Stem cells isolated from human exfoliated deciduous teeth (SHEDs) are a type of mesenchymal stem cells (MSCs), widely investigated for regenerative treatment. They are isolated from dental pulp tissues remaining in physiologically shedding human deciduous teeth. Thus, SHEDs are easy to access and not required invasive procedure to obtain cells. SHEDs are multipotent mesenchymal stem cells; however, they possess distinct properties when compared to other MSCs. In this regard, SHEDs exhibit higher proliferative rate than bone marrow‐derived MSCs and greater osteogenic differentiation potency than human dental pulp stem cells. This chapter reviews the isolation technique and basic characteristics of SHEDs. Moreover, the intracellular signalling involved in the stemness regulation and differentiation ability of SHEDs is discussed, particularly on fibroblast growth factor, Notch, and Wnt signalling. Finally, the potential regenerative therapeutic application of SHEDs is also described.
- stem cells
- deciduous teeth
- basic fibroblast growth factor
- Wnt signalling
- Notch signalling
- mechanical stress
Dental pulp is a loose connective tissue residing in pulp chamber inside both deciduous and permanent teeth. It surrounds by hard tissues called dentin. Nutrients and oxygen supply are acquired from blood vessels passing through apical and accessory foramen of the teeth’s root. Dental pulp originates from cranial neural crest cells . Dental pulp tissues are composed of extracellular matrix and various cell types, e.g. fibroblasts, odontoblasts, endothelial cells, pericytes, immune cells and stem cells. When injured, cells in dental pulp tissues are capable of differentiating odontoblasts or odontoblast‐like cells, leading to the promotion of tertiary dentin formation. The formation of tertiary dentin is a mechanism which can protect the tooth vitality. Dental pulp tissues remaining in physiological shedding of deciduous teeth are the alternative source of mesenchymal stem cells, due to the ease of accessibility and minimally invasive technique to obtain tissues . Stem cells from human exfoliated deciduous teeth (SHEDs) are firstly identified by Miura et al. in 2003 . SHEDs have high proliferation potency and are multipotent mesenchymal stem cells. These cells are able to differentiate into, not only, dental pulp‐related cells, but also, other cell lineages, for example osteoblasts, adipocytes, neuronal‐like cells and endothelial cells [2–8]. Taking these advantageous properties together, SHEDs are one of the candidate cell types for tissue regeneration study.
2. SHEDs’ characteristics
SHEDs are heterogeneous population of cells isolated from dental pulp tissues remained in exfoliated deciduous teeth. Similar to those mesenchymal stem cells (MSCs), SHEDs exhibit fibroblast‐like morphology, adhere on plastic tissue culture surface, express mesenchymal stem cell surface marker and have multipotential differentiation ability (Figure 1). SHEDs have higher proliferation rate compared to dental pulp stem cells (DPSCs) and bone marrow‐derived mesenchymal stem cells (BMMSCs) [2, 9]. This could be due to the high expression of genes related to cell proliferation and extracellular matrix in SHEDs comparing with DPSCs . First, a study by Miura et al. demonstrated that SHEDs express mesenchymal surface markers, STRO‐1 and CD146 , though, the percentage of positive cells is low . Later studies utilized various surface markers for SHEDs characterization protocol. SHEDs expressed CD44, CD73, CD90, CD105 and STRO‐1 . In addition, these cells lack of CD45 expression . Besides these markers described above, SHEDs also express other surface markers for example, CD166 and SSEA4. Lack of CD34 is also reported . There is no specific surface marker to precisely identify SHEDs population.
