Stem cells biology has become a relevant field for the understanding of tissue regeneration and implementation of regenerative medicine. The ongoing research on stem cells continues to advance the knowledge on the development of living organisms, and also on the processes of how healthy cells replace damaged cells in adult organisms
Moreover, most of the knowledge regarding Mesenchymal Stem Cells (MSCs) comes from bone marrow cells. In this chapter, we will define the nomenclature for mesenchymal stem cells (MSCs), their morphology and immunophenotype, the biological markers used to identify them, the plasticity of these cells in forming different types of cells and tissues, and their high proliferative ability.
We will outline the current understanding of the differences between Bone Marrow Mesenchymal Stem Cells (BM-MSCs), and Dental Mesenchymal Stem Cells (D-MSCs). Since their isolation, D-MSCs properties have been studied for different applications including dental therapy, craniofacial regeneration and also for immunomodulatory capacity, these features will be discussed in the present chapter.
All stem cells, regardless of their source, share general properties. One is that they are unspecialized cells capable of self-renewing through cell division, even after long periods of inactivity. The second property is that, under certain experimental conditions, they can be induced to become a specific type of cell or tissue (multipotency) (Dominici et al. 2006; Sedgley and Botero 2012).
The essential property of stem cells for self-renewal, i.e., their ability to go through numerous cycles of cell division while maintaining an undifferentiated state represents opportunities for the treatment of diseases such as diabetes and heart diseases (Tatullo et al. 2017; Sedgley and Botero 2012).
Despite the general recognition of stem cell properties, the definition of their characteristics is inconsistent among investigators (Dominici et al. 2006). Because, many laboratories have developed different methodologies to isolate and expand MSCs, which results in different findings.
In this regard, the International Society for Cellular Therapy (ISCT) has stated that “multipotent mesenchymal stromal cells (MSCs) is the currently recommended designation for plastic-adherent cells isolated from bone marrow and other tissues that have often been labeled mesenchymal stem cell” (Zuk et al. 2001; Dominici et al. 2006).
Also, in 2006, the ISCT proposed the minimum characterization criteria for human MSCs. Other than their propensity for adherence to plastic when maintained under standard culture conditions, MSCs must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro differentiating conditions (Yildirim et al. 2016).
Additionally, ≥ 95% of the MSCs population must positively express a cluster of differentiation superficial antigenic markers of hematopoietic differentiation called CDs (for cluster of differentiation) such as CD105 (endoglin), CD73 (ecto-5’-nuclease), and CD90 (Thy1), as measured by flow cytometry. At the same time, these cells must lack expression (52% positive) of CD45, CD34, CD14 or CD11b, C79α or CD19 and HLA-DR (Dominici et al. 2006).
Stem cells can either be embryonic or adult (postnatal); however, a significant challenge in regenerative cell therapy is to find the most appropriate cell source (Sedgley and Botero 2012; Mason et al. 2014).
The discovery and characterization of BM-MSCs, require the search for more accessible MSCs, as a consequence, this has driven interest in dental tissues, which are rich sources of stem cells (Sedgley and Botero 2012).
The present chapter summarizes the properties of different stem cells from dental sources, called D-MSCs populations, such as defining the cluster of differentiation marker of each population of D-MSCs, in vitro multipotency, population doubling and immunomodulatory properties. The D-MSCs were also grouped according to their position in the tooth/dental tissue.
Moreover some ethical questions raised by recent advances in biological research regarding D-MSCs will included here.
Dental Mesenchymal Stem Cells (D-MSCs)
The discovery and characterization of BM-MSCs led to the characterization of different MSCs populations from other tissues, all based on the standard criteria for BM-MSCs (Liu et al. 2015).
Sources of MSCs, other than BM-MSCs, include a variety of tissues, such as umbilical cord blood, synovium, liver, adipose tissue, lungs, amniotic fluid, tendons, placenta, skin and breast milk (Fig. 6.1) (Liu et al. 2015; Miura et al. 2003).
