Neural crest-derived dental stem cells—Where we are and where we are going

Neural crest-derived dental stem cells—Where we are and where we are going

Journal of Dentistry, 2014-09-01, Volume 42, Issue 9, Pages 1043-1051, Copyright © 2014 Elsevier Ltd

Abstract

Objectives

There are five types of post-natal human dental stem cells that have been identified, isolated and characterized. Here, we review the information available on dental stem cells as well as their potential applications in dentistry, regenerative medicine and the development of other therapeutic approaches.

Data

Data pertinent to dental stem cells and their applications, published in peer-reviewed journals from 1982 to 2013 in English were reviewed.

Sources

Sources were retrieved from PubMed databases as well as related references that the electronic search yielded.

Study Selection

Manuscripts describing the origin, retrieval, characterization and application of dental stem cells were obtained and reviewed.

Conclusions

Dental stem cell populations present properties similar to those of mesenchymal stem cells, such as the ability to self-renew and the potential for multilineage differentiation. While they have greater capacity to give rise to odontogenic cells and regenerate dental pulp and periodontal tissue, they have the capacity to differentiate into all three germ line cells, proving that a population of pluripotent stem cells exists in the dental tissues.

Clinical Significance

Dental stem cells have the capacity to differentiate into endoderm, mesoderm and ectoderm tissues. Consequently they do not only have applications in dentistry, but also neurodegenerative and ischemic diseases, diabetes research, bone repair, and other applications in the field of tissue regeneration.

Introduction

There are two kinds of adult dental stem cells identified today: mesenchymal stem cell (MSC)-like cells and epithelial stem cells. This review will focus on the former as the latter have only been discovered in mouse incisors, not in humans yet. Researchers hypothesize that they may be unique to rodents because they have the special characteristic of continuous eruption throughout their lives. Reports of MSC-like cells isolated from different human dental tissues ( Fig. 1 ) began in the year 2000 and research studies about the subject have become more popular with time, with publications about the subject increasing every year. Postnatal dental pulp stem cells (DPSCs) were the first population of dental-tissue derived stem cells to be identified. These cells were isolated by Gronthos et al. in 2000 from human dental pulp. In 2003, Miura et al. discovered stem cells from human exfoliated deciduous teeth (SHED), followed by Seo et al. who, a year later, were able to isolate periodontal ligament stem cells (PDLSCs). In 2005, Morsczeck et al. identified dental follicle precursor cells (DFPCs), which were later characterized as stem cells. The last population of dental stem cells to be discovered was the stem cells from the apical papilla (SCAP) in 2006, by Sonoyama et al.

Locations of dental stem cells. DFSCs, dental follicle stem cells; DPSCs, postnatal dental pulp stem cells; SHED, stem cells from exfoliated deciduous teeth; PDLSCs, periodontal ligament stem cells; SCAP, stem cells from apical papilla.
Fig. 1
Locations of dental stem cells. DFSCs, dental follicle stem cells; DPSCs, postnatal dental pulp stem cells; SHED, stem cells from exfoliated deciduous teeth; PDLSCs, periodontal ligament stem cells; SCAP, stem cells from apical papilla.

As these dental tissue-derived stem cells were characterized, they were often compared to bone marrow-derived mesenchymal stem cells (BMMSCs). They are similar in that they can differentiate into three different cell lineages: adipogenic, neurogenic, and osteo/odontogenic; however, they differ in that dental stem cells seem to be committed to an odontogenic fate, more so than to an osteogenic one. So far, STRO-1+ and CD146+ staining has been widely used to identify dental stem cell niches. Shi and Gronthos found that DPSCs are located in perivascular and perineural sheath regions, while Chen et al. localized PDLSCs in small clusters in the perivascular region, where Sonoyama et al. also found SCAP.

It has been hypothesized that dental tissue-derived stem cells may be restricted in their differentiation potential because dental tissue is more static than bony tissue in that it does not undergo constant remodelling. When compared to BMMSCs, SCAP and DPSCs present weaker adipogenesis potential, while the latter is also weaker in chondrogenesis. However; since dental stem cells have a neural crest origin, they have stronger neurogenesis capabilities than BMMSCs. It is believed that stem cells derived from this tissue could have properties analogous to those of the neural crest.

Neural crest

The neural crest is a group of transient embryonic cells which was originally identified in vertebrate embryos between their epidermis and neural tube. While these cells originate in the ectoderm, on the outer edges of the neural tube, they migrate into various parts of the body, giving rise to a wide variety of tissues. The neural crest derives into four types: cardiac, vagal, cranial, and trunk neural crest. These, in turn, form and contribute to a number of organs such as the heart, the enteric ganglia of the gut, nerves, and skin, respectively. Even though positional information seems to determine cell fate, all of these cell populations have different potentials, being susceptible to environmental and extracellular signals that play a key role in their differentiation.

Even though a large number of cells and tissues can be derived from the neural crest, proving its multipotency, it was not immediately clear if its cells were in fact stem cells. It was not until 1992 when the term Neural Crest Stem Cell (NCSC) was first used by Stemple and Anderson. In their in vitro study, they found that murine neural crest cells give rise to different types of cells such as smooth muscle, neurons and glia. Most importantly, they found that the cells had the ability to renew themselves, which is an exclusive property of stem cells. Recently, Dupin et al. found a cell population in the cephalic neural crest with a higher degree of multipotency, giving rise to not only smooth muscle, neurons and glia but also to chondrocytes, melanocytes and osteoblasts.

The undifferentiated state and self-renewal capacity of early NCSCs has been preserved by activating the bone morphogenic protein (BMP) and Wnt signalling pathways concurrently in vitro ; however, the mechanisms remain unclear. It is believed that these pathways target Sox10, which has also been shown to retain the multipotency of NCSCs in vitro through smooth muscle and neuronal differentiation inhibition and the modulation of their fates both in vitro and in vivo . In 2008, Teng et al. found that Foxd3 is required for the preservation of the neural crest population in vivo , modulating Sox10. Its overexpression; however, upregulates HNK-1 and Cad-7 in embryonic chicken neural tubes, which are markers of migratory neural crest.

In 2002 Kruger et al. discovered multipotent, self-renewing cells derived from the neural crest in adult organisms. The potency of these cells was similar to that from the embryonic NCSCs. Shortly after, it was found that their differentiation depended not only on their origin, but also on specific spatial factors and their corresponding response was intrinsic to the cell at specific times. This spatiotemporal property allows the NCSCs to react appropriately according to specific cues.

Dental pulp stem cells (DPSCs)

The first type of human dental stem cells identified was DPSCs in 2000 by Gronthos et al. These cells are morphologically similar to fibroblasts, very proliferative and clonogenic. While no specific biomarker has been discovered to identify this population, they have been found to express STRO-1 and CD146, which are also expressed in MSCs and BMMSCs. Undifferentiated DPSCs also expressed Oct4, Nanog, and Sox-2, and presented the cytoskeletal proteins Nestin and Vimentin, all of which are characteristic of undifferentiated embryonic stem cells (ESCs). Once DPSCs were isolated, many researchers studied their potential, trying to compare it to BMMSCs. In one of these studies, Gronthos et al. determined that these cells were able to self-duplicate in vivo . Over the years, many parallels have been drawn between DPSCs and BMMSCs as they express protein in vitro in similar patterns. When Yamada et al. performed gene expression studies, they found that from the genes that were up-regulated in DPSCs and down-regulated in BMMSCs, most were involved in cell signalling, metabolism, or communication. In 2001, Shi et al. discovered that the biochemical pathways implicated in the process of differentiation of DPSCs into odontoblasts are very much like the ones involved in the differentiation of BMMSCs into osteoblasts.