Up to date, MSCs can be isolated from many tissue types. Though, there is no specific marker to clearly identify these cells. According to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, the minimum criteria to identify MSCs are as follow . First, the isolated MSCs should adhere to plastic tissue culture plate . Second, MSCs must express several specific surface markers, namely CD105, CD73 and CD90 . They also should not express CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA‐DR . Finally, MSCs have to be able to differentiate into osteoblasts, adipocytes and chondroblasts
2.1. Isolation technique
Two methods have been utilized for SHEDs isolation, namely an enzymatic digestion and a tissue explant. The enzymatic digestion is performed by digesting minced remaining pulp tissues from deciduous teeth, normally with type I collagenase and dispase mixed enzyme solution [12–14]. For tissue explant, minced pulp tissues are placed on the tissue culture dishes, allowing the outgrowth of the cells from the tissues . Enzymatic digestion technique leads to more heterogeneous population of isolated cells than those obtained from tissue outgrowth protocol . A study illustrated that there is no significant difference regarding cell morphology and proliferation between cells isolated using enzymatic digestion and tissue outgrowth . Enzymatic digestion‐derived SHEDs had higher mineralization ability
2.2. Differentiation potential of SHEDs
Studies have shown that SHEDs possess multi‐differentiation potency similar to MSCs. Those lineages include odontogenic/osteoblastic, adipogenic, neurogenic and angiogenic differentiation .
2.2.1. Odontogenic/osteoblastic differentiation potential
The ability of SHEDs to differentiate into odontoblastic lineage is widely known [2, 15, 16]. Primitively, SHEDs were characterized by their
Evidence suggested that SHEDs might have the preference towards the odontoblastic lineage due to its origin. SHEDs can be induced to become functional odontoblasts
2.2.2. Neurogenic differentiation potential
Neurogenic potential of SHEDs is expecting due to their neural crest embryonic origin. Several research studies focusing on differentiating dental stem cells to be used for neurodegenerative disease therapy. These cells are prone to undergo neurogenic differentiation both
2.2.3. Angiogenic differentiation potential
Angiogenic potential of SHEDs is another aspect of interest for the benefit of connective tissue regeneration. The rapid and effective induction of vasculation is required for sufficiently supply of oxygen and nutrients as well as removing the toxic waste from the newly synthesized tissues. Unstimulated SHEDs expressed VEGFR1 and NP‐1, the known important receptors in angiogenesis and VEGFR1 signalling play an important role in VEGF‐induced capillary tube formation by SHEDs as shown by VEGFR1 gene silencing . SHEDs cultured in the tooth slice/scaffolds in combine with VEGF expressed several endothelial differentiation markers such as VEGFR1, VEGFR2, platelet endothelial cell adhesion molecule‐1 (PECAM‐1) and vascular endothelial cadherin (VE‐Cadherin). When transplanted in immunodeficient mice, SHEDs actually lined the new blood vessels within the tooth slice/scaffolds close to the blood vessels of host . Similar results were observed when SHEDs seeded in human tooth slice/scaffolds and transplanted into immunodeficient mice differentiate into human blood vessels that anastomosed with the mouse vasculature and VEGF induced the angiogenic differentiation of SHEDs through Wnt/β‐catenin signalling . Another study also showed that SHEDs can differentiate into VEGFR2‐positive and CD31‐positive endothelial cells
2.2.4. Adipogenic differentiation potential
Several studies have reported that SHEDs can be induced into adipogenic lineage [6, 32–34]. After cultured in an adipogenic medium, SHEDs’ morphology changed from spindle‐like to polygonal shapes and lipid vacuoles were observed, along with the increased in PPARγ2 and LPL mRNA . However, the studies evaluated the adipogenic potential of SHEDs
2.3. Immunomodulatory property
Like other MSCs, SHEDs exhibit immunomodulatory properties. Though, the potency and mechanism are not exact the same to those of BMMSCs [10, 35]. SHEDs significantly reduced the percentage of IL17+IFNγ cells population in CD4+ T cells
3. Basic fibroblast growth factor signalling in SHEDs
Basic fibroblast growth factor (bFGF) is a member in fibroblast growth factor family . It binds to fibroblast growth factor receptors (FGFR) and further initiates intracellular signalling . bFGF has been shown to participate in the regulation of stemness maintenance and cellular differentiation. In human DPSCs, bFGF promotes pluripotent stem cell marker expression, corresponding with the increase of colony‐forming unit . Furthermore, bFGF inhibits osteogenic differentiation by SHEDs, human DPSCs and human periodontal ligament stem cells (PDLSCs) when supplemented in osteogenic induction medium (Figure 2) [5, 40]. In this regard, alkaline phosphatase enzymatic activity and mineralization are markedly decreased under bFGF‐treated condition compared with the control [5, 40]. On the contrary, bFGF enhances the expression of neurogenic marker, βIII‐tubulin, via FGFR and PLCγ when human DPSCs are cultured in a neurogenic induction medium supplemented with bFGF .
