Dental Pulp Stem Cells Derived From Adult Human Third Molar Tooth: A Brief Review

The fields of regenerative medicine and stem cell-based tissue engineering have the potential of treating numerous tissue and organ defects. The use of adult stem cells is of particular interest when it comes to dynamic applications in translational medicine. Recently, dental pulp stem cells (DPSCs) have been traced in third molars of adult humans. DPSCs have been isolated and characterized by several groups. DPSCs have promising characteristics including self-renewal capacity, rapid proliferation, colony formation, multi-lineage differentiation, and pluripotent gene expression profile. Nevertheless, genotypic, and phenotypic heterogeneities have been reported for DPSCs subpopulations which may influence their therapeutic potentials. The underlying causes of DPSCs’ heterogeneity remain poorly understood; however, their heterogeneity emerges as a consequence of an interplay between intrinsic and extrinsic cellular factors. The main objective of the manuscript is to review the current literature related to the human DPSCs derived from the third molar, with a focus on their physiological properties, isolation procedures, culture conditions, self-renewal, proliferation, lineage differentiation capacities and their prospective advances use in pre-clinical and clinical applications.


INTRODUCTION
Dental pulp stem cells (DPSCs) are a unique population of cells embedded within the pulp cavity of the impacted third molars. DPSCs were initially isolated and characterized by Gronthos et al. (2000). Subsequently, several investigators have reported DPSCs' isolation, characterization, differentiation, and banking (Atari et al., 2012;Ferro et al., 2012a;Tirino et al., 2012). In comparison to other adult stem cells, DPSCs are noted for their high recovery rate from the disposable dental pulp after occlusion management. Their isolation procedure involves non-invasive techniques and has no notable ethical constraints. Significantly, DPSCs' stemness, viability, proliferation, and differentiating capabilities are not compromised after cryopreservation Pilbauerova et al., 2021b). Therefore, DPSCs have the potential to be a promising personalized patient-specific stem cells source for regenerative therapy. In this review article, we will discuss the tooth anatomy and dental stem cells with a particular interest on the current advances in adult human DPSCs including their origin, biological characteristics, heterogenicity, differentiation, and immunomodulatory potentials, as well as paracrine effects and pre-clinical and clinical applications.

ANATOMICAL STRUCTURE OF THE TOOTH
Teeth are viable organs made up of well-organized structures with numerous but defined specific shapes (Magnusson, 1968). Odontogenesis or teeth generation undergoes several complex developmental stages that are yet to be fully defined (Smith, 1998;Zheng et al., 2014;Rathee and Jain, 2021). Remarkably, the tooth tissues originate from different cell lineages. The enamel develops from cells derived from the ectoderm of the oral cavity, whereas the cementum, dentin, and pulp tissues are derived from neural crest-mesenchyme cells of ectodermal and mesodermal origins ( Figure 1A; Miletich and Sharpe, 2004;Thesleff and Tummers, 2008;Caton and Tucker, 2009;Koussoulakou et al., 2009). The lineage diversities may explain the observed differences in tissue topography and physiological function. The enamel-producing cells and associated metabolites are lost during tooth eruption, whereas pulp cells are longevous and have the capacity to undergo remodeling and regeneration (Simon et al., 2014).
The dental pulp is a highly vascularized connective tissue, consists of four zones, namely (1) the peripheral odontogenic zone, (2) intermediate cell-free zone, (3) cell-rich zone, and (4) the pulp core ( Figure 1A, insert). Adjacent to the dentin layer, the peripheral odontogenic zone contains the specialized columnar odontoblast cells that produce dentin (Gotjamanos, 1969;Sunitha et al., 2008;Pang et al., 2016;Ghannam et al., 2021). Besides the extensive vascular and neuronal network, the cell-free zone or basal layer of Weil contains a small number of cells whereas, high density of specialized cells is observed in the underlying zones. The cell-rich zone contains fibroblasts, macrophages, capillaries and proliferating mesenchymal cells that can differentiate into odontoblasts. The pulp core is populated by multiple populations of dental mesenchymal cells, mesenchymal-like stem cells, macrophages and dendritic cells that maintain the dentin-pulp complex functionality and homeostasis (Berkovitz, 1989;Abd-Elmeguid and Yu, 2009;Farges et al., 2015).

