marker genes during the trajectory
Smmhc^+^_MSC expressed MSC markers (Thy1, Prrx1) and perivascular progenitor markers, including Acta2, CD146, Ng2 and Pdgfrb.^19–21^ We also found that Smmhc^+^_MSC were negative for markers of endothelial cells (Cd34, Cdh5, Cd31 and vWF), or myogenic cells (m-cadherin, myogenin, Cdhr5, Myf5, and Pax7) (Fig. 3d). The expression pattern suggested that Smmhc^+^_MSC may harbor the perivascular progenitor cell identity that may be integral to the origin of the OMSCs. It supported the tenet that an ancestor of the MSCs natively belongs to a subset of perivascular cells.^22^ We compared the Smmhc^+^_MSC with previously described OMSCs populations, including Gli1^+^, Axin2^+^, Prrx1^+^, PTHrP^+^, Lepr^+^, αSMA^+^, Ifitm5^+^, Ctsk^+^, and Fat4^+^ cells. Smmhc^+^_MSC represent a distinct population with partial overlap with other OMSCs subclusters, ranging from 7% to 23%. The highest overlap was observed with the Prrx1 (15.9%) and Acta2 (23.2%) populations (Fig. 3e). We next compared the differential gene expression (DEGs) profile of Smmhc^+^ and Smmhc^-^ cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the PI3K-Akt, cGMP-PKG, MAPK, TGF-β, and Wnt pathways were highly upregulated in Smmhc^+^_MSC (Fig. 3f). These pathways are critical regulators of craniofacial bone development, governing progenitor proliferation, osteogenic differentiation, and extracellular matrix remodeling,^23,24^ thereby highlighting the essential role of Smmhc^+^ progenitors in orofacial bone formation and homeostasis.
To further investigate the characteristics of stromal cell types in Smmhc^+^_MSC, we re-clustered this population and projected to t-SNE coordinates (Fig. 3g). Two subtypes of Smmhc^+^_MSC were identified with comparable percentage (Fig. 3i). Smmhc^+^ MSC_1 expressed stromal cell markers such as Mfap5, Prrx1, and Pdgfrb, as well as Col3a1, Col5a3, which have been associated with extracellular matrix formation.^25,26^ Smmhc^+^ MSC_2 expressed Sorbs2, Pcp4l1 and Myl9, which have previously been associated with cytoskeleton.^27,28^ It was also positive for Cd200, a marker reported to be expressed by skeletal stem cells.^29^ Mef2c was highly enriched in Smmhc^+^ MSC_2 and was known to be expressed in matrix-producing osteoblasts on the bone surface as well as in mesenchymal cells within the trabeculae of orofacial bone. This transcription factor has been implicated in regulating bone and cementum formation^30^ (Fig. 3h). Gene ontology (GO) analysis further confirmed that Smmhc^+^ MSC_1 were enriched with genes involved in regulating extracellular space, extracellular region, positive regulation of cell migration, and ossification processes, while Smmhc^+^ MSC_2 were enriched with genes associated with cytoplasm, protein binding, membrane and cytoskeleton processes (Fig. 3j, k).
We next performed trajectory-based differential expression analysis to delineate the transcriptional programs underlying distinct Smmhc^+^_MSC subtypes (Fig. 3l). Pseudotime values were calculated to establish a developmental trajectory, revealing that Smmhc^+^ MSC_1 occupied the earliest pseudotime position, which subsequently progressed toward Smmhc^+^ MSC_2. Gene module analysis identified sets of transcripts whose expression patterns shifted in a coordinated manner during the transition. Notably, a cluster of genes associated with the maintenance of multipotency and stem cell structural feature, such as Smmhc, Mylk, Acta2, and Myl9 showed a progressive upregulation along the pseudotime axis (Fig. 3m). These findings indicate that Smmhc^+^ MSC_1 represent a primitive progenitor-like state, while Smmhc^+^ MSC_2 exhibit enhanced cytoskeletal organization and lineage commitment potential as differentiation progressed. Overall, Smmhc^+^_MSC possess stromal cell properties and can modulate extracellular matrix organization and cytoskeletal dynamics, thereby influencing stem cell fate decisions and ultimately regulating osteogenic activity.
