**2. Dental-related stem cell biology: From the classification to the characterization & differentiation potential**

According to the literature, there are several types of stem/progenitor cells existed in dental tissue. In this section, each of these cells will be described in terms of their main characteristics.

### **2.1. Dental Pulp Stem Cells (DPSCs)**

The presence of stem cells in dental pulp tissue primarily have been reported in 1985 by Yamamura [3, 4, 6] (Figure 1). Later on, Caplan et al. have demonstrated that these cells presented osteogenic and chondrogenic potential in vitro, and could also differentiate into dentin, in vivo. In 2000, Gronthos et al. have isolated dental pulp stem cells from adult human dental pulp, which had the ability to regenerate a dentin-pulp-like complex [7]. Interestingly, some recent works have found the presence of stem cells in inflamed pulp with capacity to form mineralized matrix both in vitro and in vivo. These findings make dental pulp as an interesting tissue source of putative stem cells, even in diseased form.[8]

DPSCs are similar to MSCs in some ways: they are of fibroblastic morphology with selective adherence to solid surfaces, having good proliferative potential and capacity to differentiate in vitro, and the ability to repair tissues in vivo. It's interesting to note that DPSCs could differentiate into not only osteoblasts, chondrocytes & adipocytes, but also myocytes, neurons and hepatocytes lineages in vitro [4]. DPSCs are characterized by their negative expression of hematopoietic antigens (*e.g.*, CD45, CD34, CD14), and positive expression of stromal-associ‐ ated markers (*e.g.*, CD90, CD29, CD73, CD105, CD44) (Table 1). They also express multipotent marker (STRO-1) and extracellular matrix proteins, such as collagen, vimentin, laminin, and fibronectin. [9-11]. Interestingly, some of the pluripotent stem cell markers, such as Oct4, Nanog, Sox2, Klf4, SSEA4 & c-Myc have been reported to express on DPSCs [12-14]. More recently, it was demonstrated that core transcription factor of the reprogramming Oct4, Nanog, Klf4 and c-Myc become significantly down-regulated following the DPSC differentiation [4].

Apart from stemness markers, DPSCs are also shown to express bone markers, such as bone sialoprotein, osteocalcin, alkaline phosphates (ALP), and type I collagen. This indicates their differentiation commitment into bone tissue [15]. On the other hand, the expression of dentin sialophosphoprotein (an odontoblast specific protein precursor) is not present in the cultures of hDPSCs implied that these cells represent an undifferentiated pre-odontogenic phenotype [7, 16].

From immunological perspective, it has been reported that DPSCs displayed more immnosuppressive activities than the BM-MSCs. This was obvious in inhibiting T cells response in vitro [17].

Based on some investigations, there is a sub-type of DPSCs referred to as "immature dental pulp stems cells" (IDPSCs), which have promising potential in future stem cell researches. IDPSCs were firstly, isolated from pulp tissue of the human exfoliated deciduous as well as permanent teeth [18]. These cells express both embryonic and MSC markers (see part 2.2). It has been indicated that transferring of human IDPSCs (hDPSCs) into mouse blastocysts resulted in formation of human/mouse chimera which was able to retain proliferation and differentiation capacity [19]. Furthermore, hIDPSCs possess the capacity to rapidly reprog‐ rammed into induced pleuripotent stem cells (iPSc) which are able to produce primary hIDPSC-iPSC colonies even under feeder-free conditions [20].

#### **2.2. Dental Stem cells from Human Exfoliated Deciduous teeth (SHED)**

In 2003, Miura et al. have reported to isolate a stem cell population from the living pulp remnants of exfoliated deciduous teeth. These authors have termed the cells as stem cells from human exfoliated deciduous teeth (SHED) [21] (Figure 1). These cells which are believed to be of the neural crest origin are heterogeneous fibroblast-like population possessing an extensive proliferating capacity than either DPSCs or BM-MSCs [22]. In terms of surface epitopes, it has been found that they express markers of MSCs (STRO-1, CD146, SSEA4, CD90, CD73, CD 105, CD106 and CD 166) and lack of hematopoietic/endothelial markers (CD34, CD31) (Table 1). Under an appropriate culture conditions, SHED are able to differentiate into the variety of cell types, including neural cells, angiogenic endothelial cells, adipocytes, osteoblasts, and odontoblasts [23-25]. In vivo transplantation of SHED have been reported to result in formation of bone and dentin like-tissue. [18, 21, 26-29]. There are some studies suggested that SHED is different from IDPSCs in terms of expression of stem cell markers (see part 1.1)[18, 30]. Moreover, some research works have been reported that SHED would possess immunomo‐ dulatory function as seen in BM-MSCs [28].