STEM CELLS IN THE TOOTH COMPARTMENTS
Like other organs, tooth compartments harbor a niche of heterogeneous stem/progenitor cell populations of the embryonic stem cells; however, the developmental stage for most dental stem cells has not been established yet and their precise role remains poorly understood (Kaukua et al., 2014;Krivanek et al., 2017). Several studies have indicated that in mild tooth trauma and post-inflammatory recovery, these cells regenerate dentin barrier to protect the pulp from infectious agents and demonstrate an immunomodulatory capacity, either via secreting proinflammatory cytokines or through crosstalk with immune cells (Lesot, 2000;Tomic et al., 2011;Hosoya et al., 2012;Leprince et al., 2012;Li et al., 2014).
The various sources of dental progenitor cells include the DPSCs (Gronthos et al., 2000), stem cells from human exfoliated deciduous teeth (SHED) (Miura et al., 2003), periodontal ligament stem cells (PDLSCs) (Seo et al., 2004), dental follicle stem cells (DFSCs) (Morsczeck et al., 2005), stem cells from apical papilla (SCAP) (Sonoyama et al., 2006(Sonoyama et al., , 2008, and gingival stem cells (GING SCs) (Mitrano et al., 2010; Figure 1B). Like bone marrow-derived mesenchymal stem cells (BM-MSCs), dental progenitor/stem cells exhibit self-renewal capacity and multilineage differentiation potential. In vitro studies have shown that dental stem cells generate clonogenic cell clusters, possess high proliferation rates and have the potential of multi-lineage differentiation into a wide spectrum of cell types from the three germ layers or, at least in part, express their specific markers under the appropriate culture conditions ( Figure 1C). Despite being similar at a coarse level, the transcriptomic and proteomic profiles of oral stem cells reveal several molecular differences including differential expression of surface marker, structural proteins, growth hormones, and metabolites; indicating prospective developmental divergence (Hosmani et al., 2020;Krivanek et al., 2020), and also suggest that dental stem cells might be the optimal choice for tissue self-repair and regeneration.

DENTAL STEM CELL LINEAGE TRACING
The plasticity and multi-potential competency of oral stem cells owe to the fact that the dental pulp contains neuro-mesenchymal components. Genetic lineage tracing studies have identified the perivascular pericytes and glial cells as immediate ancestors of the dental stem cells. Utilizing mouse incisors' regenerative capacity as a model for recovery from pulp/dentin injury, Feng et al. (2011) identified the pericytes NG + cells as oligodendrocyte FIGURE 1 | Oral tissue and dental stem cells. (A) A schematic outline for the tooth development. Ectoderm cells contribute to the formation of the tooth enamel only, whereas, neural crest cells generate the rest of the tooth tissues. (B) Sources of oral tissue and dental stem cells. GMSCs, gingiva-derived mesenchymal stem cells; DFPCs, dental follicle precursor cells; SCAP, stem cells from apical papilla; PDLSCs, periodontal ligament stem cells; DPSCs, dental pulp stem cells; SHED, stem cells from human exfoliated deciduous teeth, ABMSCs, alveolar bone-derived mesenchymal stem cell. (C) A schematic illustration of the dental stem cells multilineage differentiation potential. Created with BioRender.com.
Frontiers in Cell and Developmental Biology | www.frontiersin.org progenitors. Post trauma, pericytes NG + cells proliferate rapidly and partially contribute to the generated odontoblasts (Pang et al., 2016). Later, Zhao et al. (2014) reported the role of neurovascular sensory cells in activating periarterial Gli1 + cells through sonic hedgehog signaling pathways, which were sufficient to maintain homeostasis and injury repair of the incisor mesenchyme. Notably, lineage tracing studies revealed that Gli1 + cells contributed to the entire pericytes NG + cell population, but not vice versa (Zhao et al., 2014). Therefore, periarterial Gli1 + cells are believed to be the sole source of odontoblast derivation. These observations were further supported experimentally by combining a clonal color-coding technique with tracing of peripheral glia cells, in addition, quantification analysis revealed that both pericytes and glial cells contribute equally to the dynamics of tooth organogenesis, homeostasis, and growth (Kaukua et al., 2014;Sharpe, 2016;Shi et al., 2020).