Smmhc+ MSCs contribute to orofacial tissue development
To investigate the spatial and temporal dynamics of Smmhc^+^ MSCs during orofacial development, we generated an inducible lineage tracing model using Smmhc^CreER^;Rosa26^Ai14^ transgenic mice. In this system, administration of tamoxifen induces Cre-mediated recombination, enabling precise labeling of Smmhc^+^ cells and their progeny via Rosa26^Ai14^ (tdTomato) expression. This approach allowed us to trace the biological behaviors of Smmhc^+^ cells over defined developmental process. Tamoxifen was first administered intraperitoneally at postnatal day 3 (P3) to label Smmhc^+^ cells and their descendants at an early postnatal stage. Mandibles were collected at P21 and analyzed (Fig. 4a). Notably, Rosa26^Ai14^^+^ cells were distributed across multiple regions of the mandibles, including the alveolar bone, dental pulp, and PDL (Fig. 4b). This expression pattern suggested that Smmhc^+^ MSCs contribute to various craniofacial compartments and may play diverse roles during development.Fig. 4. The spatial and temporal dynamics of Smmhc^+^ MSCs by lineage tracing. a Schematic of tamoxifen induction at postnatal day 3 (P3) and subsequent sample harvest at P21. b Lineage tracing of Smmhc^CreER^; Rosa26^Ai14^ mice at P21. c Immunofluorescence co-staining for tdTomato with Osx, COL1, Periostin and CD31 in 3-week-old mandibles. d Schematic of tamoxifen induction at P42 and sample harvest at P63. e Lineage tracing of Smmhc^CreER^;Rosa26^Ai14^ mice at P63. f Immunofluorescence co-staining for tdTomato with OSX, COL1, Periostin and CD31 in 9-week-old mandibles. Scale bars: 200 μm (b, e); 100 μm (1–4, c, 5–8, f). n = 6
To further assess the lineage fate of Smmhc^+^ MSCs, we performed immunofluorescence double staining. Rosa26^Ai14^^+^ cells co-expressed the osteogenic markers Osterix (OSX) and Collagen type I (COL1) in alveolar bone, indicating that Smmhc^+^ MSCs can differentiate into osteoprogenitors and osteoblasts. Rosa26^Ai14^^+^ cells also co-localized with Periostin, a marker of PDL fibroblasts, suggesting a contribution of Smmhc^+^ MSCs to the PDL lineage. Moreover, a subset of Rosa26^Ai14^^+^ cells was found in close proximity to CD31^+^ endothelial cells, arranged along the vasculature, indicative of their perivascular progenitor cell potential (Fig. 4c).
To further evaluate the spatial distribution and differentiation potential of Smmhc^+^ MSCs at later stages of postnatal development, tamoxifen was administered at P42, and mandible were analyzed at P63 (Fig. 4d). Lineage tracing revealed a more restricted localization of Smmhc^+^ MSCs compared to earlier developmental stages (Fig. 4e). Rosa26^Ai14^-labeled cells were detected in alveolar bone, PDL, and dental pulp, and co-expressed with OSX, COL1, and Periostin (Fig. 4f). These cells were more broadly distributed within the dental pulp but less abundant in periodontal tissues and predominantly confined to perivascular regions, with relatively sparse distribution in osteoblasts and osteocytes. These findings suggest a spatial and functional restriction of Smmhc^+^ MSCs in adult stage, possibly reflecting a transition toward a quiescent, perivascular progenitor state.
We next validated the stemness and multilineage differentiation potential of Smmhc^+^ MSCs in vitro. Rosa26^Ai14^-positive OMSCs were isolated and cultured from Smmhc^CreER^;Rosa26^Ai14^ mice. Colony-forming unit (CFU) assays revealed that Smmhc^+^ cells exhibited clonogenic potential, indicating a capacity for self-renewal (Fig. S1a). Flow cytometry analysis revealed that Smmhc^+^ MSCs accounted for (39.23 ± 9.44)% of total OMSCs (Fig. S1b). To assess their multipotency, Smmhc^+^ cells were subjected to lineage-specific differentiation under osteogenic, chondrogenic, and adipogenic induction. Under osteogenic conditions, Smmhc^+^ cells showed upregulated expression of Osx, demonstrating their osteogenic potential. When exposed to chondrogenic medium, Smmhc^+^ cells expressed the chondrogenic marker Sox9, confirming their ability to differentiate into chondrocytes. Furthermore, adipogenic induction resulted in the formation of lipid-laden adipocytes, as evidenced by Perilipin expression (Fig. S1c). The behavior of Smmhc^+^ cells support their classification as a subset of OMSCs with functional stemness and differentiation competency.
Specific ablation of Smmhc+ MSCs reduces orofacial bone volume
Next, we investigated the functional role of Smmhc^+^ MSCs during early development, tamoxifen was intraperitoneally administered to P3 mice to activate Cre recombinase and induce diphtheria toxin receptor (DTR) expression. Subsequently, diphtheria toxin (DT) was administered from P7 to P9 to selectively ablate Smmhc^+^ MSCs. Mandibles were collected at P16 for histological analysis (Fig. 5a). DT-induced ablation was achieved by binding to DTR on Smmhc^+^ MSCs, triggering targeted apoptosis. qRT-PCR analysis confirmed efficient depletion of Smmhc^+^ MSCs after DT treatment (Fig. S2a, b). The mutant mice showed comparable body size (Fig. 5b, f). Using micro-computed tomography (micro-CT), three-dimensional (3D) reconstructions were performed to visualize the morphology and internal architecture of the mandibles (Fig. 5c). Quantitative analysis revealed a significant reduction in total bone