In Vitro Characterization of Dental Pulp Stem Cells Cultured in Two Microsphere-Forming Culture Plates

Objective: Cancer, characterized by uncontrolled cell proliferation and invasion into surrounding tissues, is a leading cause of global mortality. Traditional two-dimensional (2D) cell culture systems fail to adequately replicate the tumor microenvironment (TME). In contrast, three-dimensional (3D) culture systems, which better simulate cell–cell and cell–extracellular matrix (ECM) interactions, have become powerful tools in biomedical research. This study aims to compare the spheroid formation capacity of A549 lung cancer cells using three different 3D culture methods: ultra-low attachment (ULA) plates, agarose hydrogel, and the hanging drop technique. The primary objective is to identify the most effective spheroid formation method for A549 cells and to provide findings that can guide future biomedical research, particularly in cancer modeling, drug screening studies, and investigations of the tumor microenvironment.Materials and Methods: A549 cells were cultured using three different 3D culture methods: ultra-low attachment plates, agarose hydrogel, and the hanging drop method. In the ultra-low attachment method, spheroid formation was observed at cell densities of 5,000, 10,000, and 30,000 cells/ml. In the agarose hydrogel method, agarose concentrations of 1%, 1.5%, and 2% were used to evaluate cell aggregation and spheroid stability. In the hanging drop method, cells aggregated under the influence of gravity. Spheroid diameter and area were analyzed using ImageJ software.Results: In this study, the spheroid formation capacity of A549 lung cancer cells was evaluated using three different three-dimensional (3D) culture methods. The ultra-low attachment (ULA) plate method allowed cell aggregation; however, the resulting structures were not large or compact enough to be classified as spheroids. The hanging drop method showed that cells formed small clusters by day 3 but failed to develop a compact and stable spheroid structure by day 7. The agarose hydrogel method, particularly at a 2% agarose concentration, demonstrated the highest spheroid formation capacity compared to the other methods. In this method, spheroid formation began at 72 hours depending on cell density, with significant growth observed at a density of 30,000 cells/ml (p < 0.0001). Trypan Blue staining results indicated that 2% agarose and cell densities of 10,000–30,000 cells/ml provided the highest cell viability. Specifically, 4,800 viable cells were counted at a density of 30,000 cells/ml, while 3,600 viable cells were observed at 10,000 cells/ml. These findings suggest that the agarose hydrogel method, especially at 2% agarose concentration and higher cell densities, offers optimal spheroid formation and cell viability for A549 lung cancer cells.Conclusion: This study demonstrated that the agarose hydrogel method effectively promoted stable and organized spheroid formation in A549 lung cancer cells. Notably, the 2% agarose concentration was identified as the most effective condition for maintaining cell viability and optimizing spheroid size. In contrast, the ultra-low attachment (ULA) plate and hanging drop methods exhibited limited spheroid formation capacity, resulting in less compact and disorganized structures. These findings emphasize the critical role of three-dimensional (3D) cell culture methods in biomedical research, particularly for experimental tumor modeling and drug screening studies. In this context, the agarose hydrogel method, with its high spheroid formation capacity and ability to support cell viability, emerges as a promising 3D culture model that warrants further exploration in cancer research.

Three-dimensionally (3D) cultured dental pulp stem cells (DPSCs) reportedly exhibit superior multi-lineage differentiation capacities and have a higher expression in regeneration-related gene categories compared to conventionally cultured DPSCs. This study aimed to evaluate the effects of various mineral trioxide aggregates (MTAs) on DPSCs cultured in 3D, assessing their cell viability and tissue mineralization properties. We examined the morphology, cell viability, alkaline phosphate (ALP) activity and qualitative alizarin red S staining assay of the DPSCs that reacted with various MTAs, which included ProRoot (PRM), Biodentine (BIO), and Well-Root PT (WRP), in two different culture plates, an ultra-low attachment plate (ULA) and a conventional monolayer plate (2D). As a control, MTA-free and IRM samples were prepared. None of the MTA groups affected the microsphere-forming characteristics of DPSCs that had been cultured in ULA. The DPSCs that were cultured in ULA showed high cell viability in all MTA groups compared to IRM. The mineralization potential was favorable in all MTA groups, with a significantly higher ALP activity among the DPSCs that were cultured in ULA. Among MTAs, the PRM group showed substantially higher ALP activity than the other MTA groups. In conclusion, our results indicate that 3D-cultured DPSCs with various MTAs showed comparable viability and mineralization capacity similar to those cultured without reacting with MTA cement.