DENTAL PULP STEM CELLS ISOLATION PROCEDURES AND CULTURE CONDITIONS
Dental pulp stem cells constitute merely 5% of the pulp cells and they were first isolated and characterized by Gronthos et al. (2000). The quality of the isolated DPSCs primarily impacts their regenerative potential. The culturing method and accurate characterization are pivotal steps for the isolation of high-quality DPSCs. Following extraction of the third molar, further procedures include mechanical extraction of the soft pulp connective tissue, maceration, enzymatic digestion of extracellular matrix proteins (ECM), and cell growth in plastic tissue/cell culture plates. The various isolation and culture procedures used for the human DPSCs have been best reviewed by Rodas-Junco and Villicana (2017).
Here, we also describe the standard procedure used in our clinic and laboratory. Briefly, immediately after extraction, the third molar is thoroughly rinsed with ethanol and sterile distilled water. Using a cylindrical turbine bur, an incision is made between the enamel and the cement at the point of molar fracture. The fragmented tooth is refreshed in PBS in sterile tubes and rushed to the laboratory. Using aseptic techniques, the tooth is transferred to a petri dish and dental pulp tissue is isolated using a sterile nerve-puller file-15 and forceps, chopped into fine fragments and digested by collagenase type I for 60 min at 37 • C. Single cell suspension is prepared by first passing cells through an insulin syringe and then passing through cell strainer with 40 µm APD, followed by centrifugation. The cell fraction is washed with sterile PBS, counted and cells are seeded in culture medium. For primary culture establishment, cells are seeded in fibronectincoated culture dishes. At 60% confluency, DPSCs are passed at a cell density of 80-100 cells/cm 2 . DPSCs expansion medium consists of 60% DMEM-low glucose and 40% chick fibroblast basal medium MCDB-201, supplemented with a myriad of factors such as Insulin-Transferrin-Selenium (ITS), linoleic acid bovine serum albumin (LA-BSA), dexamethasone (dex), ascorbic acid 2-phosphate (Asc-2P), antibiotics (Penicillin/Streptomycin), human Platelet-Derived Growth Factor (hPDGF)-BB, human Epidermal Growth Factor (hEGF), human Leukemia Inhibitory Factor (hLIF), Chemically Defined Lipid Concentrate (CDLC), and β-mercaptoethanol (Atari et al., 2012;Martinez-Sarra et al., 2017;Nunez-Toldra et al., 2017a,b;Al Madhoun et al., 2018;Faruqu et al., 2020).

DENTAL PULP STEM CELLS MARKERS
Dental pulp stem cells are a heterogeneous mixture of cell populations with no distinct cell surface antigens (Kawashima, 2012). DPSCs display characteristics that are much similar to those of MSCs such as the abilities for self-renewal and multilineage differentiation. According to the minimal criteria defined by the International Society of Cellular Therapy (ISCT) for the human MSCs, these cells adhere to plastic, express CD29, CD44, CD49a-f, CD51, CD73 (SH3), CD90, CD105 (SH2), CD106, and CD166, and lack the expression of the hemopoietic surface antigens including CD11b, CD14, CD19, CD34, CD45, CD79a, and human leukocyte antigen-DR isotype (HLA-DR) (Dominici et al., 2006;Sonoyama et al., 2006;Huang et al., 2009;Wu et al., 2015). DPSCs express a wide spectrum of other surface markers also as shown in Table 1. However, notable complexity and divergence in their expression levels have been reported by several groups (Laino et al., 2005;Yamada et al., 2010;Hilkens et al., 2013;Niehage et al., 2016;Alraies et al., 2020) which could be attributed, at least in part, to their heterogenicity. DPSCs can be enriched by using different isolation procedures and cell culture conditions. For example, their surface marker expression may vary depending on the serum concentrations and/or the addition of growth factors to the basal culture media. Martens et al. (2012) have documented expression of the neural markers (nestin, vimentin, synaptophysin, S100, and βIII-tubulin) on undifferentiated DPSCs that were cultured in media containing 10% FBS. Longoni et al. (2020) reported fibrous cartilage tissue conversion (expression of aggrecan, glycosaminoglycan, elevated expression of collagen type I, and limited expression of collagen type II) of DPSCs using chondro-inductive growth factors such as insulin-like growth factor (IGF)-1, transforming growth factor (TGF)-β3, and bone morphogenetic protein (BMP)-2, -6, -7. Notably, Zhang et al. (2008) have reported adipogenic, myogenic, and odontogenic plasticity of the DPSCs using respective lineagespecific pre-inductions media in vitro.
Antibody-based methods, proteomics and RNA transcriptomics are the main procedures used for DPSCs immunophenotyping. Besides the MSCs markers, DPSCs possess the embryonic stem cell-specific markers ( Table 1). In addition, DPSCs express a variety of antigens associated with cell adhesion, growth factors, transcription regulation and multiple lineage-specific markers related to perivascular tissue, endothelium, immunogenic, neuronal and osteo/odontogenic tissues ( Table 1). It is also noteworthy to mention that DPSCs express Major Histocompatibility Complex (MHC) class I antigens, but they do not express the immune co-stimulating molecules such as MHC class II antigen HLA-DR, CD40, CD80, and CD86 (Wada et al., 2009;Bhandi et al., 2021;Pilbauerova et al., 2021a).
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HETEROGENEITY OF DENTAL PULP STEM CELLS
The heterogeneity of the DPSCs subpopulations isolated from different donors is mainly influenced by donor health, age, genetic, and environmental factors (Kellner et al., 2014;Wu et al., 2015;Alraies et al., 2017;Kobayashi et al., 2020;Longoni et al., 2020). Alternatively, the intra-population heterogeneity refers to the DPSCs subpopulations found within the preparation from a single individual. The evidence demonstrating that DPSCs populations are functionally heterogeneous comes largely from their surface antigen profile or expression patterns of a variety of markers that are associated with progenitors of different lineages. DPSCs show surface expression of STRO-1, CD13, CD29, CD44, CD73, CD90, CD105, CD146, and CD166 -a profile which is reminiscent of BM-MSCs (Table 1), while DPSCs lack the expression of hematopoietic (CD34 and CD45) and monocytic (CD14) markers (Yamada et al., 2010). Additionally, DPSCs express various pluripotency markers, such as Oct-3/4, Nanog, and Sox-2, i.e., the stemness-related markers observed in embryonic stem cells (Table 1) which explains, at least in part, their self-renewal potential (Atari et al., 2011(Atari et al., , 2012Ferro et al., 2012b;Faruqu et al., 2020). As neuronal crest-derived cells, DPSCs express several neural stem cell markers, including nestin, neuronal nuclei antigen, vimentin, synaptophysin, musashi-1, Galactosyl-ceramidase, S100 calcium binding protein B, neurofilament heavy (NFH) chain, class III β-tubulin, and neurofilaments (Table 1). Thus, DPSCs are comprised of the progenitor cells that are marked by diverse characteristics, such as clonal heterogeneity, multi-lineage differentiation, self-renewal capacity, and phenotypic complexity. Notably, specific conditions and media components used may act as a source of potential phenotypic and functional changes in the freshly extracted DPSCs. The isolation procedures may also influence their heterogeneity. Whereas, DPSCs isolation by enzymatic digestion provides a large number of cells at low passage rate, the tissue explants enable the isolation of a more homogeneous cell population (Bronckaers et al., 2013;Raoof et al., 2014). Furthermore, DPSCs heterogeneity is impacted by the culture media components, serum concentration, and growth factors supplements, all of which have been wellreviewed by Rodas-Junco and Villicana (2017). Moreover, longterm and large-scale expansion in culture may also impact the heterogeneity, survival, and differentiation potential of DPSCs. The selection and expansion of different DPSCs subpopulations driven by specific culture conditions, media supplementation and 2D/3D culture systems may collectively alter the cellular profile, homeostasis, plasticity, and regenerative potential, as well as immunomodulatory properties of DPSCs (further elaborated below).