After their original characterization, DPSCs have been differentiated into dentinogenic, neurogenic, odontogenic, chondrogenic, myogenic, osteogenic, and adipogenic tissues. Gronthos et al. arrived at the conclusion that these cells have the potential for multilineage differentiation when they discovered that their morphologies and gene expression are very similar to those of adipocytes and neuronal cells. Interestingly, DPSCs have preserved their self-renewal capability and have formed dentine-like and pulp-like tissue as well as bone-like tissue, once transplanted into animal models. When cultured in vitro , DPSCs can be differentiated into odontoblasts with their characteristic accumulation of mineralized nodules and polarized cell bodies. In the last few years, investigators have used DPSCs for neural tissue and corneal regeneration, and the treatment of myocardial infarction as well as cerebral ischaemia.

DPSC colonies grow at different rates and they are composed of a heterogeneous group of cells, with different morphologies and sizes. Huang et al. compared different isolation methods for DPSCs and determined that they yielded different population lineages and found that enzyme digestion provided higher proliferation rates. They also established that treated dentine could potentially stimulate odontoblast differentiation and that using a collagen matrix may be detrimental in pulp tissue regeneration given that pulp cells cause collagen contraction. In a consequent study, the same group isolated human DPSCs through enzyme separation and seeded them onto mechanically and chemically treated dentine where they spontaneously formed mineral structures when cultured. After 16 days, cells presented odontoblast-like morphology which consisted of cytoplasmic processes spreading into the dentinal tubule and seemed to be of remarkably pure population.

Gronthos et al. in 2000, and Batouli et al. three years later, expanded DPSCs ex vivo and implanted them with hydroxyapatite/tricalcium phosphate (HA/TCP) into immunocompromised mice. The implanted mixture resulted in the formation of an ectopic structure with properties similar to those of pulp and dentine. In the latter study, investigators discovered that the heterogeneous DPSCs were able to generate vascularized pulp-like tissues along with odontoblast-like cells. These cells were found to express dentine sialophosphoprotein (DSPP), involved in the production of dentine, which resulted in the thickening of dentine. In a different study, Zhang et al. used different scaffolds to look into the performance of DPSCs when seeded into them in vitro and in vivo . The three scaffolds used were: hydroxyapatite/beta-tricalcium phosphate (HA/β-TCP), a collagen matrix, and a titanium web with varying textures. DSPP-expressing tissue grew in all three settings; however, it was more similar to connective tissue than to dentine. Graziano et al. also tested a convex hydroxyapatite scaffold, a flat titanium scaffold and a concave poly-lactide-coglycoide (PLGA) scaffold in search of the best substrate for osteodifferentiation. They found that PLGA seeded scaffolds resulted in thicker tissue that was composed of mature cells expressing the correct proteins. Additionally, another group found that nanohydroxyapatite (nHA) enhances the differentiation of DPCSs into odontoblast-like cells.

In 2005, investigators were able to determine that DPSCs were also found in people over 30 years of age and that they seemed not to be different from those found in younger patients. Three years later, a subpopulation of dental pulp stem cells with osteogenic potential was identified. The cells, which were termed ‘osteoblasts derived from human pulpar stem cells’ (ODHPSCs), had lower levels of expression of numerous genes compared to osteoblasts. The investigators concluded that this difference may contribute to the histological differences observed in the tissues formed by each of the stem cell populations.

Interestingly, Alongi et al. examined if DPSCs derived from inflamed pulps (DPSCs-IPs) could be used for dental tissue regeneration. Inflamed pulp is routinely discarded after pulpectomies and presents a possible source of stem cells as normal pulp is rarely available. They were able to isolate, culture and implant DPSCs-IPs which showed tissue regeneration potential in vivo , though they seemed to lose some of their stem cell properties according to in vitro studies. Still, this new source of DPSCs presents a very promising population of cells.

Stem cells from human exfoliated deciduous teeth (SHED)

SHED were discovered in 2003 by Miura et al. They isolated these clonogenic, proliferative cells using a technique very similar to the one Gronthos et al. used to isolate DPSCs. The protocol, however, differed in two important aspects: the pulp cells used came from the crown of exfoliated deciduous teeth, and the cells initially clustered into colonies instead of growing as individual cells. After separating colonies, though, SHED grew as individual fibroblast-like cells. It is important to note that these cells can also be derived from naturally exfoliated teeth. SHED proliferate faster and have greater population doublings than DPSCs which in turn are faster than BMMSCs. They are highly proliferative and develop clusters in the shape of spheres with various cytoplasmic processes, expressing glial and neuronal cell surface markers, like nestin, when cultured in neurogenic medium. The cell clusters can be dissociated and grown in culture as individual fibroblastic cells. In vitro , SHED have also demonstrated the potential to undergo adipogenic and osteogenic differentiation. Miura et al. studied the potential of SHED to differentiate into neural tissue in a murine study by injecting them into the dentate gyrus of the hippocampus. The cells survived for over 10 days expressing neural markers and they have been shown to express both neuronal and glial cell markers. This unique expression profile may be attributed to the neural crest origin of dental pulp. Still, in a study by Kerkis et al. SHED could be traced in numerous organs and tissues 90 days after injection, which is suggestive of a highly plastic differentiation.

Like DPSCs, SHED express STRO-1 and CD146 as well as Oct4 and tumour recognition antigens and stage-specific embryonic antigens. STRO-1 and CD146 are markers for cells in the vicinity of the pulp’s blood vessels hinting at the possibility that these cells come from the perivascular environment.

In murine models, transplanted SHED formed dentine-like structures that were in part composed of odontoblast-like cells. These findings suggest an important odontoinductive differentiation potential. Interestingly, unlike DPSCs, SHED did not form dentine–pulp complexes, indicating a difference in odontogenic potential. Another important difference is their osteogenic differentiation potential. While DPSCs can differentiate into osteocytes or osteoblasts, SHED are not able to. Instead, SHED have a remarkable characteristic: they are able to induce the differentiation of recipient murine cells into osteoblasts in vivo . When transplanted into immunocompromised mice, ex vivo expanded SHED differentiated into odontoblast-like cells expressing human markers associated with dentine, and the regenerated tissue expressed DSPP. Based on these observations, their higher proliferation rates and their potential for odontogenic differentiation and promoting osteogenesis, SHED are believed to be an immature form of DPSCs. In this way, SHED have been found to mend calvarial defects in a murine model that resulted in bone formation. Another interesting property of SHED is that instead of displaying the regular fibroblastic morphology, they show multicytoplasmic processes. They also have been shown to possess chondrogenic and myogenic potentials. These findings suggest a new function of deciduous teeth: the induction of bone in permanent teeth. SHED may be able to generate bone tissue because the roots of deciduous teeth go through resorption along with the generation of new bone around it.