To evaluate the biocompatibility and viability of nonionic triblock copolymer Pluronic F-127 as a cell scaffold for osteogenic differentiation of dental pulp stem cells(DPSC). DPSC were obtained via enzymatic digestion method and purified bylimited dilution method. The freeze dried hydrogel of 20% Pluronic F-127 was prepared and itsstructurewas observed usingscanning electron microscopy(SEM). After the encapsulation of cells of passage 3 in Pluronic F-127, the effects of hydrogel on the proliferations of DPSC were assessed with methyl thiazolyl terazolium(MTT) after one day and 3, 5, 7 days of incubations, respectively. On day 14, osteogenic abilities of DPSC encapsulated in the hydrogel were estimated by means of alizarin red S, immunocytochemical staining and real-time quantitative PCR(RT-qPCR). DPSC were isolated and cultured successfully in the present study. SEM observations showed that porous structures which might be suitable for cell culture. A570 values of MTT were then normalized. A570 values of the cells in 2D cultures were 0.30±0.06, 0.30±0.17, 0.35±0.04 and 0.25±0.06 and A570 values of DPSC in 3D cultures were 0.36±0.06, 0.54±0.18, 0.70±0.10 and 0.32±0.10 on day 1, 3, 5 and 7, respectively. A570 value peaks were found on day 5 in both groups. The proliferation of 3D cultured DPSC was higher than that of 2D cultured cells(P<0.05). After 14 days of osteogenic induction, there were no calcium nodules observed in the control group and the numbers of calcium nodulesin the 2D and 3D groups had no significant difference(P>0.05). Inmmunocytochemical staining demonstrated strong expression of osteoblast marker Runt-related transcription factor 2(RUNX2), type Ⅰ collagen(Col-Ⅰ) and relatively low expression of osteocalcin(OCN). Moreover, RT-qPCR showed no differences between the relative expression of ALP, RUNX-2, OCN in the 2D and 3D groups (P>0.05), but a higher relative expression of Col-Ⅰ was observed in the 3D group(P=0.023). Pluronic F-127 is a promising cell scaffold or cell carrier for the osteobalst differentiation of dental pulp stem cells.

Conventional in vitro assays are based on cells grown on two-dimensional (2D) substrates, which are not representative of the true in vivo cell environment. In tissue environments, cells interact with neighboring cells and the extracellular matrix (ECM). Three-dimensional (3D) cell culture methods allow cells to grow in structures more resembling the in vivo environment. Cells can develop cell-cell and cell-ECM interactions in 3D. The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. Cells and neutralized collagen are mixed and subsequently incubated at 37°C to allow the formation of a cell-seeded hydrogel. Specialized RAFT™ Absorbers are placed on top of the hydrogels. The RAFT™ Absorbers gently remove abundant medium, thus compacting the cell/collagen hydrogel. Additional epithelial or endothelial cells may be added as overlays on top to study co-cultures or more complex cultures. Another commonly used 3D cell culture model is spheroids. Spheroids are compact aggregates of cells that are generated without the addition of exogenous ECM – e.g. in so-called ultra-low attachment (ULA) plates or by the hanging-drop method. While 3D cultures more accurately mimic the in vivo cell environment, it might be difficult to analyze cells in 3D due to the dense, tissue-like structure of these cultures compared to 2D cell monolayers. This presentation explains how to measure cell viability in both RAFT™ 3D cell cultures and in spheroid cultures with the ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay. The ViaLight™ Assay is based on the bioluminescent detection of cellular ATP as a measure of cell viability. Only minor modifications of the standard two-step protocol that is used for 2D cell cultures are required for using the assay for 96-well and 24-well RAFT™ 3D cell cultures as well as for spheroid cultures in 96-well ULA plates. Two different cell types were used in this study: Normal Human Dermal Fibroblasts (NHDFneo) and the colon cancer cell line HCT-116. Both cell types efficiently form spheroids in ULA plates. Also in RAFT™ 3D cultures HCT-116 cells build compact aggregate-like structures, whereas NHDFneo grow in as individual cells interspersed in the collagen scaffold. By elongating the first step of the Vialight™ Assay, the lysis step, from 10 minutes to 30 minutes, a linear performance of the assay for cell numbers of up to 96,000 cells in the 96-well format and 480,000 cells in the 24-well format could be obtained for RAFT™ cultures. For spheroid cultures an extension of the lysis time to 60 minutes was required to obtain efficient lysis of spheroids with a diameter of up to 400μm, as confirmed by CalceinAM and propidium iodide staining. In summary this presentation shows that analyzing cells in 3D cultures can easily and routinely be done by slightly adjusting standard 2D cell culture assays like the ViaLight™ Assay. Citation Format: Stefanie Buesch, John Langer, Sabine Schaepermeier, Lubna Hussain, Jeffrey Bergeron, Volker Vogel, Jenny Schroeder. Analyzing cell viability in 3-D tissue models with the ViaLight™ plus cell proliferation and cytotoxicity bioassay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 607.

