Characteristics, Classification, and Application of Stem Cells Derived from Human Teeth

Since mesenchymal stem cells derived from human teeth are characterized as having the properties of excellent proliferation, multilineage differentiation, and immune regulation. Dental stem cells exhibit fibroblast-like microscopic appearance and express mesenchymal markers, embryonic markers, and vascular markers but do not express hematopoietic markers. Dental stem cells are a mixed population with different sensitive markers, characteristics, and therapeutic effects. Single or combined surface markers are not only helpful for understanding the subpopulation of mixed stem cell populations according to cell function but also for improving the stable treatment effect of dental stem cells. Focusing on the discovery and characterization of stem cells isolated from human teeth over the past 20 years, this review outlines the effect of marker sorting on cell proliferation and differentiation ability and the assessment of the clinical application potential. Classified dental stem cells from markers and functional molecules can solve the problem of heterogeneity and ensure the efficacy of cell therapy strategies including dentistry, neurologic diseases, bone repair, and tissue engineering.


Introduction
Due to the advantages in immune privilege, ethical approval, and easy accession, mesenchymal stem cells are receiving increasing attention in medical research. Mesenchymal stem cells (MSCs) can be isolated from multiple human organs or tissues, such as umbilical cord blood, bone marrow, adipose tissue, and brain tissue. Dental stem cells are a kind of mesenchymal stem cells and can be obtained by specific methods, separating tissues around human teeth. So far, eight types of dental stem cells were successfully isolated, including dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), apical papilla stem cells (SCAP), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFSCs), gingival mesenchymal stem cells (GMSCs), human tooth germ stem cells (TGPCs), and alveolar bone mesenchymal stem cells (ABMSCs) [1][2][3].
Obtaining the stability of the stem cell transplantation treatment effect has always been the core issue of clinical treatment [4][5][6]. The most fundamental problem is how to control the quality and efficacy of cell populations in regener-ative medicine. This means that the safety of stem cell treatments can only be guaranteed by addressing cell population heterogeneity. However, even the subgroup of stem cells isolated from human teeth has significant differences in cell properties, such as proliferation and differentiation. Therefore, how to identify and characterize dental stem cells is an essential point in basic research, but the systematic discussion is lacking. This review attempted to combine the surface markers with the differentiation ability of eight types of dental stem cells and understand the feature of preclinical applications.
2.1. CD34, CD45, and c-Kit. CD34, CD45, and c-kit (CD117) are the markers of hematopoietic stem cells (HSCs). It has been found that CD34 is related to the short-term reproduction capacity and the formation of cell clones [17][18][19]. After planted in dental scaffolds, CD34 + DPSCs showed excellent biocompatibility, high proliferation viability, osteogenic differentiation, and high abundance of VEGF. This indicated that CD34 could be used as a marker to select DPSCs for subgroups that have the potential to become dental materials. CD45 is involved in proliferation and myogenic capacity [20,21]. c-Kit is a receptor for stem cell factor (SCF) and c-kit+ HSCs can proliferate in response to hematopoietic factors [22,23]. In enrichment analysis, these membrane molecules (CD34, CD45, and c-kit) are involved in cell adhesion, vasculature and bone development, cell proliferation, and stem cell differentiation. Populations of cells expressing CD34 and CD45, c-kit were identified as the first landmark in the evaluation of DPSCs. The c-kit+/CD34+/CD45-DPSCs that has high proliferation characteristics can not only differentiate into osteoblasts but also produce active autologous fibrous bone (LAB) tissue in vitro, which can form layered bone after being transplanted into immunodeficient rats [24].
2.3. CD90 and CD271. CD90 is positive in mesenchymal progenitor cells but negative in skin fibroblasts. Based on the active screening of CD90, it was found that CD45-/CD90+ population showed a higher floating fat fraction and had a significant correlation with the frequencies of CFU-F [29][30][31]. CD271 expression is a screening criterion for hematopoietic progenitor cells. The CD34+/CD271+ population has high cloning activity [32]. CD90+/CD271+ DPSC subpopulations are able to form high-density cell populations. These subpopulations have the potential for long-term proliferation and multilineage differentiation. Hence, they can  Stem Cells International promote the formation of new bones and treat skull defects [33]. The cell surface hydrolase tissue nonspecific alkaline phosphatase (TNAP) (also known as MSCA-1), coexpressed with CD73 and CD90, is a potentially useful marker for DPSC selection, especially for mineralized tissue regenerative therapies [34]. CD271 is regarded as a marker of oral cells originating from the neural crest. This surface marker shows adipogenesis, cartilage formation, myogenic, and osteogenic potential of DPSCs [33,35]. CD271-DPSCs display higher proliferation rates compared with CD271+ DPSCs [36].
3.1. CD105. CD105 (also known as Endoglin) is one of the positive markers of HSCs and skeletal-muscle-derived stem cells. CD105 can also promote the production of bone marrow and red blood cells, and regulate hematopoietic development of specific lineages. Besides, CD105+ MSCs have the adipogenesis and osteogenesis potential [46,47]. In addition, CD105 is closely related to hereditary hemorrhagic telangiectasia and may be involved in preeclampsia, type 2 diabetes, and several types of cancer [48][49][50][51]. CD105 was validated in its ability to allow select and enrich for SHED in many studies, and CD105+ subpopulations in SHED have higher osteogenic potential in vitro. In addition, hsa-mir-1287 regulates CD105 expression and controls osteopotential in SHED by fine-tuning hsa-mir-1287 levels [52].
3.2. CD146. CD146, also known as MUC18, is a cell adhesion molecule. CD146+ MSCs tend to form bones, and the boneforming ability will be downregulated in hypoxia [53,54]. CD146 is often used as a marker to diagnose diseases such as systemic sclerosis, colorectal cancer, and breast cancer and as a target for immunotherapeutic treatment against osteosarcoma [55][56][57]. To obtain CD146+ and CD146-subpopulations of SHED, the magnetically activated cell sorting (MACS) was applied. The CD146 + SHED favors the differentiation of osteoblasts and glial and the negative subpopulation prefers adipogenic and neuronal differentiation [58].
Priming with FGF-2 is an effective way to increase the proportion of CD146+ cells in SHED [43].
4.1. CD24. CD24 is proposed as a specific surface marker, which can distinguish SCAP from DPSCs and BMSCs [60]. CD24 is strongly expressed in SCAP regardless of whether the tissue is inflamed or not, and one of the markers for multilineage stem cells in screening stable proliferation and differentiation populations [62,63]. Moreover, CD24 shows a high abundance in the early stages of root development. It was found that high-CD24 expressing SCAP showed poor renewal ability but showed stronger osteogenic differentiation ability than low-CD24 expressing SCAP [64].