In SHEDs, long‐term culture
Regarding osteogenic differentiation, bFGF attenuated osteogenic differentiation. In this regard, bFGF attenuated alkaline phosphatase enzymatic activity and mineralization in SHEDs after osteogenic induction [5, 43]. The inhibition of endogenous bFGF in SHEDs either by a chemical inhibitor for FGFR or lentiviral shRNA against bFGF resulted in the enhancement of osteogenic differentiation . It was also demonstrated that bFGF attenuated alkaline phosphatase mRNA expression and mineral deposition via FGFR and MEK signalling pathway .
Several possible mechanisms were reported. Firstly, bFGF might attenuate osteogenic differentiation in SHEDs via decreasing Notch signalling . Notch signalling activation led to the enhancement of mineralization in SHEDs . Treatment with bFGF attenuated Notch receptor, ligand and target gene expression which may participate in bFGF attenuated osteogenic differentiation in SHEDs . Secondly, bFGF inhibited matrix metalloproteinase (MMP) expression, for example
4. Wnt signalling in SHEDs
Canonical Wnt signalling also has a significant role in tooth development and stem cells self‐renewal through β‐catenin [46, 47]. Inactivation of β‐catenin in the mesenchyme of developing tooth results in arrested tooth developmental at the bud stage . Various studies established the influence of canonical Wnt signalling pathway to promote the osteogenic differentiation of dental stem cells, i.e. DPSCs, PDLSCs, stem cells from apical papilla (SCAPs) and dental follicle stem cells (DFSCs) [49–52]. However, the effect of the canonical Wnt/ β‐catenin on SHEDs is very limited. The involvement of Wnt/β‐catenin on SHEDs‐mediated mineralized tissue regeneration was investigated with the addition of basic fibroblast growth factor (bFGF) . Treatment with bFGF attenuated SHEDs‐mediated mineralized tissue regeneration via activation of ERK 1/2 pathway and consequently inhibited Wnt/β‐catenin pathway, leading to osteogenic deficiency of SHEDs .
Activation of β‐catenin by LiCl in SHEDs led to the significant decrease of colony formation by SHEDs . In addition, LiCl enhanced subG0 population in SHEDs .
5. Notch signalling in SHEDs
Notch signalling controls various function of stem cells, ranging from stemness maintenance to cell‐specific differentiation . It is a highly conserved pathway, firstly identified in Drosophila. Notch signalling is initiated by the binding between membrane‐bound Notch receptors and ligands of neighbouring cells [56–58]. Further, Notch receptors are cleaved by a γ‐secretase enzyme, leading to the release of Notch intracellular domain (NICD) [56–58]. Subsequently, NICD translocates into nucleus and forms complex with other transcriptional molecules, resulting in the activation of Notch target genes [56–58]. Common Notch signalling target genes are Hes and Hey families [56–58]. In the canonical Notch signalling pathway, four receptors and five ligands are identified [56–58]. The four types of Notch receptors are Notch1, Notch2, Notch3 and Notch4. Five ligands are Delta‐like‐1 (Dll‐1), Delta‐like‐3 (Dll‐3), Delta‐like‐4 (Dll‐4), Jagged1 and Jagged2 [56–58].