BackgroundMagnesium (Mg2+)-enriched microenvironment promotes odontogenic differentiation in human dental pulp stem cells (DPSCs), but the regulatory mechanisms remain undefined. The aim of this work was to assess magnesium’s function in the above process and to explore the associated signaling pathway.MethodsDPSCs underwent culture in odontogenic medium with the addition of 0, 1, 5, or 10 mM MgCl2. Intracellular Mg2+ levels in DPSCs were evaluated flow cytometrically using Mag-Fluo-4-AM. Mg2+-entry was inhibited by TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB). RNA-Sequencing was carried out for assessing transcriptome alterations in DPSCs during odontogenic differentiation associated with high extracellular Mg2+. KEGG pathway analysis was performed to determine pathways related to the retrieved differentially expressed genes (DEGs). Immunoblot was performed for assessing magnesium’s role and exploring ERK/BMP2/Smads signaling.ResultsMg2+-enriched microenvironment promoted odontogenic differentiation in DPSCs via intracellular Mg2+ increase. Consistently, the positive effect of high extracellular Mg2+ on odontogenic differentiation in DPSCs was blocked by 2-APB, which reduced Mg2+ entry. RNA-sequencing identified 734 DEGs related to odontogenic differentiation in DPSCs in the presence of high extracellular Mg2+. These DEGs participated in many cascades such as MAPK and TGF-β pathways. Consistently, ERK and BMP2/Smads pathways were activated in DPSCs treated with high extracellular Mg2+. In agreement, ERK signaling inhibition by U0126 blunted the effect of high extracellular Mg2+ on mineralization and odontogenic differentiation in DPSCs. Interestingly, BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly decreased by U0126, indicating that BMP2/Smads acted as downstream of ERK.ConclusionsMg2+-enriched microenvironment promotes odontogenic differentiation in DPSCs by activating ERK/BMP2/Smads signaling via intracellular Mg2+ increase. This study revealed that Mg2+-enriched microenvironment could be used as a new strategy for dental pulp regeneration.