In 2008 Gotlieb et al. seeded two types of scaffolds with SHED. One was made of collagen, the other made of open-cell polylactic acid. The results showed that cell adherence was possible and did not vary between the scaffolds nor did it change with the addition of different growth factors. According to Nakamura et al. SHED have better proliferative potential than DPSCs and BMSCs due to their higher levels of TGF-β2 and FGF2. The group suggests that this is why SHED are potentially the most promising population of cells to be used in tissue engineering and regeneration. In the year 2010, Wang et al. used these cells in their first clinical application, showing promising results in the treatment of Parkinson’s disease.

Periodontal ligament stem cells (PDLSCs)

Given the heterogeneous nature of the periodontal ligament and its capacity to remodel continuously, it had been speculated that it contained progenitor cells. In 2004 this theory led to the discovery of PDLSCs using a protocol similar to the one used to ascertain both DPSCs and SHED; except, this time investigators used the periodontal ligament instead of pulp. PDLSCs can be obtained from extracted teeth and their properties seem to vary depending on the harvest location: cells extracted from the alveolar bone surface are better able to regenerate the alveolar bone as compared to cells obtained from the root surface. Subpopulations of PDLSCs present embryonic stem cell (Nanog, Oct4, Klf4 and Sox2) and neural crest (Sox10, Slug, p75 and Nestin) markers, which indicates that they have the potential to differentiate into osteogenic, cardiomyogenic, chondrogenic (derived from mesoderm) and neurogenic (derived from ectoderm) lineages. What is more, preliminary data suggests that PDLSCs can differentiate into insulin producing cells, which would signify that they can also differentiate into the endodermal lineage. Therefore, these cells could potentially differentiate into tissues from all three germ layers. The pluripotency of PDLSCs was confirmed by a recent study which identified and isolated a novel group of pluripotential stem cells from human periodontal ligament based on selection of connexin 43 (Cx43) expressing cells.

PDLSCs are clonogenic and present with fibroblast-like morphology. In 2002, Shi et al. discovered that telomerases were highly expressed in PDLSCs, which might explain their high proliferative properties. Studies have shown that these cells express scleraxis in addition to STRO-1 and CD146. Scleraxis is a transcription factor specific to tendons and is highly expressed in PDLSCs, as opposed to DPSCs or BMMSCs. This was not surprising as the periodontal ligament is morphologically similar to tendon. Gay et al. did a study comparing PDLSCs to BMSCs. They found that PDLSCs express alkaline phosphatases (ALP) 7 days earlier than the other cells, but that both populations express bone sialprotein (BSP) at the same time. Lindroos et al. found that PDLSCs present the same bone-related surface markers as BMMSCs, suggesting that they might be a promising candidate for bone regeneration. In addition, PDLSCs have the capacity to differentiate into both osteoblasts and cementoblasts, suggesting that the function PDLSCs is to maintain homeostasis and regenerate periodontal tissue.

PDLSCs have been used to regenerate a cementum-like structure alongside collagen fibres with thin cells similar to the structure of the periodontal ligament. This was achieved by expanding the cells ex vivo and then implanting them in a murine host. It is important to note that the collagen fibres attached to the cementum-like structures much like it happens with Sharpey’s fibres in natural physiological attachment. These results suggest that there may be multiple subpopulations of PDLSCs, ones able to differentiate into cementoblast-like cells and others able to form collagen in vivo . In 2008, a study showed that PDLSCs are capable of giving rise to cementum-like cells, when implanted along with noncollagenous dentine proteins. In the past few years, PDLSCs were also found to present osteogenic, chondrogenic, and adipogenic features when cultured under the right conditions. In 2012, Song et al. compared stem cells derived from the periodontal ligament of permanent teeth to those from deciduous teeth and found that the former are more promising for use in periodontium reconstruction.

Dental follicle precursor cells (DFPCs)

The dental follicle controls osteogenesis and osteoclastogenesis and differentiates into the periodontium during the eruption process. Since the periodontium is composed of various different cell types, it was speculated that the dental follicle contained stem cells. This theory was proven correct in 2005 by Morsczeck et al. who isolated these cells using the same protocol that Gronthos et al. used five years earlier to discover and culture DPSCs. Much like DPSCs, dental follicle precursor cells (DFPCs), also termed dental follicle stem cells (DFSCs), had a fibroblast-like morphology and showed potential to undergo neurogenic, adipogenic, and osteogenic differentiation in vitro . DFPCs express markers such as nestin and Notch-1, as well as various neural cell markers and cementoblast markers after the respective inductions.

Stem cells of apical papilla (SCAP)

In 2006 Sonoyama et al. were the first to isolate stem cells from the dental apical papilla, which is the soft tissue at the root of developing permanent teeth. The apical papila is involved in the development of the root and later becomes the pulp. They termed these cells Stem Cells of Apical Papilla (SCAP). Like DPSCs, SCAP are clonogenic and have a fibrolast-like morphology, but they are more proliferative than DPSCs. These cells express the same surface markers as the other dental stem cells (STRO-1 and CD146); however, they also express a unique marker: CD24. Expression is regulated, though, when they undergo osteogenic stimulation. SCAP have been shown to differentiated in vitro into neurogenic and chondrogenic tissue; and, like DPSCs, have been able to form dentine-like structures containing odontoblast-like cells in immunocompromised animal models.

Ex vivo expanded SCAP, like SHED and DPSCs, have odontogenic potential in vitro . SCAP have also been found to undergo adipogenic differentiation under the right induction environment in vitro . Another interesting feature of these cells is that they express neural markers even when not exposed to neurogenic stimulation. However, when stimulated, more neural markers are expressed. When transplanted into immunocompromised mice in an adequate carrier matrix such as HA, these cells are capable of forming a dentine/pulp-like complex like the one previously described for DPSCs and they have also been found to regenerate alveolar bone and periodontal ligament in vivo. SCAP are a great example of cells from a developing tissue that may be a better source of stem cells for tissue regeneration than cells derived from mature tissues.

Dental stem cells in tissue engineering and regeneration

Dental pulp regeneration and bio-root engineering

Even though dental implants have greatly improved in the past decade, issues with the technology are still present. The main obstacles to overcome are their unnatural cylindrical shape and the subpar attachment to the alveolar bone due to the absence of periodontal ligament. Also, there is still a lack of consistency in the root formation and tooth size, as well as complete eruption into functional occlusion. In order to solve these problems, many in vivo studies have been performed where investigators tested the development of tooth-like structures by seeding isolated stem cells into scaffolds. As of now, the field of tooth regeneration still has many other obstacles to overcome.

To tissue-engineer or regenerate a whole tooth, the use of all different types of dental stem cells may be required. On the other hand, for the repair of only part of it, one or two types may suffice, depending on which tissues need to be regenerated. Cordeiro et al. used a tooth slice model to create pulp-like tissue by seeding SHED onto biodegradable scaffolds. The cells differentiated into cells resembling odontoblasts and endothelial cells. Conversely, Prescott et al. used the same model to implant a collagen scaffold seeded with DPSCs and supplemented with dentine matrix protein (DMP-1). This study resulted in the regeneration of pulp-like tissue. Furthermore, by expanding human SCAP and DPSCs ex vivo , Huang et al. have been able to form a uniform dentine-like structure on the dentinal wall of emptied root canal space in immunocompromised mice, as well as regenerating vascularized pulp-like tissue within it. While the pulp tissue obtained is very similar to the natural human pulp, the dentine-like tissue formed is not. Dentinal tubules are scarce, the tissue is more cellular than the naturally occurring tissue and it is formed asynchronously. Nevertheless, the cells remained viable and regenerated the tissue.