As for the maxillocraniofacial complex, the search for MSC-like cells led to the discovery of a unique population of MSCs from human dental tissues (Liu et al. 2015). Dental Mesenchymal Stem Cells (D-MSCs) have a variety of clinical benefits, and are expected to be applied in different areas of regenerative medicine.
Next the different sources of D-MSCs and their characteristics will be described, based on their position in the tooth/dental tissue.
Dental Pulp Stem Cells (DPSC)
At the beginning of 2000, several human dental stem/progenitor cells were isolated and characterized. The D-MSCs populations come from a variety of dental tissues; however, they share common characteristics, such as the capacity for self-renewal and the ability to differentiate into at least three distinct lineages (Fig. 6.1) (Sedgley and Botero 2012). Table 6.1 summarizes surface markers that have been identified in D-MSCs.
In the last few decades, several studies have reported that human tooth germs contain multipotent cells that give rise to dental and periodontal tissues (Yalvac et al. 2010). The dental pulp, particularly from third molars, have shown to be a significant stem cell source (Arthur et al. 2009).
Dental Pulp Stem Cells (DPSC) were first isolated from human permanent third molars in 2000, and the cell population was clonogenic and highly proliferative (Gronthos et al. 2000; Gronthos et al. 2002).
The cell population isolated by enzymatic digestion from dental pulp tissue was found to express several surface markers, such as CD73, CD90, and CD 105, but not CD 14, CD34, or CD45 (Liu et al. 2015). Table 6.2 summarizes the surface markers found in the D-MSCs discussed here.
Later on, studies made by Gronthos et al. confirmed that MSCs derived from dental pulp fulfilled the criteria needed for stem cells, including their ability to differentiate into adipocytes, neural cells and odontoblasts; together with their ability of self-renewal capabilities (Gronthos et al. 2002).
Besides, DPSCs have been reported to differentiate into osteoblasts, chondrocytes and myoblast-like cells, and demonstrate axon guidance (Arthur et al. 2009). Fast population doubling time, immunosuppressive properties and the ability to form a dentin-pulp-like complex have also been reported for DPSC (Liu et al. 2015).
Also, our research group has isolated and characterized DPSCs from third molars. The cells can differentiate into adipogenic, osteogenic and chondrogenic lineages, and were found to express surface markers, such as CD150, CD90, CD4 and HLA-ABC. This is in concordance with the minimum criteria to be considered and classified as mesenchymal stem cells (Fig. 6.2).
CD105 Endoglin, TGFB receptor; CD90 Thy-1, T cells activator; CD73 Ecto-5 nucleotidase hydrolyses AMP to adenosine and phosphate; CD45 lymphocyte common antigen, lymphocyte signaling; CD34 gp 105-120, transmembrane phosphoglycoprotcin; CDR PECAM-1, leukocyte transcndothelia1 migration; HLA-ABC major histocompatibility antigen, L-selectin; HLA-DR major histocompatibility antigen, VCAM-1.
Stem Cells from Human Exfoliated Deciduous Teeth (SHED)
Tooth development is a well-orchestrated process, which includes shedding of the deciduous teeth, followed by the eruption of permanent teeth. The transition from deciduous teeth to permanent adult teeth is a dynamic process, that coordinates development and eruption of permanent teeth with the resorption of the deciduous teeth roots (Liu et al. 2015).
Researchers took advantage of such a process isolating a distinct population of multipotent stem cells from the remnant pulp of exfoliated deciduous teeth and expanding them ex vivo; unexpectedly providing a unique and accessible tissue source of MSCs (Miura et al. 2003).
Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) are highly proliferative stem cells isolated from exfoliated deciduous teeth, and are capable of differentiating into a variety of cell types under defined culture conditions, including osteoblasts, odontoblast, adipocytes and neural cells (Morsczeck et al. 2005; Miura et al. 2003). It has also been demonstrated that SHED can induce dentin and bone formation (Morsczeck et al. 2005).
Furthermore, SHEDs express a variety of neural cell markers, such as Nestin and GFAP; and can form sphere-like clusters, and multicytoplasmic processes when cultured under neurogenic conditions (Miura et al. 2003; Sedgley and Botero 2012).