volume in the Smmhc^+^ MSCs-ablated mice compared to controls (Fig. 5d). Bone volume fraction (BV/TV%) and trabecular thickness (Tb.Th) were significantly decreased in Smmhc^CreER^;iDTR mice, indicating reduced bone mass and thinner trabeculae. Furthermore, trabecular separation (Tb.Sp) was markedly increased with a trend towards decrease in trabecular number (Tb.N) in the mutants, suggesting a greater distance between individual trabeculae (Fig. 5d). These data demonstrated that early depletion of Smmhc^+^ progenitors disrupted orofacial bone development by impairing both bone volume and microstructural organization.Fig. 5. Smmhc^+^ MSCs specific ablation affected orofacial tissue development and the maintenance of homeostasis. a Schematic diagram of tamoxifen and diphtheria toxin (DT) injections in mice at P3. b Representative gross images at P16 of iDTR and Smmhc^CreER^;iDTR mice. c Micro-CT images of manibles at P16. d Analysis of trabecular bone morphometric parameters: Bone Volume/Tissue Volume (BV/TV), Trabecular Thickness (Tb.Th, mm), Trabecular Separation (Tb.Sp, mm), and Trabecular Number (Tb.N, 1/mm); n = 3. e Schematic diagram of tamoxifen and DT injections in mice at P49. f Representative gross phenotype at P67 of iDTR and Smmhc^CreER^;iDTR mice. g Micro-CT images of manibles at P67. h Analysis of trabecular bone morphometric parameters; n = 4. i, j HE staining of mandibles from iDTR and Smmhc^CreER^;iDTR mice at P16 and P67; n = 3. k Analysis of periodontal ligament width and dentin thickness at P16 and P67 of iDTR and Smmhc^CreER^;iDTR mice; n = 3 (P16), n = 5 (P67). l, m Periostin and Dmp1 staning of iDTR and Smmhc^CreER^;iDTR mice at P67; n = 3. Scale bars: 1 mm (c, g), 200 μm (i),100 μm (j), 25 μm (l, m) Significance is determined using unpaired two-sided Student’s t-tests in (d, h, k). Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1
To further assess the role of Smmhc^+^ MSCs during postnatal bone remodeling, an ablation strategy was applied at later point: tamoxifen was administered at P49, followed by DT injection from P51 to P53, and mandibles were harvested at P67 (Fig. 5e). Deletion of Smmhc^+^ cells during early development compromises survival, whereas ablation after 6 weeks has no effect (Figure S2c). Micro-CT analysis revealed a significant reduction in mandibular bone volume in mutants. Notably, bone loss was particularly evident in the alveolar bone region in mutants (Fig. 5g). (BV/TV)% and Tb.Th were significantly reduced in the Smmhc^+^ MSCs-ablated mice, while Tb.Sp and Tb.N were significantly increased (Fig. 5h). These findings indicated that Smmhc^+^ MSCs play a critical role in the maintenance of orofacial bone homeostasis. The depletion resulted in disrupted bone microarchitecture and compromised skeletal integrity.
Hematoxylin and eosin (HE) staining showed that early ablation of Smmhc^+^ MSCs led to impaired tooth root development and a marked reduction in bony tissue (Fig. 5i). At later stage, Smmhc^+^ MSCs ablation did not affect root development but caused disrupted trabecular bone structures with significantly reduced bone mass (Fig. 5j). These histological changes were consistent with the micro-CT findings, particularly the reduction in bone volume and increase in Tb.N, further indicating a shift toward thinner trabeculae.
Since Smmhc^+^ MSCs were detected in PDL and dental pulp, we next examined the structural changes in these tissues in Smmhc^CreER^;iDTR mice. Micro-CT analysis revealed increased PDL width at P16, followed by a slight decrease at P67 (Fig. 5k). H&E and Periostin staining showed that the overall PDL architecture in mutants remained comparable to that of controls (Fig. 5i, l). Dentin thickness was unchanged at P16 but was reduced at P67 (Fig. 5k). Immunoflorecent staining for Dmp1 further demonstrated decreased expression and disrupted organization of dentinal tubules in mutants (Fig. 5m). These findings indicated that Smmhc^+^ MSCs play an essential role in supporting dentinogenesis and maintaining PDL integrity.
Ablation of Smmhc+ MSCs impairs osteogenic differentiation
Histomorphometry analysis revealed significant reduction in mineral apposition rate (MAR), mineral surface/bone surface (MS/BS) and bone formation rate/bone volume (BFR/BV) in Smmhc^CreER^;iDTR mice, suggesting impairment in dynamic bone formation following the ablation of the Smmhc^+^ cells (Fig. 6a, b). To investigate the function of Smmhc^+^ MSCs on osteogenic differentiation in vivo, we examined the expression of osteogenesis-related markers at developing and mature stages. Immunofluorescence staining revealed a significant reduction in OSX^+^ and COL1^+^ osteoblasts in both developmental and homeostasis stages following Smmhc^+^ MSCs depletion, indicating an essential role of Smmhc^+^ MSCs in promoting osteoblast differentiation (Fig. 6c–f). To elucidate the underlying molecular mechanisms, we isolated mandibles from Smmhc^CreER^;iDTR and control mice and performed qRT-PCR analysis. The results showed that key osteogenic genes including Osx, Runx2 and Alp were significantly downregulated in Smmhc^CreER^;iDTR mice, Col1 and Opn also showed a decreasing trend (Fig. 6g). Downregulation of Osx and Runx2 suggested impaired differentiation and maturation of osteoblasts,^31,32^, and reduced expression of Alp and Opn could compromise osteoblast function and bone matrix production.