Abstract

Tooth development is a continuous, highly orchestrated process that serves as an ideal model for dissecting the cellular and molecular mechanisms of organogenesis. In recent years, advances in single-cell transcriptomics have provided unprecedented insights into cellular heterogeneity, lineage trajectories, and molecular signaling networks during mouse and human tooth development, greatly enhancing our understanding of odontogenesis. Crucially, these single-cell-level studies of tooth development provide both a theoretical foundation and advanced strategies for dental tissue regeneration.

1 Introduction

The formation of teeth represents a paradigm of organogenesis, in which complex epithelial-neural crest mesenchymal interactions and tightly coordinated developmental signaling events give rise to the highly organized structures of enamel, dentin, cementum, pulp, and periodontal ligament (Bei, 2009). However, dental caries (Selwitz et al., 2007), periodontitis (Michaud et al., 2017), pulp and periapical inflammation (Galler et al., 2021), as well as traumatic injuries (Tsai et al., 2017) frequently result in the loss of dental and periodontal tissues, and severe cases can lead to tooth loss, thereby compromising essential functions such as mastication, speech, airway maintenance, and the structural support of facial soft tissues (Wang et al., 2024). Currently, therapeutic approaches such as root canal treatment (RCT), periodontal scaling, guided tissue regeneration (GTR), and dental implantation represent the main clinical strategies for managing these oral diseases (Del Fabbro et al., 2016; Liu et al., 2022). Despite these advanced therapeutic approaches, the complete restoration of dental, pulpal, and periodontal function remains a major challenge (Zeichner-David, 2006; Balic, 2018). Stem cell-based, biomimetic material-driven, and bioactive factor-mediated tissue engineering strategies offer promising avenues for the functional regeneration of dental, pulpal, and periodontal tissues, and potentially even the whole tooth (Zhai et al., 2019).

Over the past decades, our understanding of tooth development has advanced substantially, with the roles of multiple key cell populations elucidated through mouse genetic models. Studies on these populations and their fate-determining mechanisms have provided new insights and potential strategies for tooth regeneration. For example, a Runx2+/Gli1+ subpopulation identified in adult mouse incisors regulates the proliferation and differentiation of transit-amplifying cells (TACs), as well as the rate of incisor growth, via insulin-like growth factor (IGF) signaling (Chen H et al., 2020), thereby providing insights into the maintenance and regenerative capacity of the local microenvironment during tooth development. He et al. (2021) identified an Lhx6+ dental mesenchymal subpopulation that regulates root furcation formation by modulating Wnt signaling activity within the local microenvironment, providing important insights into the morphogenesis of dental roots and informing the development of root regeneration strategies. Despite sustained efforts to dissect the cellular and molecular basis of tooth formation (Ahtiainen et al., 2016), the extent of cellular heterogeneity and the dynamic changes in key developmental signals during odontogenesis remain incompletely explored.

Single-cell RNA sequencing (scRNA-seq) enables the profiling of genetic information at the individual cell level, offering significant advantages in identifying complex and rare cell populations, investigating intercellular interactions, and tracing the developmental trajectories of distinct cell types (Hwang et al., 2018). At present, scRNA-seq has found extensive applications in dental and oral research, encompassing tooth, pulp, and periodontal biology (Wu et al., 2022). Particularly in the field of tooth development, scRNA-seq has been used to construct single-cell atlases of developing dental tissues in both humans and mice (Jiang et al., 2025). Collectively, these investigations not only furnish deeper and more nuanced insights into the cellular and molecular mechanisms underlying tooth development but also propel advances in dental regenerative strategies.

Against this background, in the present review, we summarize how heterogeneous cellular populations within dental tissues contribute to tooth development and discuss the potential implications of these findings for regenerative dentistry.