DENTAL PULP STEM CELLS CROSSTALK WITH MICROENVIRONMENT IN HOMEOSTASIS
Dental pulp stem cells are located within a heterogenic niche. Homeostatic regulation of the DPSCs niche, DPSCs' proliferation and differentiation implicate a complex network of bioactive molecules, growth factors, ECM, and key signaling pathways (Scheller et al., 2008;Mitsiadis et al., 2011;Tsutsui, 2020;Deng et al., 2021). Nevertheless, the signals that regulate DPSC fate are not only the biochemical cues, but also the biophysical cues (mechanical signals) that play a crucial role in influencing DPSC fate since orthodontic mechanical tension or stresses are exerted to teeth and transmitted into the dental pulp tissue by jaw movement during the process of normal mastication (Tatullo et al., 2016). Thus, DPSCs are mechanosensitive cells by default with the capacity to recognize mechanical signals and transform these stimuli into various cellular responses to sustain niche homeostasis (Han et al., 2008(Han et al., , 2010Hata et al., 2013). Importantly, Marrelli et al. (2018) have presented an excellent review of the mechanobiology and mechanoresponsiveness of DPSCs, deciphering how the mechanical stimuli might regulate behavior, fate, and homeostasis of DPSCs. These studies may enhance our understanding and improve approaches to the DPSC-based tissue engineering applications.

DENTAL PULP STEM CELLS POTENTIAL ROLE IN TISSUE REPAIR AND FLOURISHMENT
Dental pulp stem cells could be valuable source for cell therapy and advancing the current regenerative medicine strategies. DPSCs plasticity to surrounding environment has made them a notable source for disease treatment, though the full understanding of DPSCs tissue repair mechanisms is still in their preliminary stages. In this section, we will review the current advances in DPSCs in vitro differentiation potentials and the capability to secrete growth factors that may contribute to their role in tissue repair.

Differentiation Potential of Dental Pulp Stem Cells
Due to their potential to differentiate into several cell lineages (Figure 1C), DPSCs have received extensive attention in the field of regenerative medicine and tissue engineering. DPSCs have the potential to differentiate into endodermal (respiratory and gastrointestinal tracts, liver, pancreas, thyroid, prostate, and bladder lineages), mesodermal (adipogenic, osteogenic, and chondrogenic lineages) and ectodermal (skin and neural lineages) (Yamada et al., 2019). In addition, DPSCs were shown to differentiate into myocytes, cardiomyocytes, hepatocyte-like cells, melanocytes, and active neurons ( Figure 1C; Stevens et al., 2008;Patil et al., 2014). As a thumb rule, a substantial improvement in the efficacy DPSCs differentiation was observed using defined conditioned media.
Several recent reviews have documented the current knowledge and understanding of DPSCs' differentiation into vital lineages including their angiogenic and neurogenic potential (Ratajczak et al., 2016;Mattei et al., 2021), odontogenic and chondrogenic potential (Nuti et al., 2016;Ching et al., 2017;, and periodontal and dental tissue regeneration (Hu et al., 2018;Zhai et al., 2019); Therefore, we will focus in this section in summarizing the current knowledge regarding DPSCs hepatogenic and pancreatic β-cells differentiation capacities.