Notch signalling participates in odontogenesis, dental pulp repair and regeneration. Mice lacking of Jagged2 expression exhibited defective enamel formation of incisors and malformation of molars . The expression of Notch receptors and ligands was upregulated in response to calcium hydroxide, a material for direct pulp capping treatment . Human DPSCs over‐expressing Jagged1 exhibited the reduction of osteogenic differentiation ability and mineralization
Studies illustrated that indirectly immobilized Notch ligands, Jagged1 or Dll‐1, on tissue culture surface increased
It has been shown that bFGF inhibited the mRNA expression of Notch signalling components. In this regard, bFGF significantly reduced the mRNA levels of
6. Mechanical stress influences SHEDs’ behaviours
Dental pulp tissues are surrounded by hard tissues, namely dentin. During inflammation, an interstitial fluid pressure increases [65, 66], causing biological changes in local cells and tissues. In addition, fluid movement in dentin‐pulp complex during normal occlusal force may expose cells to mechanical stimuli . Mechanical forces are shown to regulate biological functions in many cell types, for example osteoblasts, osteocytes, periodontal ligament cells and dental pulp cells. Different types and magnitude of force lead to different cell responses. In human DPSCs, uniaxial cycle stretching inhibited odonto/osteogenic differentiation but increased cell proliferation [68, 69], while cyclic hydrostatic pressure synergistically enhanced BMP‐2‐induced DSPP expression by human DPSCs
7. Potential application of SHEDs in regenerative therapy
SHEDs are the good candidate for the stem cells used in regenerative therapy due to their high plasticity as well as ability to cross lineage boundaries and differentiate into several specialized cells. Current progresses have been made for tissue engineering‐based therapies involving a large number of tissues. However, dentin-pulp complex and neuronal tissue seem to be the most promising aspects for the application of SHEDs in regenerative therapy.
The first evidence to show that SHEDs can differentiate to become the functional odontoblasts with the ability to generate the mineralized tissue resemble to dentin was shown in mice . SHEDs were seeded within a scaffold in a tooth slice and implanted into the dorsum of mice. Dental pulp‐like tissue was observed in the central area of the pulp chamber of the tooth slice . The expression of odontoblastic differentiation markers such as DSPP and DMP‐1 was detected . Remarkably, the newly deposited dentin was observed and suggested that SHEDs can differentiate into fully functional odontoblasts
In addition to dentin-pulp complex regeneration, SHEDs also show the potential to be used in neuroregeneration. Stem cell therapy is the promising therapeutic options for treating the neurodegenerative diseases due to the limited regenerative capacity of the specialized cells in the nervous system. The neural crest cell in origin makes SHEDs the candidate cell model for neuron tissue regeneration. These cells are prone to undergo neurogenic differentiation both
In a focal cerebral ischemia rat model induced by permanent middle cerebral artery occlusion, intranasal administration of supernatants from the medium used to culture SHEDs significant decreased in the motor disability score and significantly reduced in the infarct volume . Moreover, positive signals for neuronal nucleus, neurofilament H, doublecortin and rat endothelial cell antigen in the peri‐infarct area were observed in the rats treated with SHEDs conditioned media compared to the DMEM control from approximately 140 mm3 in DMEM control to 50 mm3 in SHEDs conditioned medium . These results suggest that SHEDs might secrete some compounds that positively influence the recovery of the brain lesion in focal cerebral ischemia .