2 Tissue architecture underlying tooth development

Similar to most ectoderm-derived organs, tooth morphogenesis is driven by continuous, finely tuned epithelial–mesenchymal interactions. The process is initiated by the thickening of the oral epithelium, which subsequently forms the dental lamina endowed with odontogenic potential. Remarkably, at this stage, recombining the dental lamina epithelium with non-dental mesenchyme is sufficient to autonomously initiate tooth formation (Lumsden, 1988; Mina and Kollar, 1987). Subsequently, the dental lamina epithelium invaginates into the underlying cranial neural crest (CNC)-derived mesenchyme to form the tooth bud, with dental mesenchymal cells condensing around the epithelial tooth germ. The epithelium then transfers its odontogenic potential to the mesenchymal compartment, orchestrating the subsequent stages of tooth development (Kollar and Baird, 1970; Thesleff and Sharpe, 1997). The epithelial component differentiates into enamel under the influence of various signaling factors, with its folding patterns determining both the shape and number of cusps (Jernvall and Thesleff, 2000). The mesenchymal compartment further differentiates into the dental papilla and dental follicle. Situated beneath the enamel, the dental papilla gives rise to dentin and contributes to root development and elongation (Yao et al., 2024). As root formation progresses, the portion of the dental papilla enclosed by the enamel and dentin of the crown continues to mature into the dental pulp, a highly vascularized and innervated structure (Yao et al., 2024). The dental follicle, enveloping the developing enamel, dentin, and dental papilla, is responsible for forming cementum, the lamina dura (alveolar bone), and the periodontal ligament, and serves as a critical effector tissue for tooth eruption (Zhou et al., 2019).

Overall, tooth morphogenesis exemplifies how dynamic epithelial-mesenchymal crosstalk orchestrates complex tissue patterning, offering a conceptual framework for understanding organogenesis and guiding regenerative strategies.

3 Morphogenesis and regenerative of enamel

Enamel, located coronally to the dentin, is the hardest tissue in the human body. In certain animals, such as mouse incisors, enamel epithelial stem cells continuously give rise to functional ameloblasts, enabling the regeneration of enamel (Binder et al., 2020). This feature renders mouse incisors an ideal model for studying tooth development. However, substantial differences remain between mouse and human dentition, including variations in cusp shape and number, as well as differences in the timing and sequence of molar development (Kavanagh et al., 2007). In humans, the enamel lacks a stem cell niche, rendering it incapable of self-repair once damaged (Fugolin and Pfeifer, 2017).

Beyond the ameloblast lineage, enamel-forming organs also comprise multiple supporting cell populations, including stellate reticulum cells and inner and outer enamel epithelial cells (Wu et al., 2024). These supporting cells are considered essential for maintaining ameloblast function (Harada et al., 2006; Maas and Bei, 1997). However, prior to the advent of scRNA-seq, the roles of these supporting cells in ameloblast differentiation and functional maturation remained largely unclear. Recent scRNA-seq studies (Alghadeer et al., 2023; Krivanek et al., 2020; Sharir et al., 2019) have begun to elucidate the molecular signatures and differentiation trajectories of heterogeneous epithelial populations during human and mouse tooth germ development, and have proposed novel strategies for human enamel regeneration (Alghadeer et al., 2023).

3.1 Cellular architecture underlying human enamel formation

Alghadeer et al. (2023) employed scRNA-seq to investigate human fetal tooth development between 9 and 22 gestational weeks, identifying 13 epithelial cell populations involved in enamel formation: oral epithelium (OE), dental epithelium (DE), enamel knot (EK), outer enamel epithelium (OEE), inner enamel epithelium (IEE), cervical loop (CL), inner stratum intermedium (SII), outer stratum intermedium (SIO), inner stellate reticulum (SRI), outer stellate reticulum (SRO), pre-ameloblasts (PA), early ameloblasts (eAM), and secretory ameloblasts (sAM).

Pseudotime analysis indicated that the OE directly differentiates into DE, which subsequently gives rise to the EK and stellate reticulum lineages (SIO and SRI); meanwhile, the OEE lineage generates SII, SIO, IEE, PA, eAM, and sAM cells (Alghadeer et al., 2023) (Figure 1). In another scRNA-seq study of human tooth germs at 17–24 gestational weeks, it was found that OEE differentiates into IEE, which subsequently gives rise to an activated leukocyte cell adhesion molecule (ALCAM)+ stem cell population with the capacity to generate stratum intermedium cells (Shi et al., 2024). In addition, the EK has been identified as an indispensable signaling center during human tooth formation, playing a pivotal role in crown morphogenesis (Alghadeer et al., 2023). Supporting cells, such as SII (via Hedgehog and Wnt pathways) and SIO (via the TGF-β pathway), appear to exert precise signaling influences on specific neighboring epithelial cell populations within the ameloblast lineage (Alghadeer et al., 2023).