Dental pulp stem cells (DPSCs) demonstrate high proliferative and multilineage differentiation potential. As previously reported, the helioxanthin derivative 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) has been demonstrated to induce the osteogenic differentiation of DPSCs. However, the mechanism of osteogenesis induced by TH in DPSCs remains unknown. The objective of this study was to identify functional extracellular vesicle (EV) microRNAs (miRNAs), and the principal genes involved in the TH-induced osteogenesis of DPSCs. DPSCs were derived from dental pulp extracted from the third molars of three healthy subjects, and were cultured with or without TH. miRNAs were extracted from DPSC-derived EVs. The gene expression patterns of mRNA and miRNA were compared using RNA-Seq and miRNA-Seq. To investigate miRNA/mRNA interacting networks, functional analyses were performed by Ingenuity Pathway Analysis. Alkaline phosphatase (ALP) staining demonstrated that treatment with TH resulted in enhanced ALP activity in DPSCs after 7 days. The expression levels of ALP and type 1 collagen alpha 1 were significantly higher in TH-induced DPSCs on day 7. RNA-Seq and miRNA-Seq analyses identified 869 differentially expressed genes (DEGs) and 18 miRNA-DEGs. Gene Ontology analysis of the mRNA-Seq results showed that TH induced several biological activities associated with signal transduction, cell adhesion, and cell differentiation. Integrated miRNA-mRNA analyses showed that these miRNAs contain the targeting information of 277 mRNAs of the DEGs. Among them, 17 target genes known to be involved in the differentiation of osteoblasts, and 24 target genes known to be involved in the differentiation of bone cells were identified. Quantitative real-time PCR showed that WNT5a expression in DPSCs was upregulated by 48 h of TH treatment. Upstream regulator analysis indicated that WNT3a, FOS, and RAC1 may be responsible for gene expression changes in DPSCs after TH treatment. EV miRNA regulatory networks might play crucial roles in TH-induced osteogenic differentiation of DPSCs. Our results presented herein offer valuable insights that will facilitate further research into the mechanism of osteogenesis of DPSCs, which is expected to lead to the clinical application of TH-induced DPSCs for bone regeneration. Furthermore, EVs derived from TH-induced DPSCs might be useful as therapeutic tools for bone defects.

To identify the basic characteristics and gene expression profiles of supernumerary teeth derived stem cells (SNTSCs) and compare them with those of normal dental pulp stem cells (DPSCs). Flow cytometry was conducted to identify the protein expression of stem cell markers. Cell proliferation, migration and differentiation abilities of both SNTSCs and DPSCs were determined by CCK8, transwell and differentiation assays, respectively. Gene expression profiles were studied by RNA sequencing analyses. After knocking down the expression of certain differential expression genes (DEGs), the function of DEGs was investigated by CCK8 and transwell assays. Statistical differences were determined using a two-tailed t-test and P values below 0.05 were considered significant. Supernumerary teeth derived stem cells were capable of differentiating into adipocyte, chondrocyte and osteoblast lineage cells, and compared to ordinary DPSCs, SNTSCs had a significantly higher cell proliferation rate (P<0.01) and significantly lower migration rate (P<0.01). RNA-seq results revealed the differential expression genes (DEGs) between SNTSCs and DPSCs. A principal component analysis (PCA) and cluster analysis revealed that the gene expression patterns of SNTSCs and DPSCs were different from each other. A total of 12861 genes were differentially expressed at a significant P value (P≤0.01), and 5292 of these increased in SNTSCs and 7569 decreased. Further study on the selected DEGs revealed that FUT11, FAM155A and BRD2 inhibited the cell proliferation rate of SNTSCs, and FUT11 and GLUD1 inhibited the cell migration rate, whilst FAM155A promoted the migration rate. The biological characteristics and gene expression profile of SNTSCs was revealed. The stem cell properties of SNTSCs were similar to normal DPSCs but they had a high cell proliferation rate and may have greater potential for cell differentiation.

Sivelestat-loaded nanostructured lipid carriers modulate oxidative and inflammatory stress in human dental pulp and mesenchymal stem cells subjected to oxygen-glucose deprivation

目的观察泊洛沙姆188（P188）对体外三维（3D）培养诱导脐血单个核细胞向巨核细胞分化的影响。方法将分离的脐血单个核细胞分别接种于细胞瓶和细胞培养袋中，后者采用WIGGENS摇床模拟生物反应器进行3D培养。在巨核细胞诱导培养基中加入P188体外培养14 d，观察细胞形态、计数细胞数并计算细胞存活率，采用流式细胞术观察巨核细胞表面标志表达情况。结果与采用传统的细胞培养瓶二维（2D）培养诱导巨核细胞相比，2D+P188培养组巨核系CD41+、CD41+/CD61+、CD61+细胞数明显增加（P值均<0.01）；在3D培养中加入P188，细胞体积变大，核形状不规则，胞质含紫红色颗粒，细胞分化更接近成熟。2D培养、3D培养及3D+P188培养组组间巨核细胞表面标志CD41、CD41/CD61、CD61表达水平差异有统计学意义（P值均<0.01）。LSD-t检验两两比较显示，与2D培养相比，3D培养诱导巨核细胞存活率及细胞数均降低（P值分别为0.018、0.027），3D+P188培养组细胞数、细胞存活率与2D和3D培养组比较差异均无统计学意义（P值均>0.05）。而3D培养组巨核细胞CD41/CD61表达水平为（36.30±1.27）%，高于2D培养组的（23.95±1.34）%（P=0.002），3D+P188培养组CD41/CD61表达水平更高[（59.45±1.20）%]。结论3D培养有利于巨核系祖细胞诱导分化，但细胞存活率低，加入P188，细胞生存状态好，且诱导效率更高。