Differentiation of Dental Pulp Stem Cells Into Hepatocytes
Implementing defined, serum-free, and stepwise differentiation protocols that mimic the developmental stages of hepatocytes during embryogenesis were found to be sufficient for inducing hepatogenesis (summarized in Table 2). Using this approach, Ishkitiev et al. (2010) were the pioneers to demonstrate the hepatogenic differentiation potential of DPSCs. Initially, they developed the Ishkitiev et al. (2010) developed the twostage conditioned media that contained low percentage of fetal bovine serum (FBS) but was enriched with essential hepatogenic inducers (see Table 2). Later, they used a serum-free conditioned medium to generate the hepatocytelike cells from a CD117 + DPSCs subpopulation (Ishkitiev et al., 2012). The latter hepatogenesis protocol utilized three developmental stages, i.e., cell specification, differentiation, and maturation to generate cells with phenotypical, and functional characteristics similar to hepatocytes. This approach was further improved and implemented in later studies ( Table 2; Ferro et al., 2012a;Kumar et al., 2017). Recently, we implemented a similar approach to differentiate a pluripotentlike subpopulation of DPSCs into hepatocyte-like cells with detailed characterization of each differentiation stage and associated markers (Gil-Recio et al., 2020).

Differentiation of Dental Pulp Stem Cells Into Pancreatic Insulin-Producing Cells
There are several reports (summarized in Table 2) describing the differentiation of DPSCs into glucose-responsive pancreatic insulin-producing β-cells (IPCs). Using a threestep differentiation procedure, IPCs induction and functionality were confirmed by insulin secretion and C-peptide expression in a glucose-dependent manner ( Table 2; Govindasamy et al., 2011;Carnevale et al., 2013;Sawangmake et al., 2014). Interestingly, another study demonstrated that DPSCs-derived IPCs were physiologically functional and, as expected, reversed hyperglycemia to the normal level in streptozotocin (STZ)induced diabetic mice (Kanafi et al., 2013). Furthermore, Matei et al. (2017) observed that hydrogen sulfide exposure increases insulin and C-peptide secretions, protects against glucotoxicity, and enhances the expression insulin and PI3K/AKT pathway. As a proof of principle, Ishkitiev et al. (2013) confirmed that CD117 + DPSCs subpopulation generated a heterogeneous population of cells that expressed pancreatic-specific endocrine and exocrine markers ( Table 2). Notably, comparative studies between 2D and 3D culture systems revealed that IPCs in 3D models mimic in vivo cell growth and possess phenotypical structures like native pancreatic islets (Yagi Xu et al., 2019). Importantly, Xu et al. (2020) documented that coating IPCs with Matrigel, a basement membrane matrix, improves cell survival after orthotopic injection into the pancreatic parenchyma of Sprague Dawley (SD) rats (Table 3).

PARACRINE ACTIVITY OF DENTAL PULP STEM CELLS: SECRETOME AND EXOSOMES
Currently, accumulating evidence indicates that a great deal of therapeutic benefit of primary DPSCs exists in their paracrine activity, that is, the ability to modulate their microenvironment through the release of bioactive molecules. These factors can be released directly into the surrounding microenvironment known as secretome or they can be embedded within the membrane-bound extracellular nanovesicles (∼30-150 nm in diameter), known as exosome (Thery et al., 2002). These factors include cytokines, chemokines, growth factors, angiogenic mediators, hormones, and regulatory nucleic acid molecules (Wei et al., 2020). In general, secretome and exosomes participate in the processes of tissue replenishment, cellular homeostasis, anti-inflammation, immunomodulation, and other functions (Tang et al., 2021).
Dental pulp stem cells secrete angiogenic growth factors including VEGF, bFGF, and PDGF which are sufficient to mediate the formation of a network of tubular structures of endothelial cells, an indicator of angiogenic stimulation . Furthermore, conditioned media from cultured DPSCs promoted wound healing, angiogenesis and soft-tissue regeneration in a mouse model of excisional wound healing (Yang et al., 2013).