STRO-1 and CD146.
One of the evidences for the presence of stem cells in the apical papilla was the positive expression of STRO-1 [65]. The positive expression of CD146 in SCAP is still controversial. The CD146+/STRO-1+ SCAP cell population was observed morphologically bigger with greater granularity in flow cytometry. Filtering by multiple marker combinations, the STRO-1+/CD146+ subpopulation was found to have a better potential for embryonic, mesenchymal, and odontogenic differentiation compared with the STRO-1-/CD146+ subpopulation [66,67].

Periodontal Ligament Stem Cells
A cell population was collected by dissolving the periodontal ligament from a third molar of a 16-19-year-old human tooth to obtain a dispersed cell suspension, which could express MSC markers and differentiate into dentin cells, adipocytes, and collagen cells under special support nourishment. After transplantation, the cells were able to help rodents repair periodontal tissue, so the population was named as periodontal ligament stem cells (PDLSCs) (Figure 1)
6.1. CD68 and CD117. CD68 and CD117 are markers expressed by endothelial cells, and CD68 is a human macrophage marker [86]. CD117+ DFSCs may be involved in the signal transmission between cells during tooth germ development [87]. The expression of CD68 in DFSCs does not mean the presence of inflammatory cells. On the contrary, CD68+ DFSCs maintain a local hematopoietic stem niche. CD117 +/CD68+ DFSCs can be used to identify endothelial progenitor cells and is related to a myeloid and progenitor phenotype in the human dental follicle. Meanwhile, the subsets are able to form neovessels and play a hematopoietic role [88].
7.1. CD39 and CD73. CD39 and CD73 are coexpressed on GMSCs and regulate T cells, which play a catalytic role to promote immune regulation and reduce inflammation [96,97]. CD39 and CD73 are involved in the formation of osteoclasts in GMSCs and significantly attenuate the severity of arthritis in vivo and in vitro [98]. Simultaneously, in the experiment of cotransplantation of GMSCs and PBMC in the NOD/SCID mouse, it was found that GMSCs suppressed the immune response through the CD39/CD73 pathway to relieve graft-versus-host disease (GVHD). It shows that CD39/CD73 can be used as a marker to evaluate the therapeutic effect of GMSCs on autoimmune diseases [99,100].
8.1. STRO-1. STRO-1 is positively expressed in TGPCs, and the subpopulation shows remarkable osteogenic differentiation ability. SRTO-1+ TGPCs exhibit strong mineralization with high expression of the osteogenic gene including OCT4, SOX2, MYC, and NANOG. STRO-1 can be used to evaluate the feasibility of TGPCs application in bone regeneration material [105,106].
ABMSCs are able to inhibit the activation and proliferation of monocytes and T cells after cocultured with different types of immune cells including THP-1 monocytes, macrophages, and peripheral blood mononuclear cell (PBMCs). Therefore, ABMSCs are a viable cell source for treating inflammation [111].