SHEDs have a higher proliferation rate than DPSCs and BM-MSCs, suggesting that they represent a more immature population of multipotent stem cells (Morsczeck et al. 2005). As for their gene expression profile, SHEDs are also different from DPSCs and BM-MSCs. Genes related to cell proliferation and extracellular matrix formation such as, Transforming Growth Factor (TGF-β), Fibroblast Growth Factor (FGF2), TGF-β2, collagen type I (Col I), and Col III, show increased expression in SHEDs compared to DPSCs (Sedgley and Botero 2012).
The continuously accumulating evidence suggests that SHEDs from exfoliated deciduous teeth might be an excellent source for stem cell-based therapies, including autologous stem cell transplantation and tissue engineering (Miura et al. 2003).
Periodontal Ligament Stem Cells (PDLSC)
The periodontal ligament (PDL) is a soft connective tissue embedded between the cementum and the alveolar bone socket. Early evidence showed that PDL not only played a vital role in supporting teeth, but it also contributed to tooth nutrition, homeostasis and the regeneration of periodontal tissue (Liu et al. 2015).
McCulloch reported the presence of progenitor/stem cells in the periodontal ligament of mice in 1985. Subsequently, the isolation and identification of multipotent MSCs in human periodontal ligaments were first reported in 2004 (Seo et al. 2004).
In this regard, explants cultures or enzymatic digestion treatment of the PDL made it possible to obtain a population of PDLSCs; and the postnatal multipotent stem cells can be expanded in vitro to generate a cementum/PDL-like complex (Liu et al. 2015).
In their study, Seo et al. (Seo et al. 2004) demonstrated that PDLSCs were similar to other MSCs regarding their expression of STRO-1/CD146, and suggested that PDLSCs might also be derived from a population of perivascular cells. Moreover, later works showed that PDLSCs’ differentiation could be promoted by Hertwig’s epithelial root sheath cells in vitro (Sonoyama et al. 2007). Besides, the lineages of differentiation for PDLSCs are cementoblast-like cells, adipocytes and fibroblasts that secrete collagen type I (Sedgley and Botero 2012).
Similar to BM-MSCs, PDLSCs can undergo osteogenic, adipogenic and chondrogenic differentiation (Sedgley and Botero 2012; Sonoyama et al. 2007).
PDLSCs, like DPSCs, also show a higher number of populations doubling than those of BM-MSCs; however, the mechanisms contributing to the long lifespan of PDLSCs and DPSCs is still unclear (Seo et al. 2004).
We have been able to isolate and characterize PDLSCs, the cells can differentiate into adipogenic, osteogenic and chondrogenic lineages; and expressed surface markers, such as, CD150, CD90, CD4, and HLA-ABC in concordance with the minimum criteria to be considered and classified as MSCs (Fig. 6.2).
Dental Follicle Stem Cells (DFPCs)
Tooth development represents a unique and temporal series of events; in which the dental follicle is an ectomesenchymal tissue that surrounds the developing tooth germ preceding its eruption. This tissue is thought to contain stem cells, and lineage-committed progenitor cells form cementoblasts, periodontal ligament cells and osteoblasts (Morsczeck et al. 2005).
MSCs can be isolated from the dental follicle of human third molars (Liu et al. 2015). Like other dental stem cells, Dental Follicle Stem Cells (DFPCs) have an extensive proliferative potential, express similar cell surface antigens, and are capable of forming hard tissue both in vitro and in vivo. They also express the recognized stem cell markers Notch-1 and Nestin and form the tissue of the periodontium, including alveolar bone, PDL and cementum (Morsczeck et al. 2005).
At the early stage of tooth development, a condense ectomensenchyme progenitor cells limiting the dental papilla and encapsulating the enamel organ is formed, that is, the dental follicle or sac (Nanci 2008). Dental follicles are loose vascular connective tissues that surround the developing tooth germ, and progenitors for periodontal ligament cells, cementoblasts and osteoblasts.