IMMUNOMODULATORY PROPERTIES OF DENTAL PULP STEM CELLS
The crosstalk between DPSCs and immune cell subsets impacts the functioning of both the innate and adaptive immune systems, implying that DPSCs have immunomodulatory propertiesan exciting field that needs to be further investigated [best reviewed in Li et al. (2014) and Andrukhov et al. (2019)]. Immunomodulatory phenotype of DPSCs is primarily attributed to in vitro cell culture approaches and conditions, such as enzymatic stimuli, soluble factors secretions, and cell-to-cell contacts. As though, all of these attributes may not precisely mimic the complexity of the in vivo microenvironment, nevertheless, the data generated are valid, at least in part, with respect to the studied immune cell subsets. Co-culture cell models have revealed that DPSCs mediate G0/G1 cell cycle arrest of the chemically-activated T cells (Table 3; Pierdomenico et al., 2005); while, other studies also show induction of differential T-cell subset responses. Co-cultures of DPSCs with CD3 + , CD4 + , or CD8 + T cells mediated differential proliferation arrest, apoptosis and/or induction of regulatory T cells (Treg) ( Table 3; Demircan et al., 2011;Zhao et al., 2012;Ozdemir et al., 2016;Kwack et al., 2017). Interestingly, proliferation inhibition of the peripheral blood mononuclear cells (PBMCs) was observed in cultures with conditioned medium from DPSCs pretreated with interferon (INF)-γ (Wada et al., 2009). Taken together, T lymphocyte activation and INF-γ production is a prerequisite for the induction of immunomodulatory DPSCs and secretion of IL-10/TGF-β1, expression of soluble factors inducing Treg formation and lymphocytes proliferation arrest (Ding et al., 2015;Kwack et al., 2017). Furthermore, co-culture of the DPSCs isolated from symptomatic irreversible pulpitis with macrophages suppresses the LPS-stimulated secretion of TNF-α, via TNF-α/IDO (indoleamine 2,3-dioxygenase) axis mechanism . In a rat model of diabetic neuropathy, DPSCs transplantation led to the anti-inflammatory M2-type macrophage polarization and ameliorated diabetic polyneuropathy (Omi et al., 2016). In addition, DPSCs were reported to express the complement cascade receptors C3aR and C5aR, and the treatment with C3a or C5a augmented DPSCs' proliferation and mobilization (Chmilewsky et al., 2014;Rufas et al., 2016).