One study, performed by Honda et al. in 2006, seeded scaffolds with single cells at bell stage from dog tooth buds and implanted them in the original tooth socket. Even though dentine was regenerated in this case, no enamel or root was observed. In another attempt, Kuo et al. used a swine model to examine the possibility of tooth regeneration. They expanded bell stage tooth bud cells ex vivo and cultured them in cylindrical scaffolds which were then implanted in the original alveolar socket. In this case, they observed both dentine and root formation along with periodontium. Sonoyama et al. took another approach by generating a bio-root instead of a whole tooth. They used autologous swine SCAP and PDLSCs to regenerate root and periodontal tissue, seeding them into an HA/TCP carrier and PDLSC-seeded gelfoam scaffolds respectively. The structure was then implanted into the socket of swine. Three months later, the implant was growing successfully and had developed a mineralized root-like tissue along with periodontal ligament space. This promising approach would potentially yield a cell-based alternative, wherein a bio-root is formed instead of using an artificial dental implant. Nevertheless, the HA residues seemed to contribute to a structural change which resulted in diminished mechanical strength. Additionally, Gebhardt et al. studied the survival of DPSCs and PDLSCs in tissue-engineered dental pulp and periodontal constructs, comparing their presence in three different kinds of scaffolds: calcium phosphate, a synthetic polymer and collagen. They discovered that the first two were notably conducive to cell survival and proliferation, with cell numbers increasing over time. The results suggest that these two materials could be used to create de novo dental constructs which would provide preformed replacement tissue when implanted into a root canal.

Another important contribution was by Elseed et al. who were the first group who sought to determine the effect of using bioactive and bio-adhesive therapies in the attachment of PDLSCs to dentine in the hopes of enhancing it so as to potentially using such therapies for the replantation of avulsed teeth after extended extra-oral periods. They used slabs from human dental lower roots which were either left untreated or coated with bio-adhesive, human recombinant transforming growth factor-beta1 (hrTGF-B1), or human recombinant bone morphogenic protein-2 (hrBMP-2). These dentine slabs were then cultured in contact with confluent PDLSC cultures for 4, 24 and 72 h, and analyzed using scanning electron microscopy (SEM). PDLSCs attached to all root dentine samples, treated and untreated, showing that bioactive coatings are not necessary for the cells to adhere to the surface. However; the addition of BMP-2 seemed to result in a speedier process as the phenotype of the cells changes from oval to flattened earlier than in other cases, suggesting the stimulation of adhesion. It is important to bear in mind, nonetheless, that according to statistical analysis, results may be different in a larger data set.

While BMMSCs have been shown to repair periodontal defects by forming bone and cementum, they have failed to regenerate the periodontal ligament. PDLSCs could potentially be the solution to this problem as shown by Liu et al. They treated a minipig with autologous PDLSCs and obtained a satisfactory regeneration of the ligament and alveolar bone.

Diabetes treatment

Recently, Govindasamy et al. obtained islet-like cell aggregates (ICAs) from DPSCs derived from deciduous teeth. They established that the cells were islets through positive staining of dithiozone. Additionally, the cells expressed the islet markers C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. In vitro , they were able to show functionality as, after 10 days, these aggregates responded to glucose by releasing insulin and C-peptide increased in a dose dependent fashion. Overall their study proved that DPSCs are capable of differentiating into the pancreatic cell lineage. An important point to consider is that although there are numerous similarities between the BMMSC- and DPSC-derived insulin-producing cells in vitro , there is an important difference: the latter are mature insulin-secreting ICAs, while the former only give rise to immature islets incapable of producing insulin.

Liver function recovery

Ishkitiev et al. were able to obtain hepatocyte-like cells from dental stem cells. They used magnetic separation and CD117 antibodies to obtain pure SHED and DPSC cultures and differentiated them into the hepatic lineage using serum-free medium (SFM). Large amounts of these cells were positive for the stem cell surface marker CD117 as well as endodermal, mesodermal, ectodermal, mesenchymal, and embryonic markers. Most of the CD117-positive cells showed the hepatic-specific markers HNF-4a, albumin, aFP, CPS-1 and, IGF-I. Additionally, these cells changed their morphology to hepatocyte-like ovoids and contained high amounts of glycogen in their cytosol and urea concentration in the media increased considerably after differentiation. Both populations are good candidates for creating hepatocyte-like lineages ; however, SHED seem to be a better hepatic progenitor source as they are believed to be a more immature stem cell population and have been shown to have neurogenic differentiation capabilities.

Cardiac function improvement

In a murine study of acute myocardial infarctions, DPSCs transplanted into the affected zone caused partial repair of the area four weeks later. The mouse hearts showed higher numbers of vessels and a smaller infarct area, which help improve cardiac function overall. Interestingly, the implanted DPSCs never differentiated into muscle cells, which indicates that their effects were inductive, probably through the secretion of cytokines and growth factors. This could be a breakthrough in the use of DPSCs to treat ischaemic diseases.

Bone repair

As early as 2005, there have been reports of DPSCs causing osteogenesis both in vitro and in vivo . SHED have also been proven to have the ability to generate bone tissue, by inducing host cells to produce osteoblasts. Since PDSCs and SHED are more proliferative and obtained less invasively than BMMSCs, they are a good alternative for use in bone regeneration. Interestingly, a study used SHED/β-TCP to successfully reconstruct defects in the mandibles of minipigs, achieving bone regeneration in a large animal model for the first time. Since every investigator has used a scaffold of their own preference, no optimal method for bone regeneration has been determined using dental stem cells.

Neural tissue regeneration

SHED are capable of differentiating into neurons, while DPSCs are able to obtain neuronal morphology and express gene and protein markers specific to neuronal cells, both in vitro and in vivo . Researchers were also able to generate a sodium current under the effects of inductive media. In a more recent study, Arthur et al. used an animal model to show that axons in the trigeminal ganglion migrated in the direction of implanted DPSCs as a result of them expressing CXCL12. These findings suggest that DPSCs may be capable of inducing neuroplasticity in the host. These advances show how promising dental stem cells can be in the field of neuronal regeneration, providing a less invasive option.

Conclusion

It has only been thirteen years since the first human dental stem cells were discovered. Although we have learned much about them in this short time, there is still even more to uncover. We do know; however, that due to their multilineage potential which allows the differentiation into the three germ layers, they are very good candidates for inducing tissue regeneration. All dental stem cells mentioned here have been demonstrated to regenerate dental pulp and periodontal tissue. Function of those regenerative dental tissues needs to be further evaluated. DPSCs and SHED have been shown to mend small bone defects. With further research to optimize the conditions for bone regeneration of DPSCs and SHED, they can potentially be used for treating larger trauma injury or even for construction of entire bone implants. SHED have also been differentiated into neuron-like cells while DPSCs have been shown to induce neuroplasty, showing great possibilities to address neurodegenerative diseases and nerve damage due to traumatic injuries. Moreover, DPSCs have also differentiated into islet-like cells, giving a new alternative route for diabetes research, and when injected into hearts resulted in increased function and vasculature due to paracrine effects, which is a valuable option for treating ischaemic diseases. In addition, DPSCs and SHED were shown to differentiate into hepatocyte lineage, indicating another potential cell sources for treating liver disease. Importantly, they are derived from an easily accessible, abundant source. Although the amount of cells that can be derived from one donor or tooth may be small, their proliferative capacity may allow us produce an adequate number of cells for therapeutic uses. Still, dental stem cell research is currently in early stages and it is far from the clinics. Preclinical evaluation in animal models needs to be performed in order to verify the capability of dental stem cells to regenerate functional tissues or restore the function of diseased/injured tissues. Also specific markers need to be determined to better isolate and track stem cell populations in dental tissues. Overall, dental stem cells have the potential to develop solutions to different clinical problems such as dental implants, heart failure, diabetics, and even Parkinson’s disease. While many challenges remain, dental stem cells could propel the field of personalized regenerative medicine forward and therefore should be studied further.