^33,34^ These data indicated the critical role of Smmhc^+^ MSCs in maintaining orofacial homeostasis by enhancing osteogenic differentiation.Fig. 6. Ablation Smmhc^+^ MSCs impaired osteogenic differentiation. a Double calcein labeling in the alveolar bone region of iDTR and Smmhc^CreER^;iDTR mice at P67. n = 4. b Histomorphometry analysis of dynamic bone formation parameters: mineral surface/bone surface (MS/BS, %), mineral apposition rate (MAR, um/day) and bone formation rate/bone volume (BFR/BV,%/day) at P67. n = 4. c, d Immunofluorescence staining of OSX and COL1 in mice injected with tamoxifen at P3 and DT during P7-P9, harvested at P16. e, f Immunofluorescence staining of OSX and COL1 in mice injected with tamoxifen at P49 and DT during P51-P53, harvested at P67. g Gene expression of osteogenesis-related factors (Osx, Alp, Runx2, Col1 and Opn) in orofacial bone, n = 3. Significance is determined using unpaired two-sided Student’s t tests. Data are mean ± SEM. **P < 0.01, ***P < 0.001. Scale bars: 25 μm (a), 100 μm (c–f)
Deletion of Smmhc+ MSCs reduces osteoclastic activity
To investigate the potential mechanisms underlying the reduction in orofacial bone volumn observed in Smmhc^CreER^;iDTR mice, we further examined osteoclast-related phenotypes. Osteoclasts are critical for bone resorption and remodeling, and changes in their number or activity directly affect orofacial bone mass.^35^ We performed tartrate-resistant acid phosphatase (TRAP) staining and immunofluorescence staining for Cathepsin K (CTSK), a key protease expressed by active osteoclasts.^36,37^ TRAP staining revealed a marked decrease in TRAP^+^ cells in the orofacial bone of Smmhc^+^ MSCs-ablated mice (Fig. 7a). Consistently, CTSK immunostaining showed a significant reduction in osteoclast number and distribution compared to control mice (Fig. 7b). These findings suggested that depletion of Smmhc^+^ MSCs non-cell autonomously affected osteoclast function, thereby contributing to the reduced bone resorption in the orofacial bone. To further explore molecular changes associated with osteoclast activity, qRT-PCR was performed on mandibular bone tissues from Smmhc^CreER^;iDTR and iDTR mice. The results demonstrated a significant downregulation of Ctsk, Mmp9, and Rank, which are essential for bone matrix degradation and osteoclast function,^38,39^ indicating suppressed osteoclastic activity and impaired bone resorption. Osteoblasts secrete a wide range of regulatory factors, including M-CSF, Rankl, Opg, Sema3A, Wnt5a, and Wnt16, to fine-tune osteoclast activity through paracrine mechanisms.^40^ Smmhc^CreER^;iDTR mice exhibited decreased Rankl levels at both the transcript and protein levels (Fig. 7c, d). No significant changes were detected in M-csf, Sema3a, Wnt5a, or Wnt16 expression in the mutants (Figure S3). The RANK/RANKL signaling axis is the central regulatory pathway for osteoclast differentiation and activation,^41,42^ and its suppression is likely to contribute to decreased osteoclast numbers. Although Opg, a decoy receptor that antagonizes RANKL activity,^43^ also showed a downward trend, the Rankl/Opg ratio was comparable between mutants and control littermates (Fig. 7c). These results indicated that Smmhc^+^ MSCs play a paracrine role in regulating osteoclast differentiation and activity. Their ablation leads to downregulation of key osteoclastic genes and signaling pathways, ultimately contributing to impaired bone remodeling and decreased bone mass.Fig. 7. Smmhc^+^ MSCs-specific ablation downregulated orofacial bone resorption. a TRAP staining of mandibles from iDTR and Smmhc^CreER^;iDTR mice at P16. b CTSK staining of mandibles from iDTR and Smmhc^CreER^;iDTR mice at P67. c Gene expression of Rank, Rankl, Opg, Rankl/Opg, Ctsk, and Mmp9 in orofacial bone, n = 3. Significance is determined using unpaired two-sided Student’s t tests. Data are mean ± SEM. P < 0.05, **P < 0.01. d RANKL staining in orofacial bone from iDTR and Smmhc^CreER^;iDTR* mice at P67; n = 3. e Graphic abstract. Scale bars: 100 μm (a, b, d)
Together, these findings demonstrated that the orofacial bone marrow harbors multiple distinct mesenchymal progenitor populations, each exhibiting unique temporal and spatial distributions and potentially specialized roles in diverse biological processes. The inherent heterogeneity of OMSCs may contribute differentially to orofacial bone development, remodeling, and homeostasis. We have identified a novel subpopulation of OMSCs, marked by Smmhc expression, which represented a functionally distinct and critical subset involved in regulating orofacial bone development and maintaining skeletal homeostasis. Ablation of these Smmhc^+^ MSCs leads to a marked impairment in osteogenic differentiation and exerts non-cell autonomous effects on osteoclast activity, resulting in significant bone loss and compromised bone architecture. These findings underscored the central role of Smmhc^+^ MSCs in coordinating the balance between bone formation and resorption within the orofacial skeletal niche. Given their essential function and regulatory capacity, Smmhc^+^ MSCs may serve as a promising cellular target for stem cell-based therapeutic strategies treating craniofacial bone defects and disorders (Fig. 7e).