Study type Description/approach Methods & outcome References
Pre-clinical comparative study using a canine bone defect model Comparative study aimed to investigate the cell-based bone engineering efficacy and determined the association between the osseointegration of dental implants and tissue-engineered bone by using DPSC, BM-MSC, and periosteal cells (PC) All premolars and the first molar were extracted from 3 dogs. In each animal, 6 bone defects, 3 on either side, were created after 4 weeks. Different materials were implanted in the defects and allowed to heal. Dental implants were placed in the defects after 8 weeks. After 8 more weeks, bone regeneration was assessed by histology and histomorphometry. It was concluded that DPSC had the highest osteogenic potential compared to BMSC and PC, proving them as a valuable cell source for tissue-engineered bone around dental implants Ito et al., 2011;Martens et al., 2014 Clinical study investigating bone regeneration effects Bone regeneration capacity, comparative study between DPSCs, DTSC and BM-MSCs, on hydroxyapatite-coated osseointegrated dental implants, by using tissue engineering technology In vitro, human DPSCs and DTSCs expressed osteogenic marker genes including alkaline phosphatase, Runx2, and osteocalcin. In vivo, the prepared bone defect model was implanted with graft materials. After 8 weeks, the dental implants were installed and after 16 weeks, sections were assessed histologically and histometrically, confirming the presence of well-formed mature bone and neovascularization. Stem cells with platelet-rich plasma (PRP) can generate bone which might be useful for osseointegrated hydroxyapatite-coated dental implants with improved levels of bone-implant contact Yamada et al., 2010 Pilot clinical study to assess the safety, efficacy, potential, and feasibility of autologous transplantation of mobilized DPSCs in pulpectomized teeth Five patients with irreversible pulpitis were enrolled and followed up for up to 24 weeks after mobilized DPSC transplantation Mobilized DPSCs were procured from discarded teeth and then expanded. The quality of mobilized DPSCs at 9 or 10 passages was assessed by karyotyping. Mobilized DPSCs were transplanted with GCSF in atelocollagen into pulpectomized teeth. No adverse events or toxicity was observed. The cone beam computed tomography confirmed functional dentin formation in 3/5 patients. This study concluded that human mobilized DPSCs were safe and efficacious for total pulp regeneration in endodontics in humans Nakashima et al., 2017;Karaoz et al., 2011;Mikami et al., 2011 A clinical study using a biocomplex constructed from DPSCs and a collagen sponge scaffold for oro-maxillo-facial (OMF) bone tissue repair in patients requiring extraction of their third molars This study involved the patients with bilateral bone resorption of the alveolar ridge distal to the 2nd molar, secondary to impaction of the 3rd molar on the cortical alveolar lamina. Since this clinical condition does not permit spontaneous bone repair after extraction of the third molar, it eventually leads to loss of the adjacent 2nd molar as well DPSCs were isolated from the extracted maxillary 3rd molars and the cells were seeded onto a collagen sponge scaffold. DPSC/collagen sponge biocomplex was used to fill in the injury site. After 3 months of the autologous DPSCs grafting, alveolar bone showed optimal vertical repair and complete restoration of periodontal tissue. Histology revealed the complete bone regeneration, with optimal results after 1 year of autologous DPSCs grafting, indicating that these cells could be used for the repair and regeneration of tissues and organs D' Aquino et al., 2009;de Almeida et al., 2014 Clinical study evaluating the biological and clinical implications at 3 years following the DPSC-based transplants in human mandibles This study investigated the stability and quality of the regenerated bone and vascularization after 3 years of the grafting intervention The authors used conventional procedures, in-line holotomography, and advanced phase-imaging method using synchrotron radiation for increased sensitivity toward low-absorbing structures. It was observed that the regenerated tissue from the graft sites comprised of a fully compact bone with a higher matrix density than control human alveolar spongy bone from the same patient. Although the regenerated bone was not of the proper type found in the mandible, it had a positive clinical impact in terms of increased implant stability as well as improved resistance to physical, chemical, mechanical and pharmacological agents Giuliani et al., 2013 (Continued) Frontiers in Cell and Developmental Biology | www.frontiersin.org In 11 chronic periodontitis patients, a total of 11 isolated intra-bony defects were accessed with a minimally invasive flap and were filled with autologous DPSCs, seeded on a collagen sponge At 1 year of autologous DPSCs implants, an average clinical attachment level gain of 4.7 ± 1.5 mm, associated with a residual mean probing depth (PD) of 3.2 ± 0.9 mm and remarkable stability of the gingival margin was attained. In 63.6% of the experimental sites, complete pocket closure (PD < 3 mm) was achieved. The clinical outcomes, as supported by the radiographic analysis, showed a bone fill of 3.6 ± 1.9 mm Aimetti et al., 2018 A single center, two arm ratio 1:  Both impacted mandibular 3rd molars were extracted and resulting DPSCs seeded on a resorbable collagen matrix were implanted in 32 experimental post-extraction sockets, whereas collagen matrices alone were implanted in 32 contralateral control post-extraction sockets. At 6 months post-extraction, CT and an advanced display platform was used to record extraction socket density and compared with measurements obtained immediately after extraction. However, the investigators were unable to show that autologous DPSCs reduce socket bone resorption after inferior 3rd molar extraction Barbier et al., 2018

PRE-CLINICAL AND CLINICAL APPLICATIONS OF HUMAN DENTAL PULP STEM CELLS
Dental pulp stem cells have remarkable potential as alternative sources to multipotent MSCs and due mainly to their immunomodulatory properties as discussed earlier, DPSCs constitute a highly valuable source for cell therapy of a variety of inflammatory diseases and other disorders. Briefly, in regard to basic or pre-clinical studies in experimental animal models, the human DPSCs-based therapies, implicating cells or secretome/conditioned media, have been successfully used in several disease conditions, which has been extensively reviewed by Anitua et al. (2018) including diabetes (Govindasamy et al., 2011;Datta et al., 2017), neuropathy (Makino et al., 2019), hepatic diseases Kim et al., 2016), oculopathies (Syed-Picard et al., 2015;Kushnerev et al., 2016;Mead et al., 2016), spinal cord injury (Sakai et al., 2012;Yang et al., 2017), peripheral nerve injury (Sasaki et al., 2011;Sanen et al., 2017), AD (Nakashima et al., 2009;Mita et al., 2015;Wang et al., 2017), cerebral ischemia (Leong et al., 2012;Song et al., 2017), muscular dystrophy (Kerkis et al., 2008;Pisciotta et al., 2015;Martinez-Sarra et al., 2017), myocardial infarction (Gandia et al., 2008), Parkinson's disease (Nesti et al., 2011;Gnanasegaran et al., 2017a,b), lung injury (Wakayama et al., 2015), and stroke (Yang et al., 2009;Leong et al., 2012;Song et al., 2015). It is noteworthy that not many clinical trials have so far published their results. Of note, owing to their anti-inflammatory properties and regenerative potentials, DPSCs are also being tested for their therapeutic benefits in coronavirus disease 2019  patients. Like in acute respiratory distress syndrome (ARDS), COVID-19 patients show loss of alveolar structures and invasion/accumulation of proinflammatory M1 macrophages, resulting in the release of proinflammatory cytokines/mediators and enhanced tissue fibrosis. Interestingly, Wakayama et al. (2015) demonstrated earlier in a mouse model study of acute lung injury that intravenous infusion of DPSC/SHED or the conditioned media potentiated the anti-inflammatory effects via M2 macrophage activation and ameliorated the disease pathophysiology. Currently, few clinical trials are in progress to test out the safety and efficacy of DPSCs-based therapies in COVID-19 patients ( Table 4).