The OE directly differentiates into DE, which subsequently gives rise to the EK, SIO and SRI; meanwhile, the OEE lineage generates SII, SIO, IEE, PA, eAM, and sAM cells.

Alghadeer et al. (2023) identified Leucine Rich Repeat Containing G Protein-Coupled Receptor 6 (LGR6)+ cells localized at the CL at the junction of the OEE and IEE. Previous studies have reported that the CL region in adult mouse incisors harbors epithelial stem cells (Chang et al., 2013). LGR6+ cells at the CL can give rise to the Hertwig’s epithelial root sheath (HERS), playing a critical role in root formation, and may also contribute to ameloblast formation during the early stages of crown development (Alghadeer et al., 2023). (summarized in Table 1). However, human enamel development lacks a continuously maintained CL niche, representing a fundamental limitation for in vivo enamel regeneration. This underscores both the conservation of epithelial signaling hierarchies across species and the species-specific differences that must be carefully considered when translating regenerative strategies from mouse models to humans.

Cell typeMarkersFunctionLineage relationshipReferencesOral epitheliumNot mentionedThe outermost layer of initiating epithelial cellsDirectly differentiates into DE.Alghadeer et al. (2023)Dental epitheliumNot mentionedEarly dental epithelium, the starting point for further specializationGives rise to the EK and stellate reticulum lineage cells (SIO, SRI)Alghadeer et al. (2023)Enamel knotSHH+, WNT10A+, WNT10B+A signaling center during development, determining tooth crown shapeSecretes signaling molecules such as SHH, WNT10 A/B, regulating surrounding cellsAlghadeer et al. (2023), Shi et al. (2024)Outer Enamel epitheliumNot mentionedEpithelial layer located on the outer convex aspect of the tooth germGives rise to subsequent differentiating cells including SII, SIO, and IEE.Alghadeer et al. (2023)Inner Enamel epitheliumSP6+ (cytoplasmic), ALCAM+ (subpopulation)Epithelial cells located on the inner concave aspect, adjacent to the dental papillaDifferentiates into PA and contains ALCAM+ stem cell populationsAlghadeer et al. (2023), Shi et al. (2024)Cervical loopLGR6+The looped region at the junction of the OEE and IEE, containing stem cellsLGR6+ cells localize here; can form HERS, involved in root formationAlghadeer et al. (2023), Chang et al. (2013)Inner stellate reticulumTGF-β ligand+Support cells located on the inner side of the stellate reticulumSupports adjacent epithelial cells via the TGF-β pathwayAlghadeer et al. (2023)Outer stellate reticulumNot mentionedSupport cells located on the outer side of the stellate reticulumSupports adjacent epithelial cells via the TGF-β pathwayAlghadeer et al. (2023)Inner stratum intermediumHH ligand+, EGF+ (late stage)Inner stratum intermedium cells adjacent to IEE/PA.Supports the ameloblast lineage via HH and WNT pathways; secretes EGF at later stagesAlghadeer et al. (2023)Outer stratum intermediumTGF-β ligand+, FGF+ (late stage)Outer stratum intermedium cells adjacent to OEE.Supports the ameloblast lineage via the TGF-β pathway; secretes FGF at later stagesAlghadeer et al. (2023)Pre-ameloblastsHH ligand+, SP6+ (cytoplasmic)IEE-derived precursors poised to become mature ameloblastsSecrete HH ligands; transition into eAM.Alghadeer et al. (2023)Early ameloblastsWNT ligand+, SP6+ (nuclear translocation)Early stage of ameloblast differentiationTransition into sAM); secrete WNT ligandsAlghadeer et al. (2023)Secretory ameloblastsAMBN+, AMELX+, SP6+ (nuclear)Functionally mature ameloblasts that secrete enamel matrixFinal differentiation stage, responsible for enamel formationAlghadeer et al. (2023)

Epithelial cell subpopulations in human tooth development and their lineage relationships.

3.2 Single-cell insights into molecular signaling during human enamel morphogenesis

Using scRNA-seq, Alghadeer et al. (2023) found that during the transition from OE to DE, underlying dental mesenchymal cells secrete BMP, ACTIVIN, and non-canonical WNT ligands, whereas canonical WNT ligands are produced within the OE. Similarly, during the transition from DE to OEE, DE and the EK secrete WNT ligands, whereas BMP and FGF ligands are primarily derived from the dental mesenchyme (Alghadeer et al., 2023). Moreover, Shi et al. (2024) reported that the EK secretes SHH, WNT10A, and WNT10B. These findings underscore the pivotal role of the EK as a signaling hub during the early stages of enamel development.