Discussion
Here we identified distinct stromal subclusters in the orofacial bone marrow, including EMP, LMP, CAR cell, MSC_OLC, MSC_chondrocyte, MSC_endothelial, MSC_neurological cell, pre_osteoblast, osteoblast and Smmhc^+^_MSC, each characterized by specific gene expression profiles. Understanding the cellular heterogeneity of these subpopulations holds important clinical implications for targeted regenerative therapies and orofacial bone disorders. Cell-cell communication analysis revealed that MSC_OLC, Smmhc^+^_MSC, LMP, and osteolineage cells exhibited the most active intercellular interactions. These interactions suggest a central role for these clusters in orchestrating the osteogenic niche, potentially regulating the balance between bone formation and resorption. Among the identified OMSCs subpopulations, we uncovered a previously unrecognized Smmhc^+^ progenitor cells that appears to represent an early-stage MSCs population. Trajectory analysis positioned this cluster at the root of the osteogenic differentiation continuum. In vitro cell culture experiments confirmed their stem cell properties. In vivo lineage tracing further revealed that Smmhc^+^ MSCs were capable of differentiating into osteoblasts, osteocytes, PDL cells and dental pulp cells, highlighting their functional contribution to both bone and dental tissue compartments. Functionally, ablation of the Smmhc^+^ lineage cells led to significant defects in orofacial bone development and disrupted skeletal and dental tissue homeostasis, underscoring its indispensable role in orchestrating stem cell-mediated orofacial tissue formation and bone remodeling. These findings suggest that Smmhc^+^ MSCs may act as a key regulatory hub that integrates stem cell fate decisions within the osteogenic niche.
The orofacial region harbors a highly heterogeneous population of MSCs. Recent studies delineated a broad spectrum of OMSCs subsets marked by distinct molecular signatures and spatial distributions, such as Gli1^+^, Axin2^+^, Prrx1^+^, PTHrP^+^, Lepr^+^, αSMA^+^, Ifitm5^+^, Ctsk^+^, and Fat4^+^ cells.^5,9–14^ These subpopulations exhibit diverse lineage potentials and are differentially engaged in development, tissue remodeling, and repair, underscoring the functional specialization within the OMSCs compartment. This cellular diversity appears to be tightly linked to the unique embryonic origin and dynamic mechanical environment of orofacial tissues. For instance, Gli1^+^ MSCs serve as the major source of osteoblast lineage cells in orofacial bone,^9^ while Axin2^+^ cells in the PDL remain quiescent until activated by injury.^11^ Lepr^+^ cells remain largely quiescent in the alveolar bone under physiological conditions and do not co-express osteogenic markers. However, they are activated following tooth extraction and contribute substantially to osteoblastogenesis during healing.^13^ Similarly, αSMA lineage cells are recognized as periodontal progenitors that give rise to osteoblasts during normal tissue turnover and remodeling. During root formation, PTHrP^+^ cells differentiate into osteoblasts and osteocytes in alveolar bone.^30^ Prrx1^+^ progenitors are predominately present in the bone marrow of alveolar bone surrounding incisors and at the base of molars.^44^ Several distinct skeletal stem cell populations have also been identified in the cranio-maxillofacial region by scRNA-seq. Krt14^+^Ctsk^+^ progenitors, found in regenerated tissue after maxillary sinus floor elevation, act as osteoprogenitors in maxillofacial bone regeneration.^45^ A comparative analysis of mesenchymal cells from the periosteum and bone marrow revealed that Ctsk^+^Ly6a^+^ periosteal osteoprogenitors significantly contribute to bone defect repair.^46^ Fat4^+^ cells, which are enriched in the constantly remodeled alveolar bone, display strong osteogenic differentiation potential.^14^ Recently, a single-cell atlas of the human embryonic mandible has identified cranio-maxillofacial skeletal stem cells marked by IFITM5.^5^ These cells are specifically located around the periosteum of the jawbone and frontal bone, displaying strong self-renewal and osteogenic capacity, supporting their role in intramembranous ossification.
In our current study, we identified a previously unrecognized Smmhc^+^ MSCs cluster, which occupies the root position of the OMSCs differentiation trajectory and may represent a population of early-stage progenitors. Spatial mapping and gene expression profiling revealed that Smmhc^+^ MSCs can differentiated into osteoblasts, osteocytes, PDL cells and dental pulp cells during development. At adult stage, Smmhc^+^ MSCs are primarily located around blood vessels within the orofacial bone marrow, expressing markers such as CD146, Ng2 and Pdgfrβ but negative for CD34, CD45, Vwf and CD144, as an indicator of perivascular progenitors. Previous studies suggested that perivascular cells, principally pericytes, have been identified in multiple human organs, which serve as a reservoir for progenitor cells that may contribute to the origin of MSCs and other adult stem cell populations.^22^ Mesenchymal progenitor cells reside in all assayed vascularized tissues, and are broadly conceptualized to participate in homeostasis/renewal and repair. Smmhc is mostly known to be expressed in smooth muscle cells, and in our data sets, it was generally co-expressed with αSMA, which play an important role in MSC cell fate specification.^47^ We employed double immunofluorescence staining to test the histological locations of Smmhc^+^ cells. They results confirmed the co-localization of Smmhc^+^ cells with Acta2^+^ and Pdgfrb^+^ cells in orofacial bone (Figure S4), implying that Smmhc-expressing mesenchymal cells are a class of perivascular cells.
Lineage tracing experiments revealed distinct spatial and temporal expression patterns of Smmhc^+^ MSCs during different developmental stages. In early postnatal development, Smmhc^+^ MSCs contributed broadly to various orofacial tissues, indicating active proliferation and migration. In contrast, at later stages, these cells exhibited a more restricted distribution, primarily localized to perivascular regions. This shift suggests that the activity and plasticity of Smmhc^+^ cells are temporally regulated. They are highly dynamic and multipotent during early development, but progressively adopting a more quiescent, regionally confined state in adulthood. Such a transition implies their potential role as local progenitors dedicated to maintaining orofacial tissue homeostasis.