Regenerative Application of Dental Stem Cells
Dental stem cells are a potential enhancer of regenerative medicine in the future. Oral tissue repair and regeneration are the most widely clinical applications of stem cells in clinical trials (https://clinicaltrials.gov/ct2/home), including dental pulp regeneration, periodontal disease, peripical, periodontitis, knee osteoarthritis, dental plaque, alveolar bone atrophy, root canal therapy, and endodontic disease [112][113][114]. The use of biomaterials can enhance the efficiency of osteogenic differentiation in vitro and in vivo, including hydroxyapatite-tricalcium phosphate (HA-TCP) and demineralized dentin matrix (DDM) [115]. Dental stem cells can be induced into osteogenic, adipogenic, and neural differentiation in a specific medium [116]. Dental stem cells with pretreatment are an effective material such as in neurodegenerative diseases. Neuron-like stem cell transplantation can exert a stable and significant effect in brain diseases, which involves the activation of Wnt/β-catenin signaling, Rho kinase, and AKT/GSK3β signaling pathway [117][118][119].
The safety of stem cell transplantation is the primary requirement for cell transplantation [120]. The tumorigenicity of cell transplantation must be checked in clinical trials. Although the safety of dental stem cells has not been questioned, every clinical application should ensure safety under long-term monitoring and large-scale samples [121]. Few clinical records of dental stem cells have been retrieved, so the safety of human trials needs to be constantly monitored [122].
Due to the uncertainty of the timeliness of cell transplantation in vivo, the injection method is one of the factors that affect the therapeutic effect, including systemic injection and local injection [123]. Injection in the lesion area is one of the common ways to improve the effectiveness of cell transplantation, but it is not suitable for systemic diseases and chronic diseases [124,125].
Why can cells improve symptoms and cure diseases? This is the most urgent problem to be solved in future research. The explanation of the mechanism of action can not only find safe and effective disease treatment drugs but also help to understand the pathogenesis of the disease [126]. Complex networks exist in the mechanism of various diseases, but the selection of key nodes needs to be supported by huge work.

Discussion and Conclusions
Since the beginning of the 21st century, stem cells isolated from teeth have been continuously discovered and reported. Dental stem cells exhibit outstanding neural differentiation and immune characteristics due to the neural crests origin. Ethical concerns regarding dental stem cells are not an issue, and the resolution of heterogeneity problems can help improve their performance and expand their applications [127]. One significant defect of stem cells isolated in vitro is the heterogeneity of the cell population. One of the main manifestations is the instability of the source of cellular materials, including the characteristics of surface markers and the ability to differentiate. On the other hand, the unstable treatment effect of stem cell transplantation is the biggest limitation of clinical application [128].
To supplement the practical requirement of allogeneic stem cell transplantation, the screening and identification of markers in dental stem cells have been widely carried out from different donors [129]. The analysis of stem cell contents and the composition of membrane molecules is one way to solve this problem. Proteomics has been applied to the characterization of stem cells, including twodimensional electrophoresis, iTRAQ or TMT-labeled quantitative proteomics, and nanomass spectrometry [129][130][131]. However, studies have rarely analyzed the essential differences in one individual by omics analyses. Subsequent studies should concentrate on the differential expression of cells from the same donor [132].
Stem cell subpopulation studies will enable the control of stem cell heterogeneity by flow cytometry or sorting via magnetic beads. Membrane surface markers cannot represent functional cell populations. Stem cells capable of treating diseases should be characterized by functional molecules, which may be involved in metabolic physiological processes and signaling pathways to participate in pathogenesis and development [73,105]. Surface molecules are used as markers to sort stem cell subsets. The proliferation and differentiation potential of subsets can be analyzed separately. Therefore, a sole marker or a combination of multiple markers separates a subset of cells with different characteristics [47,50,52,54,57,67].
Stem cell-derived extracellular vesicles (EVs) are favored as an alternative to cell transplantation [133,134]. EVs, including RNA, protein, and lipid metabolites, are able to circulate in the blood and cross the blood-brain barrier [135,136]. EVs are considered to be functional vesicles and participate in disease regulation and are used as drug carriers [137,138]. EVs secreted from DPSCs promote the formation of blood vessels and release of VEGF in vitro [139,140]. It is believed that EVs can become potential clinical materials.
In conclusion, dental stem cells as a dental material have become a promising tool for solving oral clinical problems. The heterogeneity of dental stem cell populations severely hampers the process of elucidating their mechanisms of action. A sole marker or a combination of multiple markers is used to identify stem cells derived from teeth, which is an effective method to solve the problem of heterogeneity. In summary, the marker of stem cells is the first stage to ensure the effectiveness of cell therapy. Sole marker and associated markers of stem cells play an essential role in cell characterization in clinical applications. The resolution of heterogeneity problems can help improve performance and expand applications of dental stem cells.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this manuscript.