During the transition from OEE to IEE, the underlying dental papilla (mesenchyme) primarily influences ameloblast differentiation through the secretion of BMPs (Alghadeer et al., 2023). Notably, interactions between pre-odontoblasts (POB), odontoblasts (OB), and epithelial cells are particularly prominent, with the secretion of FGF and BMP promoting the transition of PA into eAM or sAM (Alghadeer et al., 2023). During the late stages of ameloblast differentiation, PA) and SII secrete Hedgehog (HH) ligands, while SRI and SIO produce TGF-β ligands. Subsequently, in the final maturation from eAM to secretory ameloblasts (sAM), WNT ligands are predominantly secreted by eAM cells, EGF by SII, and FGF by SIO (Alghadeer et al., 2023). The secretion of these ligands not only promotes ameloblast maturation but may also influence mesenchymal tissue development. Overall, WNT, TGF-β, HH, FGF, and BMP signaling pathways are the most active during ameloblast differentiation, highlighting the critical role of mesenchymal cells in orchestrating epithelial tissue development. While scRNA-seq has revealed potential signaling roles of SII, SIO, and SRI cells in ameloblast differentiation (Alghadeer et al., 2023), functional validation in vivo remains sparse. Future work should aim to manipulate these populations to confirm their regulatory contributions.

During the transition from outer enamel epithelium (OEE) to inner enamel epithelium (IEE), WNT activity correlates with SP6 expression (Alghadeer et al., 2023). SP6 is initially localized in the cytoplasm of IEE and PA cells, subsequently expressed in eAM and sAM, and eventually translocates to the nucleus where it co-localizes with AMBN expression (Alghadeer et al., 2023). A previous study reported that WNT signaling can induce the expression of the transcription factor SP6 (Aurrekoetxea et al., 2016), which in turn acts on the promoters of AMBN and AMELX (Rhodes et al., 2021). This finding underscores the critical role of WNT-induced SP6 expression in orchestrating ameloblast maturation.

Across human enamel development, multiple signaling pathways coordinate ameloblast differentiation. Early OE-to-DE transitions are regulated primarily by mesenchyme-derived BMP/ACTIVIN and OE-derived canonical WNT ligands, whereas EK-derived SHH and WNT10 A/B signals orchestrate crown morphogenesis. Notably, SII and SIO supporting cells modulate TGF-β and HH pathways in a spatially restricted manner. Integrating these data, we propose a hierarchical model in which mesenchymal signals initiate lineage specification, epithelial cross-talk refines differentiation, and supporting cell niches fine-tune maturation. This framework highlights potential targets for enamel regeneration while underscoring the challenge of replicating human-specific signaling environments in vitro.

3.3 Cellular foundations underlying mouse incisor enamel formation

Histological analyses have traditionally identified four principal epithelial cell types in mouse incisors, including the IEE/OEE, SR, AM, and SI (Thesleff, 2003). Juuri et al. (2012) demonstrated that during mouse incisor development, Sox2+ stem cells, regulated by FGF8 signaling, are specifically localized to the CL. These cells give rise to Sfrp5+ progenitors, which in turn generate all epithelial lineages contributing to enamel formation, thereby driving continuous enamel development in mouse incisors. Sanz-Navarro et al. (2018) further explored the heterogeneity of Sox2+ stem cells and identified Lgr5+ cells as a subpopulation within the Sox2+ compartment, which are the first to repopulate following Sox2+ cell ablation. In addition, regulatory factors such as Pitx2, Sox2, Lef1, Irx1, and Nephronectin have been shown to be critically involved in maintaining the homeostatic balance of Sox2+ stem cells (Yu et al., 2020; Arai et al., 2017). Using lineage tracing, Biehs et al. (2013) identified Bmi1+ epithelial stem cells as key regulators of enamel development, acting through Bmi1-mediated repression of Ink4a/Arf and Hox gene expression to maintain proper enamel formation (Figure 2).