Smmhc^+^ cluster was also identified in periodontal stem cell and dental pulp populations in human teeth.^48^ The trajectory analysis of periodontal and dental pulp MSCs showed that Smmhc^+^ cells might constitute the most undifferentiated pool of MSCs within the periodontal tissue. It was notable that Smmhc^+^ cells were more abundant in dental pulp at adult stage. Previous study found the number of perivascular stem cells within the dental pulp increases as the tooth transitions from the unerupted to the erupted stage. Erupted molar pulps become a more heterogeneous tissue with broader differentiation potential and contain more abundant vasculature.^49^ These observations suggest that the emergence of multipotent dental pulp stem cells is closely associated with the blood vessels and the establishment of a perivascular niche. Similarly, αSMA perivascular cells were more abundant in dental pulp at 6 weeks compared to early stages.^50^ This is consistent with our observation that Smmhc^+^ cells become more broadly distributed within the dental pulp after eruption. It also provides a plausible explanation for why early ablation of Smmhc^+^ cells did not affect dentin thickness.
Although Smmhc^+^ MSCs exhibited tri-lineage differentiation potential under defined culture conditions, we did not observe Smmhc^+^ cells within the cartilage of the temporomandibular joint in vivo (Figure S1d). We compared Smmhc-expressing cells with Smmhc-negative cells and found that the Smmhc-expressing population showed lower expression of chondrogenic markers, including Col2a1, Mmp13, Comp, Sox9, Aggrecan, and Col10a1 (Figure S1e). GSEA further indicated that Smmhc-negative cells are enriched for pathways related to chondrocyte differentiation and development but not Smmhc-expressing cells (Figure S1f). Although Smmhc-expressing cells can undergo chondrogenic differentiation under specific in vitro culture conditions, these findings indicated that they may lack chondrogenic potential in vivo. Regarding adipogenic potential, mandibular bone marrow adipose tissue is known to be quantitatively sparse and compositionally distinct from that of long bones, and OMSCs have been reported to possess limited in vivo adipogenic capacity,^51^ highlighting their unique lineage constraints. Because mandibular bone marrow adiposity was not detectable at the time of sample collection, we were unable to assess the in vivo adipogenic potential of Smmhc^+^ cells, which represents a limitation of the current study.
MSCs in the craniofacial regions represent a unique and highly promising population for regenerative medicine due to their developmental origin, tissue specificity, and clinical accessibility.^52,53^ Unlike mesoderm-derived skeletal stem cells, OMSCs primarily originate from neural crest-derived mesenchyme, which confers enhanced plasticity and the ability to differentiate into diverse craniofacial tissues, including bone, cartilage, and dental tissue.^44,54^ Furthermore, OMSCs are in easily accessible anatomical sites such as the alveolar bone, allowing for minimally invasive harvesting.^55^ In human cases, the mandible is an adequate donor site, where bone marrow aspirates of sufficient volume (up to 10 mL) can be obtained with relatively low surgical burden.^56^ Subpopulations such as Gli1^+^ and Axin2^+^ cells have demonstrated robust osteogenic potential and injury-induced activation, further supporting their functional role in maintaining orofacial bone homeostasis and repair.^9^ In this context, our identification of the Smmhc^+^ MSCs adds new insight into the hierarchical organization of OMSCs. Given their position at the root of the OMSCs hierarchy and their perivascular localization, Smmhc^+^ cells may serve as a reservoir of multipotent progenitors poised to respond to regeneration. Pericytes have been proposed to serve as an important stem cell reservoir for bone regeneration, and specific pericyte subtypes have been shown to contribute to fracture and defect repair.^57^ Lineage tracing experiments using an inducible αSMA reporter mouse showed that a substantial portion of a long bone fracture callus arises from αSMA-expressing cells.^58^ Ablation of Smmhc^+^ cells led to reduced expression of osteoprogenitor and osteoblast markers in long bones after irradiation, indicating these cells contribute to bone formation after injury.^59^ Our data demonstrated that Smmhc-expressing cells reside in close proximity to blood vessels under homeostatic conditions, occupying a niche reminiscent of regenerative pericyte populations. We analyzed Smmhc expression under inflammation by generating chronic apical periodontitis and found expansion of the Smmhc^+^ cell population in periapical bone during inflammation, indicating the activation for reparative processes (Figure S5a). We also investigated Smmhc^+^ cell population in bone marrow under osteoporosis by scRNA-seq^18^ and found increased Smmhc^+^ cell percentage, implying their potential role during pathological conditions (Figure S5b). Thus, identifying this cell population not only provides a novel marker for orofacial mesenchymal stem cells but also highlights a potential therapeutic target.
These findings underscore the critical role of Smmhc^+^ subpopulation in maintaining orofacial skeletal integrity, likely through its involvement in sustaining the balance between bone formation and remodeling. Given the complex cellular composition of orofacial bone marrow, the identification pivotal progenitor population offers valuable insight into the heterogeneity of OMSCs and opens new avenues for regenerative strategies targeting craniofacial defects. Further exploration of the molecular signaling pathways governing Smmhc^+^ MSCs function may provide therapeutic targets to enhance stem cell-based interventions.