Propolis is a natural substance produced by honeybees that has various biological properties including, anti-inflammatory, antioxidant and antimicrobial properties. Although previous studies have evaluated the antimicrobial effects of propolis in dentistry, its effects on dental pulp stem cell (DPSC) viability, migration, and differentiation are yet not well understood. The objective of this study was to investigate the effects of Chinese propolis on viability/proliferation, migration, differentiation and cytokine expression in DPSCs. Commercially available DPSCs (Lonza) were treated with aqueous extract of propolis (AEP) or ethanolic extract of propolis (EEP), and viability/proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays and quantification of nuclear staining. DPSC differentiation into mineralizing cells was evaluated with Alizarin red staining and cell migration was assessed using Boyden Chamber Transwell inserts. Cytokine expression was measured by RT-qPCR. AEP and EEP at 0.03 and 0.1 mg/mL did not affect DPSC viability/proliferation for up to 7-days treatment. Higher doses (0.33-33 mg/mL) induced a dose dependent decrease in DPSC viability/proliferation with a more prominent effect with EEP at 7 days. Neither AEP nor EEP induced DPSC differentiation into mineralizing cells, but both AEP and EEP (0.03-0.1 mg/ml) induced a dose dependent increase in DPSC migration. In addition, EEP prevents the upregulation of IL1b and IL6 but not IL8 and CCL2 in response to lipopolysaccharide stimulation. AEP has less potent anti-inflammatory effects and prevents only IL1b upregulation. This study provides new information about the biologic properties of ethanolic and aqueous extracts of propolis and shows that propolis, at doses that do not affect cell viability, induces DPSC migration and has anti-inflammatory properties. These data highlight the potential use of propolis as an alternative intra-canal medicament for regenerative endodontic procedures.

Comparison of 2,3,5,4′-tetrahydroxystilbene-2-O-b-D-glucoside-induced proliferation and differentiation of dental pulp stem cells in 2D and 3D culture systems—gene analysis

ABSTRACTObjective:The aim of the present study was to evaluate and compare the cytotoxic effects of Biodentine and MTA on dental pulp stem cells (DPSCs) and to assess cell viability and adherence after material exposure to an acidic environment.Material and Methods:DPSCs were cultured either alone or in contact with either: Biodentine; MTA set for 1 hour; or MTA set for 24 hours. After 4 and 7 days, cell viability was measured using the MTT assay. Biodentine and MTA were also prepared and packed into standardized bovine dentin disks and divided into three groups according to the storage media (n=6/group): freshly mixed materials without storage medium (Group A); materials stored in saline (Group B); materials stored in citric acid buffered at pH 5.4 (Group C). After 24 hours, DPSCs were introduced in the wells and cell adherence, viability, and cellular morphology were observed via confocal microscopy after three days of culture. Cell viability was analyzed using repeated-measures analysis of variance test with Tukey's post hoc tests (α=0.05).Results:Biodentine expressed significantly higher cell viability compared with all other groups after 4 days, with no differences after 7 days. Notably, cell viability was significantly greater in 24-hour set MTA compared with 1-hour set MTA and control groups after 7 days. Material exposure to an acidic environment showed an increase in cell adherence and viability in both groups.Conclusions:Biodentine induced a significantly accelerated cell proliferation compared with MTA. Setting of these materials in the presence of citric acid enhanced DPSC viability and adherence.