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Neural crest-derived dental stem cells—Where we are and where we are going Vera Mayo , Yoh Sawatari , C.-Y. Charles Huang and Franklin Garcia-Godoy Journal of Dentistry, 2014-09-01, Volume 42, Issue 9, Pages 1043-1051, Copyright © 2014 Elsevier Ltd Abstract Objectives There are five types of post-natal human dental stem cells that have been identified, isolated and characterized. Here, we review the information available on dental stem cells as well as their potential applications in dentistry, regenerative medicine and the development of other therapeutic approaches. Data Data pertinent to dental stem cells and their applications, published in peer-reviewed journals from 1982 to 2013 in English were reviewed. Sources Sources were retrieved from PubMed databases as well as related references that the electronic search yielded. Study Selection Manuscripts describing the origin, retrieval, characterization and application of dental stem cells were obtained and reviewed. Conclusions Dental stem cell populations present properties similar to those of mesenchymal stem cells, such as the ability to self-renew and the potential for multilineage differentiation. While they have greater capacity to give rise to odontogenic cells and regenerate dental pulp and periodontal tissue, they have the capacity to differentiate into all three germ line cells, proving that a population of pluripotent stem cells exists in the dental tissues. Clinical Significance Dental stem cells have the capacity to differentiate into endoderm, mesoderm and ectoderm tissues. Consequently they do not only have applications in dentistry, but also neurodegenerative and ischemic diseases, diabetes research, bone repair, and other applications in the field of tissue regeneration. 1 Introduction There are two kinds of adult dental stem cells identified today: mesenchymal stem cell (MSC)-like cells and epithelial stem cells. This review will focus on the former as the latter have only been discovered in mouse incisors, not in humans yet. Researchers hypothesize that they may be unique to rodents because they have the special characteristic of continuous eruption throughout their lives. Reports of MSC-like cells isolated from different human dental tissues ( Fig. 1 ) began in the year 2000 and research studies about the subject have become more popular with time, with publications about the subject increasing every year. Postnatal dental pulp stem cells (DPSCs) were the first population of dental-tissue derived stem cells to be identified. These cells were isolated by Gronthos et al. in 2000 from human dental pulp. In 2003, Miura et al. discovered stem cells from human exfoliated deciduous teeth (SHED), followed by Seo et al. who, a year later, were able to isolate periodontal ligament stem cells (PDLSCs). In 2005, Morsczeck et al. identified dental follicle precursor cells (DFPCs), which were later characterized as stem cells. The last population of dental stem cells to be discovered was the stem cells from the apical papilla (SCAP) in 2006, by Sonoyama et al. Fig. 1 Locations of dental stem cells. DFSCs, dental follicle stem cells; DPSCs, postnatal dental pulp stem cells; SHED, stem cells from exfoliated deciduous teeth; PDLSCs, periodontal ligament stem cells; SCAP, stem cells from apical papilla. As these dental tissue-derived stem cells were characterized, they were often compared to bone marrow-derived mesenchymal stem cells (BMMSCs). They are similar in that they can differentiate into three different cell lineages: adipogenic, neurogenic, and osteo/odontogenic; however, they differ in that dental stem cells seem to be committed to an odontogenic fate, more so than to an osteogenic one. So far, STRO-1+ and CD146+ staining has been widely used to identify dental stem cell niches. Shi and Gronthos found that DPSCs are located in perivascular and perineural sheath regions, while Chen et al. localized PDLSCs in small clusters in the perivascular region, where Sonoyama et al. also found SCAP. It has been hypothesized that dental tissue-derived stem cells may be restricted in their differentiation potential because dental tissue is more static than bony tissue in that it does not undergo constant remodelling. When compared to BMMSCs, SCAP and DPSCs present weaker adipogenesis potential, while the latter is also weaker in chondrogenesis. However; since dental stem cells have a neural crest origin, they have stronger neurogenesis capabilities than BMMSCs. It is believed that stem cells derived from this tissue could have properties analogous to those of the neural crest. 2 Neural crest The neural crest is a group of transient embryonic cells which was originally identified in vertebrate embryos between their epidermis and neural tube. While these cells originate in the ectoderm, on the outer edges of the neural tube, they migrate into various parts of the body, giving rise to a wide variety of tissues. The neural crest derives into four types: cardiac, vagal, cranial, and trunk neural crest. These, in turn, form and contribute to a number of organs such as the heart, the enteric ganglia of the gut, nerves, and skin, respectively. Even though positional information seems to determine cell fate, all of these cell populations have different potentials, being susceptible to environmental and extracellular signals that play a key role in their differentiation. Even though a large number of cells and tissues can be derived from the neural crest, proving its multipotency, it was not immediately clear if its cells were in fact stem cells. It was not until 1992 when the term Neural Crest Stem Cell (NCSC) was first used by Stemple and Anderson. In their in vitro study, they found that murine neural crest cells give rise to different types of cells such as smooth muscle, neurons and glia. Most importantly, they found that the cells had the ability to renew themselves, which is an exclusive property of stem cells. Recently, Dupin et al. found a cell population in the cephalic neural crest with a higher degree of multipotency, giving rise to not only smooth muscle, neurons and glia but also to chondrocytes, melanocytes and osteoblasts. The undifferentiated state and self-renewal capacity of early NCSCs has been preserved by activating the bone morphogenic protein (BMP) and Wnt signalling pathways concurrently in vitro ; however, the mechanisms remain unclear. It is believed that these pathways target Sox10, which has also been shown to retain the multipotency of NCSCs in vitro through smooth muscle and neuronal differentiation inhibition and the modulation of their fates both in vitro and in vivo . In 2008, Teng et al. found that Foxd3 is required for the preservation of the neural crest population in vivo , modulating Sox10. Its overexpression; however, upregulates HNK-1 and Cad-7 in embryonic chicken neural tubes, which are markers of migratory neural crest. In 2002 Kruger et al. discovered multipotent, self-renewing cells derived from the neural crest in adult organisms. The potency of these cells was similar to that from the embryonic NCSCs. Shortly after, it was found that their differentiation depended not only on their origin, but also on specific spatial factors and their corresponding response was intrinsic to the cell at specific times. This spatiotemporal property allows the NCSCs to react appropriately according to specific cues. 3 Dental pulp stem cells (DPSCs) The first type of human dental stem cells identified was DPSCs in 2000 by Gronthos et al. These cells are morphologically similar to fibroblasts, very proliferative and clonogenic. While no specific biomarker has been discovered to identify this population, they have been found to express STRO-1 and CD146, which are also expressed in MSCs and BMMSCs. Undifferentiated DPSCs also expressed Oct4, Nanog, and Sox-2, and presented the cytoskeletal proteins Nestin and Vimentin, all of which are characteristic of undifferentiated embryonic stem cells (ESCs). Once DPSCs were isolated, many researchers studied their potential, trying to compare it to BMMSCs. In one of these studies, Gronthos et al. determined that these cells were able to self-duplicate in vivo . Over the years, many parallels have been drawn between DPSCs and BMMSCs as they express protein in vitro in similar patterns. When Yamada et al. performed gene expression studies, they found that from the genes that were up-regulated in DPSCs and down-regulated in BMMSCs, most were involved in cell signalling, metabolism, or communication. In 