During the development of mouse incisors, Sox2+ stem cells regulated by FGF8 signaling are specifically located in the dental follicle. Sox2+ is modulated by regulatory factors such as Pitx2, Sox2, Lef1, Irx1, Nephronectin and Bmi1+, and Lgr5+ cells are identified as a subpopulation within the Sox2+ region. They are the first cells to re-proliferate after the loss of Sox2+ cells. Sox2+ stem cells generate Sfrp5+ progenitor cells, which eventually differentiate into ameloblasts and SI, SR and OEE cells, and participate in the renewal process of the epithelial remnant tissue (ERM). It generates all the epithelial lineages involved in enamel formation, thereby promoting the continuous enamel development of mouse incisors.

Krivanek et al. (2020) utilized Smart-seq2-based single-cell transcriptomic profiling to generate a comprehensive cellular atlas of the mouse incisor epithelium. Within the Krt14+/Cdh1+ epithelial compartment, they identified 13 transcriptionally distinct subpopulations. Among these, Shh+, Enam+, Klk4+, and Gm17660+ cells corresponded to the pre-secretory, secretory, maturation, and post-maturation stages of ameloblast differentiation, respectively—thereby reconstructing the full developmental trajectory of ameloblast lineage progression. Moreover, they demonstrated that SI cells play a pivotal role in maintaining the vascular-ameloblast interface, which is essential for proper enamel formation (Krivanek et al., 2020). Consistently, Chiba et al. (2020) through scRNA-seq analysis, further confirmed the presence of AM, IEE/OEE, and SI/SR populations within the mouse incisor epithelium, and refined the classification of sAM into Dspp+ and Ambn+ subclusters, representing early and fully differentiated sAM states, respectively.

Historically, it has been proposed that the slowly cycling epithelial stem cells located in the CL of mouse incisors give rise to IEE cells, which subsequently differentiate into all epithelial lineages contributing to enamel formation (Kuang-Hsien Hu et al., 2014). Sharir et al. (2019) used scRNA-seq to reveal that, during normal enamel development in mouse incisors, most IEE and SI cells differentiate into ameloblasts, while a small subset gives rise to SR and OEE cells. However, upon injury or disruption of homeostasis, SI cells acquire the capacity to directly convert into IEE and ameloblasts (Sharir et al., 2019). Mechanistically, NOTCH1 signaling serves as a key regulator mediating this conversion, whereas Cldn10 may facilitate ion permeability in SI cells, thereby promoting enamel formation (Chiba et al., 2020).

Taken together, these studies substantially advance our understanding of the cellular mechanisms sustaining continuous enamel formation in mouse incisors and may provide mechanistic insights into the etiology of human enamel developmental disorders. However, pronounced morphological and developmental disparities between mouse and human dentition constrain the direct extrapolation of these findings, and their validation or translational application in human enamel development and regenerative strategies remains largely uncharted.

3.4 Cellular foundations underlying enamel formation in mouse molars

Using scRNA-seq, Ye et al. (2022) identified four epithelial subpopulations in the mandibular epithelium of E12 mouse embryos: (1) Cxcl14+/Tfap2b+ aboral epithelium, extending from the anterior dental lamina to the ventral mandibular region; (2) Pitx2+/Irx2+ dental epithelium; (3) Rtl3+/Col14a1+ cells located posterior to the dental epithelium, corresponding spatially to the oral–lingual axis of the mandibular epithelium; and (4) Grhl3+/Irf6+ periderm cells. Notably, the Pitx2+/Irx2+ dental epithelium could be further subdivided into four subclusters: Timp3+/Prss23+ diastema, Ednrb+/Dtit4l+ incisors, Fgf8+ molars, and Gad1+/Sp5+/Proser2+ initiation knot (IK) (Ye et al., 2022).

The IK subpopulation exhibits key developmental signaling molecules, including Shh, Dkk4, and Fgf20, and is enriched for pathways that promote cell proliferation and tooth morphogenesis (Ye et al., 2022). Incisor and molar subpopulations display highly similar transcriptional profiles, expressing a range of dental epithelial markers such as Pitx2, Irx1, and Fst (Mucchielli et al., 1997; Yu et al., 2017), and can also be distinguished using newly identified markers, including Ntrk2, Dsc3, Enc1, and Osbpl6 (Ye et al., 2022). Diastema cells, located in the gap between the incisor and molar fields, likely undergo regression, contributing to the separation between adjacent teeth (Ye et al., 2022).