Materials and methods
Animals
Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Rosa26^Ai14^) (JAX:007914) and Smmhc^CreER^ (JAX:019079) mice were purchased from Jackson Laboratory. B6-iDTR,Rosa26^iDTR^ (No:C001189) mice were purchased from Cyagen. SmmhcCre^ER^;Rosa26^Ai14^ and SmmhcCre^ER^;iDTR mice were generated by crossing Smmhc^CreER^ mice with Rosa26^Ai14^ or Rosa26^iDTR^ mice. All mice studied in this study were male and have been genotyped by the primers listed in Supplementary Table 1. Tamoxifen (MilliporeSigma) at a dosage of 0.1 mg/g was injected to the mice at P3 intra-peritoneally once. Mice at P21 were injected intraperitoneally with tamoxifen at a dosage of 2 mg/10 g body weight every 2 days for 3 times. Mice at P49 were injected intraperitoneally with tamoxifen at a dosage of 1.5 mg/10 g body weight once. Diphtheria toxin (DT) (Sigma-Aldrich) at a dosage of 0.01 µg/g was injected to the mice for 3 times. All animal experiments were performed in accordance with the standards of the Institutional Animal Care and Use Committee at the State Key Laboratory of Oral Diseases, Sichuan University (WCHSIRB-D-2023-208).
Mouse sample harvest
Mice were euthanized by cervical dislocation and subsequently perfused transcardially with 4% paraformaldehyde (PFA). Mandibles were then dissected, fixed in 4% PFA overnight at 4 °C, and stored in 70% ethanol at 4 °C prior to further processing. For reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) analysis, soft tissues, teeth and bone posterior to the condyle were removed from mandibles. The orofacial bone tissues were snap-frozen and stored in −80 °C.
Micro-CT analysis
Samples were scanned using the µCT scanner (µCT50, Scanco, Switzerland) with the following acquisition parameters: 50 kV, 200 µA, 300 ms, and an isotropic voxel size of 8.0 µm. Three-dimensional (3D) images were reconstructed from the acquired projections. Regions of interest (ROIs) encompassing normal orofacial bone were defined within the root furcation area of the mandibular first molars. Bone microstructural parameters were quantified within these ROIs to assess bone quality.
Histology and immunostaining
Following decalcification in 20% EDTA (pH 7.5), samples were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek) and sectioned at 8 μm using a CryoStar NX50 cryostat (Thermo Fisher Scientific). Sections were stained with hematoxylin (Biosharp) and eosin (Solarbio). Tartrate-resistant acid phosphatase (TRAP) staining was performed using a commercial kit (MilliporeSigma) according to the manufacturer’s instructions. For immunofluorescence staining, sections were incubated overnight at 4 °C with the following primary antibodies: anti-OSX (1:200, Abcam, ab22552), anti-COL1A1 (1:200, Abcam, ab21286), anti-PERIOSTIN (1:200, Abcam, ab14041), anti-CD31 (1:100, BioLegend, 102501), and anti-CTSK (1:100, Santa Cruz Biotechnology, sc-4835). After washing with phosphate-buffered saline (PBS), sections were incubated for 1 h at room temperature with Alexa Fluor 488-conjugated secondary antibody (1:1 000, Invitrogen, A11070). Nuclei were counterstained with DAPI (Vector Laboratories, H-1200). Images were acquired using a FluoView FV3000 laser scanning confocal microscope (Olympus).
Histomorphometry
Calcein labels was performed to evaluate dynamic mineral apposition and bone formation in mandibles of 9-week-old mice. Mice were injected with 20 mg of calcein (Sigma-Aldrich) per kg of body weight in a 2% sodium bicarbonate solution at 8 and 2 days prior to sacrifice. Then, mandibles were collected and processed for histologic sectioning and histomorphometry. 9 μm frozen sections of undecalcified samples were processed using Multipurpose Cryosection Preparation kit (Section-LAB Co. Ltd, Japan) and cutting by a Cryomicrotome at −35 °C (Leica Microsystems KK, Germany). Mineral surface/bone surface (MS/BS, %), mineral apposition rate (MAR, um/day) and bone formation rate/bone volume (BFR/BV,%/day) were measured (Osteomeasure7, v4.3.0).
Cell sorting
Mandibles were dissected from sixteen 8-week-old male C57BL/6 mice (GemPharmatech Co., Ltd.). Soft tissues, molars, incisors, and bone posterior to the condyle were removed. The isolated bone tissue was enzymatically digested in 3 mg/mL collagenase type I (Gibco) and 4 mg/mL dispase II (MilliporeSigma) for 60 minutes at 37 °C. Resuspend the single cells in MACS buffer (0.5% BSA, 2 mmol/L EDTA) and add CD45 MicroBeads (Miltenyi). Incubate the mixture for 15 min in a 4 °C refrigerator. Following incubation, wash the cells with MACS buffer. Then, apply the cell suspension onto the MACS LS column placed within the separator’s magnetic field. Collect the flow-through fraction which were CD45^−^ cells. Perform a second magnetic separation step on the collected CD45⁻ cells using anti-Ter119 MicroBeads (Miltenyi), following the procedure described above.