2001, Shi et al. discovered that the biochemical pathways implicated in the process of differentiation of DPSCs into odontoblasts are very much like the ones involved in the differentiation of BMMSCs into osteoblasts. After their original characterization, DPSCs have been differentiated into dentinogenic, neurogenic, odontogenic, chondrogenic, myogenic, osteogenic, and adipogenic tissues. Gronthos et al. arrived at the conclusion that these cells have the potential for multilineage differentiation when they discovered that their morphologies and gene expression are very similar to those of adipocytes and neuronal cells. Interestingly, DPSCs have preserved their self-renewal capability and have formed dentine-like and pulp-like tissue as well as bone-like tissue, once transplanted into animal models. When cultured in vitro , DPSCs can be differentiated into odontoblasts with their characteristic accumulation of mineralized nodules and polarized cell bodies. In the last few years, investigators have used DPSCs for neural tissue and corneal regeneration, and the treatment of myocardial infarction as well as cerebral ischaemia. DPSC colonies grow at different rates and they are composed of a heterogeneous group of cells, with different morphologies and sizes. Huang et al. compared different isolation methods for DPSCs and determined that they yielded different population lineages and found that enzyme digestion provided higher proliferation rates. They also established that treated dentine could potentially stimulate odontoblast differentiation and that using a collagen matrix may be detrimental in pulp tissue regeneration given that pulp cells cause collagen contraction. In a consequent study, the same group isolated human DPSCs through enzyme separation and seeded them onto mechanically and chemically treated dentine where they spontaneously formed mineral structures when cultured. After 16 days, cells presented odontoblast-like morphology which consisted of cytoplasmic processes spreading into the dentinal tubule and seemed to be of remarkably pure population. Gronthos et al. in 2000, and Batouli et al. three years later, expanded DPSCs ex vivo and implanted them with hydroxyapatite/tricalcium phosphate (HA/TCP) into immunocompromised mice. The implanted mixture resulted in the formation of an ectopic structure with properties similar to those of pulp and dentine. In the latter study, investigators discovered that the heterogeneous DPSCs were able to generate vascularized pulp-like tissues along with odontoblast-like cells. These cells were found to express dentine sialophosphoprotein (DSPP), involved in the production of dentine, which resulted in the thickening of dentine. In a different study, Zhang et al. used different scaffolds to look into the performance of DPSCs when seeded into them in vitro and in vivo . The three scaffolds used were: hydroxyapatite/beta-tricalcium phosphate (HA/β-TCP), a collagen matrix, and a titanium web with varying textures. DSPP-expressing tissue grew in all three settings; however, it was more similar to connective tissue than to dentine. Graziano et al. also tested a convex hydroxyapatite scaffold, a flat titanium scaffold and a concave poly-lactide-coglycoide (PLGA) scaffold in search of the best substrate for osteodifferentiation. They found that PLGA seeded scaffolds resulted in thicker tissue that was composed of mature cells expressing the correct proteins. Additionally, another group found that nanohydroxyapatite (nHA) enhances the differentiation of DPCSs into odontoblast-like cells. In 2005, investigators were able to determine that DPSCs were also found in people over 30 years of age and that they seemed not to be different from those found in younger patients. Three years later, a subpopulation of dental pulp stem cells with osteogenic potential was identified. The cells, which were termed ‘osteoblasts derived from human pulpar stem cells’ (ODHPSCs), had lower levels of expression of numerous genes compared to osteoblasts. The investigators concluded that this difference may contribute to the histological differences observed in the tissues formed by each of the stem cell populations. Interestingly, Alongi et al. examined if DPSCs derived from inflamed pulps (DPSCs-IPs) could be used for dental tissue regeneration. Inflamed pulp is routinely discarded after pulpectomies and presents a possible source of stem cells as normal pulp is rarely available. They were able to isolate, culture and implant DPSCs-IPs which showed tissue regeneration potential in vivo , though they seemed to lose some of their stem cell properties according to in vitro studies. Still, this new source of DPSCs presents a very promising population of cells. 4 Stem cells from human exfoliated deciduous teeth (SHED) SHED were discovered in 2003 by Miura et al. They isolated these clonogenic, proliferative cells using a technique very similar to the one Gronthos et al. used to isolate DPSCs. The protocol, however, differed in two important aspects: the pulp cells used came from the crown of exfoliated deciduous teeth, and the cells initially clustered into colonies instead of growing as individual cells. After separating colonies, though, SHED grew as individual fibroblast-like cells. It is important to note that these cells can also be derived from naturally exfoliated teeth. SHED proliferate faster and have greater population doublings than DPSCs which in turn are faster than BMMSCs. They are highly proliferative and develop clusters in the shape of spheres with various cytoplasmic processes, expressing glial and neuronal cell surface markers, like nestin, when cultured in neurogenic medium. The cell clusters can be dissociated and grown in culture as individual fibroblastic cells. In vitro , SHED have also demonstrated the potential to undergo adipogenic and osteogenic differentiation. Miura et al. studied the potential of SHED to differentiate into neural tissue in a murine study by injecting them into the dentate gyrus of the hippocampus. The cells survived for over 10 days expressing neural markers and they have been shown to express both neuronal and glial cell markers. This unique expression profile may be attributed to the neural crest origin of dental pulp. Still, in a study by Kerkis et al. SHED could be traced in numerous organs and tissues 90 days after injection, which is suggestive of a highly plastic differentiation. Like DPSCs, SHED express STRO-1 and CD146 as well as Oct4 and tumour recognition antigens and stage-specific embryonic antigens. STRO-1 and CD146 are markers for cells in the vicinity of the pulp's blood vessels hinting at the possibility that these cells come from the perivascular environment. In murine models, transplanted SHED formed dentine-like structures that were in part composed of odontoblast-like cells. These findings suggest an important odontoinductive differentiation potential. Interestingly, unlike DPSCs, SHED did not form dentine–pulp complexes, indicating a difference in odontogenic potential. Another important difference is their osteogenic differentiation potential. While DPSCs can differentiate into osteocytes or osteoblasts, SHED are not able to. Instead, SHED have a remarkable characteristic: they are able to induce the differentiation of recipient murine cells into osteoblasts in vivo . When transplanted into immunocompromised mice, ex vivo expanded SHED differentiated into odontoblast-like cells expressing human markers associated with dentine, and the regenerated tissue expressed DSPP. Based on these observations, their higher proliferation rates and their potential for odontogenic differentiation and promoting osteogenesis, SHED are believed to be an immature form of DPSCs. In this way, SHED have been found to mend calvarial defects in a murine model that resulted in bone formation. Another interesting property of SHED is that instead of displaying the regular fibroblastic morphology, they show multicytoplasmic processes. They also have been shown to possess chondrogenic and myogenic potentials. These findings suggest a new function of deciduous teeth: the induction of bone in permanent teeth. SHED may be able to generate bone tissue because the roots of deciduous teeth go through resorption along with the generation of new bone around it. In 2008 Gotlieb et al. seeded two types of scaffolds with SHED. One was made of collagen, the other made of open-cell polylactic acid. The results showed that cell adherence was possible and did not vary between the scaffolds nor did it change with the addition of different growth factors. According to Nakamura et al. SHED have better proliferative potential than DPSCs and BMSCs due to their higher levels of TGF-β2 and FGF2. The group suggests that this is why SHED are potentially the most promising population of cells to be used in tissue engineering and regeneration. In the year 2010, Wang et al. used these cells in their first clinical application, showing promising results in the treatment of Parkinson's disease. 