Collectively, these findings illuminate the cellular basis of early dental epithelium development in mice. Nevertheless, how these epithelial subpopulations subsequently contribute to molar enamel formation remains largely unexplored.

3.5 iPSC-derived ameloblast organoids and biomimetic enamel regeneration strategies

To model human enamel-related disorders in vitro and explore strategies for enamel regeneration, Alghadeer et al. (2023) established a serum-free, chemically defined differentiation protocol to derive human dental epithelial-like cells from induced pluripotent stem cells (iPSCs). Leveraging key developmental signaling cues identified through scRNA-seq, these iPSCs were further guided to form human ameloblast organoids. Upon co-implantation with human dental papilla stem cells beneath the renal capsule, the organoids generated mineralized structures expressing Ameloblastin, Amelogenin, and Enamelin. By integrating developmental signaling information from human enamel formation, the iPSC-derived ameloblast organoids successfully recapitulate early differentiation stages, demonstrating how single-cell insights can inform biomimetic regeneration, although their in vivo integration and functional maturation remain to be validated.

Recently, Hasan et al. (2025) reported a biomimetic approach to recapitulate the structural features of dental enamel in vitro by precisely emulating the critical signaling microenvironment of enamel development. In this study, a tunable supramolecular protein matrix was engineered using elastin-like recombinamers. Incorporation of calcium ions (Ca2+), combined with drying-induced molecular crowding, enabled the matrix to faithfully replicate the cross-β-sheet fibrillar architecture characteristic of natural amelogenin. This biomimetic matrix can be stably applied to the surface of damaged enamel, directing the epitaxial growth of highly ordered fluorapatite nanocrystals. As a result, the regenerated enamel exhibits remarkable fidelity to native tissue in both microstructural features—including prismatic and aprismatic enamel—and macroscopic mechanical properties, such as hardness, elastic modulus, and wear resistance.

Together, these findings provide a compelling proof-of-concept for human enamel regeneration and lay the groundwork for future translational applications. Despite advances in organoid and biomimetic strategies, the absence of a persistent enamel stem cell niche and the challenge of achieving seamless integration of newly formed enamel with pre-existing tooth structures, including native enamel and dentin, remain major hurdles for in vivo regeneration.

4 Cellular foundations underlying dentin-pulp and periodontal development and their regenerative approaches4.1 Single-cell transcriptomic insights into early human dental mesenchyme heterogeneity

Alghadeer et al. (2023) characterized the heterogeneity of human dental mesenchymal cells during early tooth development (9–22 gestational weeks). Six distinct mesenchymal populations were identified: dental papilla (DP), pre-odontoblasts (POB), odontoblasts (OB), sub-odontoblasts (SOB), ectomesenchyme (DEM), and dental follicle (DF). Gene Ontology (GO) analysis revealed that DP and DEM are enriched for signaling, morphogenesis, developmental initiation, and patterning pathways, suggesting their roles as progenitor populations (Alghadeer et al., 2023). POB were characterized by proliferative and fate-determining signatures; DF cells displayed enrichment for genes involved in extracellular matrix formation; SOB expressed genes associated with cell aggregation, motility, and migration; while OB exhibited gene programs linked to odontogenesis, dental tissue formation, and mineralization (Alghadeer et al., 2023).

Pseudotime analysis further confirmed the presence of two progenitor populations within the dental mesenchyme: DP and DF. DP gives rise to POB and OB, whereas DF generates sparse DF-like cells (Alghadeer et al., 2023). Both progenitors are derived from PRRX1+ DEM cells (Alghadeer et al., 2023). At 13 gestational weeks, the dental pulp is primarily derived from DP, with DEM localized at the apical region of the pulp. Intriguingly, sparse DF-like cells are already present in the early dental pulp (prior to 13 weeks), suggesting a potential contribution of DF to pulp and dentin development. As tooth development progresses and progenitor density declines, the fate of OB lineage is largely established between 13 and 20 weeks (Alghadeer et al., 2023). By 19 weeks, the pulp contains a mixed population of sparse DF-like cells and POB, with OB positioned at the incisal edge; under injury, OB can be replenished by the underlying sparse DF-like cells, whereas during normal development, OB are predominantly derived from POB (Alghadeer et al., 2023).