Studies have shown that SHEDs have remarkable neuroregenerative activity and promote functional recovery in a spinal cord injury animal model [29, 75]. Rats that received SHEDs transplantation within the lesion created at the 9th–11th thoracic vertebral levels exhibited higher scores in the locomotor rating scale compared to the bone marrow stromal cells or fibroblasts transplantation control . In addition, the rescue of hindlimb locomotor function was prominent in the rats that received SHEDs. These animals were able to move hindlimb coordinately and walk, while the bone marrow stromal cells transplantation exhibited only subtle movements . A similar trend was observed in another study, a complete recovery of hindlimb motor function was observed after implantation of neural‐induced SHEDs in a rat spinal cord injury  which suggested that preinduction of the undifferentiated SHEDs into the neural‐like cells before implantation might improve the efficiency of SHEDs in regenerating specialized neural cells. Taken together, these high neurogenic potential of SHEDs especially in animal models makes them the favourable source for stem cell regeneration treatment for neural diseases.
Dental stem cells, including SHEDs, have been extensively studied in the past decades leading to the better understanding in their unique biological properties and therapeutic potential. As SHEDs can be easily obtained with limited ethical concern, their multi‐differentiation potentials have been demonstrated, which creates great opportunities for the application in the regenerative therapy. However, despite the intriguing results, we still need further study to deepen the understanding of the mechanisms underlying the differentiation processes to attain clinical reality. Also, the potential risks for the clinically use of SHEDs or other dental stem cells should be thoroughly studied for the safety of the patients who will greatly benefit from their regenerative ability.
The authors thank for support of the Faculty of Dentistry Research Fund, Chulalongkorn University. We would like to thank Dr. Pattarin Potisomporn for the illustration in Figure 4.
|Akt||Protein kinase B|
|bFGF||Basic fibroblast growth factor|
|BMMSCs||Bone marrow‐derived mesenchymal stem cells|
|CD||Cluster of differentiation|
|COL1||Collagen type 1|
|DAPT||N‐[N‐(3,5‐Difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester|
|DFSCs||Dental follicle stem cells|
|DMEM||Dulbecco’s Modified Eagle Medium|
|DMP‐1||Dentin matrix acidic phosphoprotein 1|
|DMP||Dentin matrix protein|
|DPSCs||Dental pulp stem cells|
|ERK||Extracellular signal‐regulated kinase|
|FGFR||Fibroblast growth factor receptor|
|GFAP||Glial fibrillary acidic protein|
|Hes||Hairy and enhancer of split|
|Hey||Hairy and enhancer of split related with YRPW motif protein|
|HGF||Hepatocyte growth factor|
|HLA‐DR||Human leukocyte antigen‐antigen D related|
|LEF‐1||Lymphoid enhancer binding factor 1|
|MEK||Mitogen‐activated protein kinase kinase|
|MEPE||Matrix extracellular phosphoglycoprotein|
|MSCs||Mesenchymal stem cells|
|MSX2||Msh homeobox 2|
|MT1‐MMP||Membrane type1‐ matrix metalloproteinase|
|NICD||Notch intracellular domain|
|Nurr1||Nuclear receptor related 1 protein|
|OCT4||Octamer‐binding transcription factor 4|
|P2Y1||Purinergic receptor P2Y1|
|PCR||Polymerase chain reaction|
|PDLSCs||Periodontal ligament stem cells|
|PECAM‐1||Platelet endothelial cell adhesion molecule 1|
|Pitx3||Paired like homeodomain 3|
|PLCγ||Phospholipase C gamma|
|PPARγ2||Peroxisome proliferator‐activated receptor‐gamma 2|
|REX1||Reduced Expression Protein 1|
|RUNX2||Runt‐related transcription factor 2|
|SCAPs||Stem cells from apical papilla|
|SHEDs||Stem cells isolated from human exfoliated deciduous teeth|
|shRNA||Short hairpin ribonucleic acid|
|SOX2||Sex determining region Y‐box 2|
|SSEA4||Stage‐specific embryonic antigen‐4|
|TERT||Telomerase reverse transcriptase|
|TWIST||Twist Family BHLH Transcription Factor|
|VE‐Cadherin||Vascular endothelial cadherin|
|VEGF||Vascular endothelial growth factor|
|VEGFR||Vascular endothelial growth factor receptor|