#### **2.3. Periodontal Ligament Stem Cells (PDLSC)**

This chapter focuses on different aspects of dental-derived adult stem cells, such as their classification, biological characterization, initiating culture, cultivation systems, cryopreser‐ vation and potential applications in tissue engineering and regenerative medicine. The data are organized as three main parts, including: 1) Dental-related stem cell biology: from the classification to the characterization and differentiation potential 2) Dental-related stem cell initiation culture, culture systems and cryopreservation 3) Dental-related stem cell- based

According to the literature, there are several types of stem/progenitor cells existed in dental tissue. In this section, each of these cells will be described in terms of their main characteristics.

The presence of stem cells in dental pulp tissue primarily have been reported in 1985 by Yamamura [3, 4, 6] (Figure 1). Later on, Caplan et al. have demonstrated that these cells presented osteogenic and chondrogenic potential in vitro, and could also differentiate into dentin, in vivo. In 2000, Gronthos et al. have isolated dental pulp stem cells from adult human dental pulp, which had the ability to regenerate a dentin-pulp-like complex [7]. Interestingly, some recent works have found the presence of stem cells in inflamed pulp with capacity to form mineralized matrix both in vitro and in vivo. These findings make dental pulp as an

DPSCs are similar to MSCs in some ways: they are of fibroblastic morphology with selective adherence to solid surfaces, having good proliferative potential and capacity to differentiate in vitro, and the ability to repair tissues in vivo. It's interesting to note that DPSCs could differentiate into not only osteoblasts, chondrocytes & adipocytes, but also myocytes, neurons and hepatocytes lineages in vitro [4]. DPSCs are characterized by their negative expression of hematopoietic antigens (*e.g.*, CD45, CD34, CD14), and positive expression of stromal-associ‐ ated markers (*e.g.*, CD90, CD29, CD73, CD105, CD44) (Table 1). They also express multipotent marker (STRO-1) and extracellular matrix proteins, such as collagen, vimentin, laminin, and fibronectin. [9-11]. Interestingly, some of the pluripotent stem cell markers, such as Oct4, Nanog, Sox2, Klf4, SSEA4 & c-Myc have been reported to express on DPSCs [12-14]. More recently, it was demonstrated that core transcription factor of the reprogramming Oct4, Nanog, Klf4 and c-Myc become significantly down-regulated following the DPSC differentiation [4]. Apart from stemness markers, DPSCs are also shown to express bone markers, such as bone sialoprotein, osteocalcin, alkaline phosphates (ALP), and type I collagen. This indicates their differentiation commitment into bone tissue [15]. On the other hand, the expression of dentin sialophosphoprotein (an odontoblast specific protein precursor) is not present in the cultures of hDPSCs implied that these cells represent an undifferentiated

**2. Dental-related stem cell biology: From the classification to the**

interesting tissue source of putative stem cells, even in diseased form.[8]

**characterization & differentiation potential**

**2.1. Dental Pulp Stem Cells (DPSCs)**

pre-odontogenic phenotype [7, 16].

tissue regeneration.

96 Regenerative Medicine and Tissue Engineering

Periodontal ligament stem cells (PDLSCs) have first been introduced by Seo et al. [31] (Figure 1). Like MSCs, PDLSCs have been reported to form adherent clonogenic population of fibroblast-like cells in the culture. They express both early MSC markers such as, STRO-1 and CD146, and other MSC and pluripotent makers, such as CD44, CD90, CD105, CD73, CD26, CD10, CD29 and CD166; meanwhile, they have no expression for CD40, CD80, and CD86[31-33] (Table 1). Some investigations have revealed that PDLSCs may be positive for embryonic stem cell markers, as well, including SSEA1, SSEA3, SSEA4, TRA-1-60, TRA-1-81, Oct4, Nanog, Sox2 and Rex1, and ALP [34]. Based on some research works, SSEA4-positive PDLSCs displayed the potential to generate adipocytes, osteoblasts, chondrocytes (from mesodermal layer), neurons (from ectodermal layer), and hepatocytes (from endodermal lineage) *in vitro* [31, 34, 35]. Furthermore, it has been shown that transplantation of PDLSCs into immunocompromised rodents resulted in the generation of cementum/PDL-like structure and contributes to periodontal tissue repair [31].