5 Periodontal ligament stem cells (PDLSCs) Given the heterogeneous nature of the periodontal ligament and its capacity to remodel continuously, it had been speculated that it contained progenitor cells. In 2004 this theory led to the discovery of PDLSCs using a protocol similar to the one used to ascertain both DPSCs and SHED; except, this time investigators used the periodontal ligament instead of pulp. PDLSCs can be obtained from extracted teeth and their properties seem to vary depending on the harvest location: cells extracted from the alveolar bone surface are better able to regenerate the alveolar bone as compared to cells obtained from the root surface. Subpopulations of PDLSCs present embryonic stem cell (Nanog, Oct4, Klf4 and Sox2) and neural crest (Sox10, Slug, p75 and Nestin) markers, which indicates that they have the potential to differentiate into osteogenic, cardiomyogenic, chondrogenic (derived from mesoderm) and neurogenic (derived from ectoderm) lineages. What is more, preliminary data suggests that PDLSCs can differentiate into insulin producing cells, which would signify that they can also differentiate into the endodermal lineage. Therefore, these cells could potentially differentiate into tissues from all three germ layers. The pluripotency of PDLSCs was confirmed by a recent study which identified and isolated a novel group of pluripotential stem cells from human periodontal ligament based on selection of connexin 43 (Cx43) expressing cells. PDLSCs are clonogenic and present with fibroblast-like morphology. In 2002, Shi et al. discovered that telomerases were highly expressed in PDLSCs, which might explain their high proliferative properties. Studies have shown that these cells express scleraxis in addition to STRO-1 and CD146. Scleraxis is a transcription factor specific to tendons and is highly expressed in PDLSCs, as opposed to DPSCs or BMMSCs. This was not surprising as the periodontal ligament is morphologically similar to tendon. Gay et al. did a study comparing PDLSCs to BMSCs. They found that PDLSCs express alkaline phosphatases (ALP) 7 days earlier than the other cells, but that both populations express bone sialprotein (BSP) at the same time. Lindroos et al. found that PDLSCs present the same bone-related surface markers as BMMSCs, suggesting that they might be a promising candidate for bone regeneration. In addition, PDLSCs have the capacity to differentiate into both osteoblasts and cementoblasts, suggesting that the function PDLSCs is to maintain homeostasis and regenerate periodontal tissue. PDLSCs have been used to regenerate a cementum-like structure alongside collagen fibres with thin cells similar to the structure of the periodontal ligament. This was achieved by expanding the cells ex vivo and then implanting them in a murine host. It is important to note that the collagen fibres attached to the cementum-like structures much like it happens with Sharpey's fibres in natural physiological attachment. These results suggest that there may be multiple subpopulations of PDLSCs, ones able to differentiate into cementoblast-like cells and others able to form collagen in vivo . In 2008, a study showed that PDLSCs are capable of giving rise to cementum-like cells, when implanted along with noncollagenous dentine proteins. In the past few years, PDLSCs were also found to present osteogenic, chondrogenic, and adipogenic features when cultured under the right conditions. In 2012, Song et al. compared stem cells derived from the periodontal ligament of permanent teeth to those from deciduous teeth and found that the former are more promising for use in periodontium reconstruction. 6 Dental follicle precursor cells (DFPCs) The dental follicle controls osteogenesis and osteoclastogenesis and differentiates into the periodontium during the eruption process. Since the periodontium is composed of various different cell types, it was speculated that the dental follicle contained stem cells. This theory was proven correct in 2005 by Morsczeck et al. who isolated these cells using the same protocol that Gronthos et al. used five years earlier to discover and culture DPSCs. Much like DPSCs, dental follicle precursor cells (DFPCs), also termed dental follicle stem cells (DFSCs), had a fibroblast-like morphology and showed potential to undergo neurogenic, adipogenic, and osteogenic differentiation in vitro . DFPCs express markers such as nestin and Notch-1, as well as various neural cell markers and cementoblast markers after the respective inductions. 7 Stem cells of apical papilla (SCAP) In 2006 Sonoyama et al. were the first to isolate stem cells from the dental apical papilla, which is the soft tissue at the root of developing permanent teeth. The apical papila is involved in the development of the root and later becomes the pulp. They termed these cells Stem Cells of Apical Papilla (SCAP). Like DPSCs, SCAP are clonogenic and have a fibrolast-like morphology, but they are more proliferative than DPSCs. These cells express the same surface markers as the other dental stem cells (STRO-1 and CD146); however, they also express a unique marker: CD24. Expression is regulated, though, when they undergo osteogenic stimulation. SCAP have been shown to differentiated in vitro into neurogenic and chondrogenic tissue; and, like DPSCs, have been able to form dentine-like structures containing odontoblast-like cells in immunocompromised animal models. Ex vivo expanded SCAP, like SHED and DPSCs, have odontogenic potential in vitro . SCAP have also been found to undergo adipogenic differentiation under the right induction environment in vitro . Another interesting feature of these cells is that they express neural markers even when not exposed to neurogenic stimulation. However, when stimulated, more neural markers are expressed. When transplanted into immunocompromised mice in an adequate carrier matrix such as HA, these cells are capable of forming a dentine/pulp-like complex like the one previously described for DPSCs and they have also been found to regenerate alveolar bone and periodontal ligament in vivo. SCAP are a great example of cells from a developing tissue that may be a better source of stem cells for tissue regeneration than cells derived from mature tissues. 8 Dental stem cells in tissue engineering and regeneration 8.1 Dental pulp regeneration and bio-root engineering Even though dental implants have greatly improved in the past decade, issues with the technology are still present. The main obstacles to overcome are their unnatural cylindrical shape and the subpar attachment to the alveolar bone due to the absence of periodontal ligament. Also, there is still a lack of consistency in the root formation and tooth size, as well as complete eruption into functional occlusion. In order to solve these problems, many in vivo studies have been performed where investigators tested the development of tooth-like structures by seeding isolated stem cells into scaffolds. As of now, the field of tooth regeneration still has many other obstacles to overcome. To tissue-engineer or regenerate a whole tooth, the use of all different types of dental stem cells may be required. On the other hand, for the repair of only part of it, one or two types may suffice, depending on which tissues need to be regenerated. Cordeiro et al. used a tooth slice model to create pulp-like tissue by seeding SHED onto biodegradable scaffolds. The cells differentiated into cells resembling odontoblasts and endothelial cells. Conversely, Prescott et al. used the same model to implant a collagen scaffold seeded with DPSCs and supplemented with dentine matrix protein (DMP-1). This study resulted in the regeneration of pulp-like tissue. Furthermore, by expanding human SCAP and DPSCs ex vivo , Huang et al. have been able to form a uniform dentine-like structure on the dentinal wall of emptied root canal space in immunocompromised mice, as well as regenerating vascularized pulp-like tissue within it. While the pulp tissue obtained is very similar to the natural human pulp, the dentine-like tissue formed is not. Dentinal