Dental pulp stem cells (DPSCs) refer to a type of stem cells, which is characterized by great differentiation potential and is easy to obtain. DPSCs are able to be employed for treating immune diseases and tissue regeneration. However, the differentiation ability exhibited by aging DPSCs is reduced, thereby limiting the application. As speculated by the microarray analysis, different expressions of miRNAs might be involved in DPSC senescence, whereas comprehensive transcriptome level detection has been rare. To gain insights into the molecular mechanisms involved, RNA-sequencing, pathway enrichment and Gene Ontology Analysis were conducted on aging and young DPSCs. In this study, the differences in long non-coding RNA (lncRNA) and messenger RNA (mRNA expressions) of the aging and young DPSCs were demonstrated, and the vital factors and the relevant pathways were speculated. On the whole, 18950 mRNAs and 21854 lncRNAs were detected, among which 14 mRNAs and 7 lncRNAs were differentially expressed. Furthermore, hsa-miR-6724-5p may be a vital node in the aging process of DPSCs, and its target genes was involved in the dopaminergic synapse. In brief, the aging of DPSCs was significantly dependent of differentially expressed genes (DEGs) which is related to dopaminergic synapse. However, the specific function and internal relationship of the DEGs should be verified in depth.

Dental stem cells (DSCs), a distinct subset of mesenchymal stem cells (MSCs), are isolated from dental tissues, such as dental pulp, exfoliated deciduous teeth, periodontal ligament, and apical papilla. They have emerged as a promising source of stem cell therapy for tissue regeneration and autoimmune disorders. The main types of DSCs include dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), and stem cells from apical papilla (SCAP). Each type exhibits distinct advantages: easy access via minimally invasive procedures, multi-lineage differentiation potential, and excellent ethical acceptability. DSCs have demonstrated outstanding clinical efficacy in oral and maxillofacial regeneration, and their long-term safety has been verified. In oral tissue regeneration, DSCs are highly effective in oral tissue regeneration for critical applications such as the restoration of dental pulp vitality and periodontal tissue repair. A defining advantage of DSCs lies in their ability to integrate with host tissues and promote physiological regeneration, which render them a better option for oral tissue regenerative therapies. Beyond oral applications, DSCs also exhibit promising potential in the treatment of systemic diseases, including type Ⅱ diabetes and autoimmune diseases due to their immunomodulatory effects. Moreover, extracellular vesicles (EVs) derived from DSCs act as critical mediators for DSCs' paracrine functions. Possessing regulatory properties similar to their parental cells, EVs are extensively utilized in research targeting tissue repair, immunomodulation, and regenerative therapy-offering a "cell-free" strategy to mitigate the limitations associated with cell-based therapies. Despite these advancements, standardizing large-scale manufacturing, maintaining strict quality control, and clarifying the molecular mechanisms underlying the interaction of DSCs and their EVs with recipient tissues remain major obstacles to the clinical translation of these treatments into broad clinical use. Addressing these barriers will be critical to enhancing their clinical applicability and therapeutic efficacy. In conclusion, DSCs and their EVs represent a transformative approach in regenerative medicine, and increasing clinical evidence supports their application in oral and systemic diseases. Continuous innovation remains essential to unlocking the widespread clinical potential of DSCs.

Recently, dental stem and progenitor cells have been harvested from periodontal tissues such as dental pulp, periodontal ligament, follicle, and papilla. These cells have received extensive attention in the field of tissue engineering and regenerative medicine due to their accessibility and multilineage differentiation capacity. These dental stem and progenitor cells are known to be derived from ectomesenchymal origin formed during tooth development. A great deal of research has been accomplished for directing osteoblastic/cementoblastic differentiation and neural differentiation from dental stem cells. To differentiate dental stem cells for use in tissue engineering and regenerative medicine, there needs to be efficient in vitro differentiation toward the osteoblastic/cementoblastic and neural lineage with well-defined and proficient protocols. This would reduce the likelihood of spontaneous differentiation into divergent lineages and increase the available cell source. This review focuses on the multilineage differentiation capacity, especially into osteoblastic/cementoblastic lineage and neural lineages, of dental stem cells such as dental pulp stem cells (DPSC), dental follicle stem cells (DFSC), periodontal ligament stem cells (PDLSC), and dental papilla stem cells (DPPSC). It also covers various experimental strategies that could be used to direct lineage-specific differentiation, and their potential applications in tissue engineering and regenerative medicine.