CONCLUDING REMARKS AND FUTURE PROSPECTIVES
Dental pulp is a promising source of DPSCs, which are multipotent stem cells with potentials of self-renewal, multilineage differentiation, and immunomodulatory functions. These stem cells offer the advantage of more comprehensive clinical applications as compared to MSCs derived from other sources like the peripheral blood, adipose tissue, umbilical cord, and bone marrow. Other intriguing aspects that make the use of DPSCs more attractive is their easy access from the discarded third molar tooth and the minimal ethical concerns are involved in the procurement process, as well as the fact that cryopreserved DPSCs will retain their ability for multilineage differentiation into osteogenic, chondrogenic, dentinogenic, myogenic, neurogenic, and adipogenic lineages. Not surprisingly, DPSCs-based stem cell therapy approaches are currently being exhaustively investigated.
However, it is noteworthy that dental pulp has the element of heterogeneity involved as it is a mixture of different cell types that can differentiate into multiple lineages. Of note, first, dental pulp-derived single-cell suspensions need to be cultured to allow the development of individual clones. Next, individual clones are isolated and single cell types are enriched by extended culture expansion, followed by immunophenotyping to characterize the subpopulations based on molecular and phenotypic markers that regulate their differentiation potential into multiple lineages using either 2-or 3-dimension culture conditions in defined media which promote the desired cell lineage specification (Al Madhoun et al., 2016Madhoun et al., , 2018. Although, fluorescence-activated cell sorting techniques are applicable and improve single-cell population purity, these approaches have several limitations including the exposure of cells to electric charge which may alter their integrity (Tirino et al., 2011). DPSCs' capacities to differentiate into various lineages are driven by donor age, genetics, and epigenetic factors such as growth and differentiation factors and culture settings used . Indeed, further research toward standardization of DPSCs' isolation and culture protocols is still needed. There is also a pressing need for identifying the markers that more specifically and consistently represent DPSCs. Moreover, it is speculated that the identification of such markers will facilitate the direct purification techniques which will minimize DPSCs' exposure to culture conditions needed for their prospective medical applications. Regarding cell-banking aspects, new cryopreservation media and optimized methods may have to be established to maintain the viability and immunobiological characteristics of the DPSCs over long term use. Similarly, further studies will also be required to better understand the molecular mechanisms that regulate interactions between DPSCs and various biomaterials. The studies employing transmission electron microscopy may help characterizing different phenotypes of the heterogeneous progenitor cells that populate dental stem cell niches and a rigorous testing of the endothelial-mesenchymal transformation will be required to assess their potential of replenishing these niches.
Last but not least, DPSCs-related cellular or secretome based therapeutic interventions used in pre-clinical and clinical trials have yielded promising outcomes. However, there is a growing need for conducting more clinical trials to further establish the safety and efficacy of the DPSCs-based interventions as a powerful therapeutic tool and to lead development in regenerative medicine. Not surprisingly, major challenges still remain before the DPSCs-based interventions can be translated into clinical application to patients (Yamada et al., 2019). Nevertheless, innovated procedures have been used to develop immortalized DPSCs such as mutant baculovirus-based piggyBac system , and DPSCs transduction with CDK4 R24C (Orimoto et al., 2020), cyclin D1 or telomerase reverse transcriptase (Wilson et al., 2015). Furthermore, CRISPR gene editing technology has been recently applied to study the functional role of genetic variations in patient-derived DPSCs such as the role of TRPV4 polymorphism (c.1855C > T), a gene known to be implicated in metatropic dysplasia disease (Kang et al., 2012;Nonaka et al., 2019). Nonaka et al. (2019) studies revealed that this gain of function mutation is associated with alternations in calcium/NFATc1 signaling pathway, which in turn accelerates chondrogenic and osteogenic differentiation of DPSCs causing the congenital skeletal disease (Han et al., 2020). Taken together, these outstanding approaches highlight the importance of DPSCS as an adult stem cell model for prospective contribution in the future development of treatment strategies for human diseases.

AUTHOR CONTRIBUTIONS
AA, SS, and RA wrote first draft of the manuscript. DH prepared the figures and tables. DH, MA, and FA-M were involved in discussing, drafting, and editing the manuscript. All authors contributed to the drafting and critical review of the manuscript and approved the final draft.