PDLSCs show immunomodulatory activity by up-regulation of soluble immunosuppressive factors (TGF-β1, hepatocyte growth factor (HGF) and indoleamine 2, 3-dioxygenase (IDO) in the presence of activated peripheral blood mononuclear cells (PBMNCs). Similar to the DPSCs, PDLSCs are positive for HLA-ABC (MHC class I antigen) while negative for HLA-DR (MHC class II antigen) [32].

#### **2.4. Dental Follicle Progenitor Cells (DFPCs)**

In 2005 & 2007, Morsczeck et al. and Kémoun et al., respectively have identified unique undifferentiated lineage committed cells possessing mesenchymal progenitor features in the human dental follicle (Figure 1). The cells were referred to as "dental follicle precursor cells" (DFPCs) [36, 37]. Characteristically, DFPCs, similar to the bone marrow stem cells, are adherent and colony-forming cells. These cells have been reported to express Notch-1, CD13, CD44, CD73, CD105, and STRO-1 [1, 36] (Table 1). Human DFPCs has been believed to consist of precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Under appro‐ priate *in vitro* conditions, they are capable of differentiating into osteoblasts, cementoblasts, chondrocytes and adipocytes. Interestingly, although both DFPCs and SHED are of the neural crest origin, their neural differentiation potentials are different under the same culture conditions. It has been reported that SHED possess good differentiation potential than DFPCs in terms of the expression of Pax6 which is a marker of retinal stem cells [27].

#### **2.5. Stem Cells from the Apical Papilla (SCAP)**

Stem cells from dental apical papilla (SCAP) were first identified and characterized by Sonoyama et al. in human permanent immature teeth [38] (Figure 1). These authors described the cells as adherent clonogenic cells with mesenchymal stem cell features, which are expressed STRO-1, CD24, CD29, CD73, CD90, CD105, CD106, CD146, CD166, and ALP, and not express‐ ing CD34; CD45; CD18*;* and CD150. Among these markers, CD24 would be of a specific marker for SCAP since it's not found in the other dental stem cells (Figure 2). Excitingly, some authors have reported that SCAP display higher telomerase expression than both DPSCs and BM-MSCs [38]. Furthermore, SCAP has been shown to positively stain with several neural markers implying their possible origin from the neural crest [39]. In terms of differentiation, SCAP are capable of generating osteoblasts, odontoblasts and adipocytes in vitro. An in vivo study has demonstrated that these cells form hard tissue when being loaded onto hydroxyapatite (HA) and implanted subcutaneously in immunocompromised rats [38-40]. Moreover, SCAP have been reported to possess a significantly higher mineralization potential as well as proliferation rate than DPSCs. This finding might be of some importance for their use in dental and/or bone tissue engineering and regeneration [41].

About the possibility of immunogenicity of SCAPs, an independent study have reported that swine SCAPs are non-immunogenic and suppressed T cells proliferation in vitro [42].

#### **2.6. Stem Cells derived from Gingiva (GSCs)**

into immunocompromised rodents resulted in the generation of cementum/PDL-like structure

PDLSCs show immunomodulatory activity by up-regulation of soluble immunosuppressive factors (TGF-β1, hepatocyte growth factor (HGF) and indoleamine 2, 3-dioxygenase (IDO) in the presence of activated peripheral blood mononuclear cells (PBMNCs). Similar to the DPSCs, PDLSCs are positive for HLA-ABC (MHC class I antigen) while negative for HLA-DR (MHC