The first isolation of DFPCs was from the dental follicle of human third molars (Morsczeck et al. 2005). DFPCs come from developing tissues; and therefore, it was considered that they might exhibit greater plasticity than other DSCs. In this regard, different cloned DFPC lines have demonstrated considerable heterogeneity (Sedgley and Botero 2012; Botelho et al. 2017; Steimberg et al. 2018).
Although DFPCs and SHED cells can differentiate into neural cells, reports are inconsistent even with similar culture conditions in these cells (Morsczeck et al. 2005).
Alveolar Bone Mesenchymal Stem Cells (AB-MSC)
The alveolar bone is derived from the dental follicle and includes a condensed edge containing the tooth sockets in the bones that hold teeth Recently, the isolation and culture of human AB-MSCs was reported (Liu et al. 2015). The isolated cells exhibited the morphology of spindle-shaped fibroblast-like, accompanied by adherence to plastic plates and colony formation. These cells express the surface markers CD73, CD90, CD105 and STRO-1; however, they showed negative expression of hematopoietic markers, such as CD14, CD34 and CD45 (Liu et al. 2015).
Stem Cells from the Dental Apical Papilla (SCAP)
During tooth development, root formation begins with the apical proliferation of epithelial cells from the cervical loop. Then, proliferating epithelial cells give shape to the apical papilla, a soft tissue found at the apices of developing permanent teeth. The dental papilla contributes to tooth formation and is eventually converted into pulp tissue (Liu et al. 2015; Sonoyama et al. 2006).
A distinctive population of MSCs referred to as Stem Cells from the dental Apical Papilla (SCAPs) was isolated from the apical papilla of human immature permanent teeth SCAPs showed a higher proliferation rate and mineralization potential than DPSCs; they express typical MSCs markers, including STRO-1, CD73, CD90 and CD105. Since SCAPs represent a population of cells from a developing tissue, they might exhibit greater plasticity than other dental stem cells (Sonoyama et al. 2006).
SCAPs are found in the apical papilla, at the junction of the apical papilla and the dental pulp. Extracted human third molar and their apical papillae were the primary sources of SCAPs. These cell populations are clonogenic and can differentiate to odontoblastic/osteogenic, adipogenic or neurogenic lineage. Compared with DPSCs, SCAPs show higher proliferation rates; interestingly, differentiation of SCAPs decreases CD24 marker expression, while alkaline phosphatase expression increases (Sedgley and Botero 2012; Sonoyama et al. 2006).
Tooth Germ Progenitor Cells (TGPCs)
Tooth Germ Progenitor Cells (TGPCs) are a stem cell population identified in the dental mesenchyme of the third molar tooth germ during the late bell stage. TGPCs can be expanded in plastic culture plates and maintained for approximately 60 populations doublings, during which they retain their spindle-shaped morphology and high proliferation rate. TGPCs express the MSCs associated markers STRO-1 and CD73, CD90, CD105 and CD166, but are negative for CD34, CD45 and CD133 (Yalvac et al. 2010).
TGPCs also demonstrate a tendency for pluripotency-associated gene expression, such as Nanog, Oct4, Sox2, Klf4 and C-myc; indicating mesenchymal phenotype (Liu et al. 2015; Yalvac et al. 2010).
Gingiva-derived Mesenchymal Stem Cells (G-MSC)
The human gingiva is an oral tissue overlaying the alveolar ridge and retromolar region that is recognized as a biological mucosa barrier and a distinct component of the oral mucosa immunity (Wang et al. 2011).
The gingival tissue can often be obtained as a discarded biological sample. Recently, G-MSCs were isolated from human gingiva; the cells exhibited clonogenicity, self-renewal and multipotent differentiation capacity. These cells also possess both stem cell-like and immunomodulatory properties and display positive signals for Oct4, Sox2, Nanog, Nestin, SSEA-4 and Stro-1 (Liu et al. 2015; Wang et al. 2011).
Our research group has successfully isolated and characterized cells of the dental pulp, periodontal ligament and gingival tissue from primary cultures by explant. In all the lines, a high rate of proliferation and differentiation has been observed, as well as positive and negative percentages of the surface markers (Fig. 6.2).