In the study by Shi et al. (2024), the dental mesenchyme of human tooth germs from 17–24 gestational weeks was divided into seven subpopulations: SFRP1+/SOSTDC1+/SMOC2+ apical pulp; TNC+/DKK3+/HEY1+ DP; FRZB+/FGF3+/TWIST2+ DP; FGF3+/TWIST2+ OB and DKK3+/FBN2+ OB; IGFBP5+/SPON1+/FOXF1+ DF; and GDF10+/COL12A1+ DF. However, the interrelationships among these populations and their specific functional roles remain largely unexplored.

4.2 Single-cell profiling of postnatal human dental papilla

To date, single-cell RNA sequencing has identified eight major cell types within the postnatal human DP, including fibroblasts, odontoblasts, mesenchymal stem cells, monocytes/macrophages, lymphocytes, endothelial cells, glial cells, and proliferating cells (Ren et al., 2022). Notably, early mesenchymal stem cell populations exhibit expression of SEPTIN genes, which may endow these cells with enhanced proliferative and differentiation potential (Ren et al., 2022).

To further explore the osteogenic potential of DP-derived mesenchymal stem cells, Weng et al. (2025) employed scRNA-seq to investigate transcriptional changes during chemically induced osteogenesis, identifying a DIO2+ subpopulation with pronounced osteogenic activity. Moreover, overexpression of DIO2 enhanced the cranial bone regenerative capacity of DP mesenchymal stem cells. However, the isolation and in vitro culture system for DIO2+ cells have not yet been established, which may substantially limit the translational relevance of these findings. (summarized in Table 2). The identification of DP and DF progenitors with distinct transcriptional signatures suggests opportunities for selectively activating these populations to enhance dentin or periodontal regeneration.

Cell typeTissue localizationMarkersFunctionLineage relationshipReferencesPre-odontoblastPeripheral dental pulpWnt10a+Precursor cells for odontoblastsDerived from DP; differentiates into OB during normal developmentAlghadeer et al. (2023)OdontoblastOutermost layer of dental pulp (incisal edge)Smpd3+Functionally mature odontoblasts; responsible for dentin formationMainly derived from POB differentiation; can be generated from SOB upon injuryAlghadeer et al. (2023)Sub-odontoblastLayer beneath OBNo specific markers have been reported so farReserve cell population; can differentiate into OB upon injuryDerived from DF; present in early pulp, suggesting DF involvement in pulp/dentin developmentAlghadeer et al. (2023)Dental papilla Subtype 1 cellDental papillaTNC+/DKK3+/HEY1+Function unclearIntercellular relationships and specific functions require further discussionShi et al. (2024)Dental papilla Subtype 2 cellDental papillaFRZB+/FGF3+/TWIST2+Function unclearIntercellular relationships and specific functions require further discussionShi et al. (2024)Odontoblast Subtype 1 cellOdontoblast layerFGF3+/TWIST2+Function unclearIntercellular relationships and specific functions require further discussionShi et al. (2024)Odontoblast Subtype 2 cellOdontoblast layerDKK3+/FBN2+Function unclearIntercellular relationships and specific functions require further discussionShi et al. (2024)Dental follicle Subtype 1 cellDental follicleIGFBP5+/SPON1+/FOXF1+Function unclearIntercellular relationships and specific functions require further discussionShi et al. (2024)Dental follicle Subtype 2 cellDental follicleGDF10+/COL12A1+Function unclearIntercellular relationships and specific functions require further discussionShi et al. (2024)DIO2+ cell subpopulationApical papillaDIO2+Exhibits significant osteogenic activity; DIO2 transfection enhances calvarial regeneration capacityIsolation and in vitro culture systems not established, limiting clinical significanceWeng et al. (2025)Dental follicle stem cellsDental follicle tissueSPARC+/POSTN+Primary effector cells for periodontal tissue formation (cementum, periodontal ligament, alveolar bone)Tissue comprises endothelial cells, Schwann cells, immune cellsetc.Liu J et al., 2025, Liu N et al., 2025Dental progenitor cellsDental follicle tissuePDGFRA+Differentiate into periodontal tissue components; regulated by endothelial PDGFBB signaling to promote angiogenesisCulturing PDGFRA + cell aggregates promotes coupling of angiogenesis and osteogenesis in periodontal defects, accelerating periodontal bone regenerationLiu J et al. (2025)