In 2005 & 2007, Morsczeck et al. and Kémoun et al., respectively have identified unique undifferentiated lineage committed cells possessing mesenchymal progenitor features in the human dental follicle (Figure 1). The cells were referred to as "dental follicle precursor cells" (DFPCs) [36, 37]. Characteristically, DFPCs, similar to the bone marrow stem cells, are adherent and colony-forming cells. These cells have been reported to express Notch-1, CD13, CD44, CD73, CD105, and STRO-1 [1, 36] (Table 1). Human DFPCs has been believed to consist of precursor cells for cementoblasts, periodontal ligament cells, and osteoblasts. Under appro‐ priate *in vitro* conditions, they are capable of differentiating into osteoblasts, cementoblasts, chondrocytes and adipocytes. Interestingly, although both DFPCs and SHED are of the neural crest origin, their neural differentiation potentials are different under the same culture conditions. It has been reported that SHED possess good differentiation potential than DFPCs

Stem cells from dental apical papilla (SCAP) were first identified and characterized by Sonoyama et al. in human permanent immature teeth [38] (Figure 1). These authors described the cells as adherent clonogenic cells with mesenchymal stem cell features, which are expressed STRO-1, CD24, CD29, CD73, CD90, CD105, CD106, CD146, CD166, and ALP, and not express‐ ing CD34; CD45; CD18*;* and CD150. Among these markers, CD24 would be of a specific marker for SCAP since it's not found in the other dental stem cells (Figure 2). Excitingly, some authors have reported that SCAP display higher telomerase expression than both DPSCs and BM-MSCs [38]. Furthermore, SCAP has been shown to positively stain with several neural markers implying their possible origin from the neural crest [39]. In terms of differentiation, SCAP are capable of generating osteoblasts, odontoblasts and adipocytes in vitro. An in vivo study has demonstrated that these cells form hard tissue when being loaded onto hydroxyapatite (HA) and implanted subcutaneously in immunocompromised rats [38-40]. Moreover, SCAP have been reported to possess a significantly higher mineralization potential as well as proliferation rate than DPSCs. This finding might be of some importance for their use in dental and/or bone

About the possibility of immunogenicity of SCAPs, an independent study have reported that

swine SCAPs are non-immunogenic and suppressed T cells proliferation in vitro [42].

in terms of the expression of Pax6 which is a marker of retinal stem cells [27].

and contributes to periodontal tissue repair [31].

98 Regenerative Medicine and Tissue Engineering

**2.4. Dental Follicle Progenitor Cells (DFPCs)**

**2.5. Stem Cells from the Apical Papilla (SCAP)**

tissue engineering and regeneration [41].

class II antigen) [32].

The isolation of a stem cell population from gingiva was firstly reported by Zhang et al. in 2009 [43] (Figure 1). These authors derived the cells from the spinous layer of human gingiva and referred to them as gingival stem cells (GSCs). In terms of markers, it has been shown that GSCs are negative for CD45/CD34, but positive for CD29, CD44, CD73, CD90, CD105, CD146, STRO-1 and SSEA4 (Table 1). In addition, extracellular matrix proteins, such as collagen, vimentin, Collagen type-1, and fibronectin have been reported to express in these cells [43, 44]. Like MSCs, GSCs possess a differentiation potential into osteoblasts, adipocytes and chondrocytes in vitro [45]. Moreover, these cells have been found to be able to differentiate along endothelial as well as neural cell lineages. Furthermore, in vivo bone regeneration potential of GSCs was demonstrated by transplantation of GSCs/HA into immunocompro‐ mised mice [45]. More importantly, in a comparative study, it was demonstrated that GSCs showed stable phenotypes, maintain normal karyotype and telomerase activity in long-term cultures in comparison with BM-MSC [45].

As with other dental related stem cells, GSCs has been found to display immunomodulatory functions; they inhibit lymphocytes proliferation and express a wide range of immunosup‐ pressive factors, including Interleukin-10 (IL-10), IDO, inducible NO synthase (iNOS), and cyclooxygenase 2 (COX-2) in response to the inflammatory cytokine, IFN- γ [43].

**Figure 1.** Timeline about the highlights in the history of the isolation of dental-related stem cells (see text).


**Table 1.** Cell Surface Marker Profiles of dental-related stem cells. DPSC; Dental pulp stem cell, SHED; Stem cells from human exfoliated deciduous teeth, PDLSC; Periodontal ligament stem cells, DFPC, Dental follicle precursor cells, SCAP; Stem cells from dental apical papilla, GSC; Gingival stem cells. [1-4]