ScRNA-Seq library preparation and sequencing
scRNA-Seq libraries were constructed and preprocessed as previously described. After generate CD45^-^/Ter119^-^ single-cell suspensions, single-cell suspension was added to the 10x Chromium chip according to the instructions for the 10X Genomics Chromium Single-Cell 3’ kit (V3), with the expectation of capturing 8 000 cells. cDNA amplification and library construction were performed according to standard protocols. Libraries were sequenced by LC-Bio Technology (Hangzhou,China) on an Illumina NovaSeq 6000 sequencing system (double-end sequencing, 150 bp) at a minimum depth of 20 000 reads per cell. The scRNA-seq sequencing data were compared to reference genome using CellRanger software, and cellular and individual cellular 3’ end transcripts were identified and counted in the sequenced samples. The output CellRanger expression profile matrix was loaded into Seurat (version 4.1.0) for filtering of low-quality cells from scRNA-seq data, and the filtered data was downscaled and clustered. Filtering low cell quality thresholds: number of genes expressed per cell > 500, mitochondrial genes expressed in < 25% of cells.
Pseudotemporal trajectories were constructed using the default settings of the R package Monocle (version 2.4). Pseudotemporal ordering was performed using the reduce Dimension function, with the maximum number of components set to 2 and the reduction method specified as DDRTree. Subsequently, genes exhibiting significant variation along pseudotime were identified from the top 50 marker genes within each cluster using the differentialGeneTest function. These dynamically regulated genes were then visualized using the plot_pseudotime_heatmap function. For Monocle3 analysis, the Smmhc^+^ MSCs_cluster 1 and Smmhc^+^ MSCs_cluster 2 were integrated using the reciprocal PCA method, and the resulting Seurat object was converted into a CellDataSet (CDS) format for input into Monocle3. Root cells were identified manually and in an unsupervised manner. Graph autocorrelation analysis was used to find genes that varied over a trajectory.
Intercellular communication was analyzed using the CellChat pipeline (version 1.1.3). A new CellChat object was generated from the merged Seurat object. The paracrine/autocrine signaling interaction dataset from CellChatDB served as the reference database. Subsequently, communication probabilities were computed using a 20% truncated mean. Intercellular communication networks were then inferred and aggregated using default parameters.
Cell culture
OMSCs were isolated from Smmhc^CreER^;Rosa^Ai14^ mice. After two passages in vitro in α-MEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (PS) (HyClone) at 37 °C, 5% (v/v) CO_2_, the cell suspension was incubated with an anti-RFP-biotin (Rockland) primary antibody at 4 °C for 1 h. Cells were washed and subsequently incubated with anti-biotin (Miltenyi) magnetic microbeads at 4 °C for 15 min. MACS was then performed using the method described above.
For osteogenic differentiation assay, primary Smmhc^+^ MSCs (passages P3-P5) were plated in 8-well chamber slid. On day 2 post-plating, the medium was replaced with osteogenic induction medium in α-MEM supplemented with 10% FBS, 1% PS, 50 μg/mL ascorbic acid (SAITONG), and 50 mmol/L β-glycerophosphate disodium salt hydrate (Sigma-Aldrich). The medium was subsequently changed every other day using freshly prepared osteogenic induction medium. Cells were subjected to immunofluorescence staining on day 7. For adipogenic differentiation induction, on day 2 post-plating, the medium was replaced with Adipogenic Induction Medium A (α-MEM supplemented with 10% FBS, 1% PS, 500 mmol/L 3-isobutyl-1-methylxanthine (IBMX), 1 mmol/L dexamethasone (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), and 1 mmol/L rosiglitazone (Sigma-Aldrich)). On day 4, the medium was changed to Adipogenic Induction Medium B (α-MEM supplemented with 10% FBS, 1% PS, 10 μg/mL insulin, and 1 μmol/L rosiglitazone). Cells were subjected to immunofluorescence staining on day 6. For chondrogenic differentiation induction, chondrogenic differentiation was subsequently induced according to the manufacturer’s instructions for the OriCell™ Mouse Bone Marrow Mesenchymal Stem Cell Chondrogenic Differentiation Induction Kit (Cyagen). The medium was changed every other day using freshly prepared chondrogenic induction medium.
Flow cytometry
For preparation of flow cytometry analysis, OMSCs were digested with trypsin (HyClone), and then suspended in PBS and washed with 3% BSA. Flow cytometry was carried out using the FACSCalibur (FC500, Beckman Coulter. Each data set was analyzed by FlowJo software (FLOWJO, LLC, OR, USA).
qRT-PCR
Mandibles harvested from control and mutant mice were homogenized using a PowerLyzer^®^ 24 Homogenizer (QIAGEN) in TRIzol™ reagent (Invitrogen). Total RNA was isolated according to the manufacturers’ protocols. RNA concentration and purity were measured using a NanoDrop™ ND-1000 spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) was synthesized from 1 μg total RNA using a PrimeScript™ RT reagent kit (Takara Bio) following the manufacturer’s instructions. qRT-PCR was performed using SsoAdvanced™ SYBR® Green Supermix (Bio-Rad Laboratories) on a CFX96 thermal cycler (Bio-Rad) using the primer sets listed in Supplementary Table 2. Relative gene expression was calculated using the 2^-ΔΔCt^ method.
Statistics
Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 10.1.1 (GraphPad Software, La Jolla, CA). Comparisons between two groups were analyzed by unpaired, two-tailed Student’s t-tests. Statistical significance was defined as P < 0.05 for all analyses.
Supplementary information
Identification of Smmhc-expressing mesenchymal cells in orofacial bone at single-cell resolution
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