tubules are scarce, the tissue is more cellular than the naturally occurring tissue and it is formed asynchronously. Nevertheless, the cells remained viable and regenerated the tissue. One study, performed by Honda et al. in 2006, seeded scaffolds with single cells at bell stage from dog tooth buds and implanted them in the original tooth socket. Even though dentine was regenerated in this case, no enamel or root was observed. In another attempt, Kuo et al. used a swine model to examine the possibility of tooth regeneration. They expanded bell stage tooth bud cells ex vivo and cultured them in cylindrical scaffolds which were then implanted in the original alveolar socket. In this case, they observed both dentine and root formation along with periodontium. Sonoyama et al. took another approach by generating a bio-root instead of a whole tooth. They used autologous swine SCAP and PDLSCs to regenerate root and periodontal tissue, seeding them into an HA/TCP carrier and PDLSC-seeded gelfoam scaffolds respectively. The structure was then implanted into the socket of swine. Three months later, the implant was growing successfully and had developed a mineralized root-like tissue along with periodontal ligament space. This promising approach would potentially yield a cell-based alternative, wherein a bio-root is formed instead of using an artificial dental implant. Nevertheless, the HA residues seemed to contribute to a structural change which resulted in diminished mechanical strength. Additionally, Gebhardt et al. studied the survival of DPSCs and PDLSCs in tissue-engineered dental pulp and periodontal constructs, comparing their presence in three different kinds of scaffolds: calcium phosphate, a synthetic polymer and collagen. They discovered that the first two were notably conducive to cell survival and proliferation, with cell numbers increasing over time. The results suggest that these two materials could be used to create de novo dental constructs which would provide preformed replacement tissue when implanted into a root canal. Another important contribution was by Elseed et al. who were the first group who sought to determine the effect of using bioactive and bio-adhesive therapies in the attachment of PDLSCs to dentine in the hopes of enhancing it so as to potentially using such therapies for the replantation of avulsed teeth after extended extra-oral periods. They used slabs from human dental lower roots which were either left untreated or coated with bio-adhesive, human recombinant transforming growth factor-beta1 (hrTGF-B1), or human recombinant bone morphogenic protein-2 (hrBMP-2). These dentine slabs were then cultured in contact with confluent PDLSC cultures for 4, 24 and 72 h, and analyzed using scanning electron microscopy (SEM). PDLSCs attached to all root dentine samples, treated and untreated, showing that bioactive coatings are not necessary for the cells to adhere to the surface. However; the addition of BMP-2 seemed to result in a speedier process as the phenotype of the cells changes from oval to flattened earlier than in other cases, suggesting the stimulation of adhesion. It is important to bear in mind, nonetheless, that according to statistical analysis, results may be different in a larger data set. While BMMSCs have been shown to repair periodontal defects by forming bone and cementum, they have failed to regenerate the periodontal ligament. PDLSCs could potentially be the solution to this problem as shown by Liu et al. They treated a minipig with autologous PDLSCs and obtained a satisfactory regeneration of the ligament and alveolar bone. 8.2 Diabetes treatment Recently, Govindasamy et al. obtained islet-like cell aggregates (ICAs) from DPSCs derived from deciduous teeth. They established that the cells were islets through positive staining of dithiozone. Additionally, the cells expressed the islet markers C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. In vitro , they were able to show functionality as, after 10 days, these aggregates responded to glucose by releasing insulin and C-peptide increased in a dose dependent fashion. Overall their study proved that DPSCs are capable of differentiating into the pancreatic cell lineage. An important point to consider is that although there are numerous similarities between the BMMSC- and DPSC-derived insulin-producing cells in vitro , there is an important difference: the latter are mature insulin-secreting ICAs, while the former only give rise to immature islets incapable of producing insulin. 8.3 Liver function recovery Ishkitiev et al. were able to obtain hepatocyte-like cells from dental stem cells. They used magnetic separation and CD117 antibodies to obtain pure SHED and DPSC cultures and differentiated them into the hepatic lineage using serum-free medium (SFM). Large amounts of these cells were positive for the stem cell surface marker CD117 as well as endodermal, mesodermal, ectodermal, mesenchymal, and embryonic markers. Most of the CD117-positive cells showed the hepatic-specific markers HNF-4a, albumin, aFP, CPS-1 and, IGF-I. Additionally, these cells changed their morphology to hepatocyte-like ovoids and contained high amounts of glycogen in their cytosol and urea concentration in the media increased considerably after differentiation. Both populations are good candidates for creating hepatocyte-like lineages ; however, SHED seem to be a better hepatic progenitor source as they are believed to be a more immature stem cell population and have been shown to have neurogenic differentiation capabilities. 8.4 Cardiac function improvement In a murine study of acute myocardial infarctions, DPSCs transplanted into the affected zone caused partial repair of the area four weeks later. The mouse hearts showed higher numbers of vessels and a smaller infarct area, which help improve cardiac function overall. Interestingly, the implanted DPSCs never differentiated into muscle cells, which indicates that their effects were inductive, probably through the secretion of cytokines and growth factors. This could be a breakthrough in the use of DPSCs to treat ischaemic diseases. 8.5 Bone repair As early as 2005, there have been reports of DPSCs causing osteogenesis both in vitro and in vivo . SHED have also been proven to have the ability to generate bone tissue, by inducing host cells to produce osteoblasts. Since PDSCs and SHED are more proliferative and obtained less invasively than BMMSCs, they are a good alternative for use in bone regeneration. Interestingly, a study used SHED/β-TCP to successfully reconstruct defects in the mandibles of minipigs, achieving bone regeneration in a large animal model for the first time. Since every investigator has used a scaffold of their own preference, no optimal method for bone regeneration has been determined using dental stem cells. 8.6 Neural tissue regeneration SHED are capable of differentiating into neurons, while DPSCs are able to obtain neuronal morphology and express gene and protein markers specific to neuronal cells, both in vitro and in vivo . Researchers were also able to generate a sodium current under the effects of inductive media. In a more recent study, Arthur et al. used an animal model to show that axons in the trigeminal ganglion migrated in the direction of implanted DPSCs as a result of them expressing CXCL12. These findings suggest that DPSCs may be capable of inducing neuroplasticity in the host. These advances show how promising dental stem cells can be in the field of neuronal regeneration, providing a less invasive option. 9 Conclusion It has only been thirteen years since the first human dental stem cells were discovered. Although we have learned much about them in this short time, there is still even more to uncover. We do know; however, that due to their multilineage potential which allows the differentiation into the three germ layers, they are very good candidates for inducing tissue regeneration. All dental stem cells mentioned here have been demonstrated to regenerate dental pulp and periodontal tissue. Function of those regenerative dental tissues needs to be further evaluated. DPSCs and SHED have been shown to mend small bone defects. With further research to optimize the conditions for bone regeneration of DPSCs and SHED, they can potentially be used for treating larger trauma injury or even for construction of entire bone implants. SHED have also been differentiated into neuron-like cells while DPSCs have been shown to induce neuroplasty, showing great possibilities to address neurodegenerative diseases and nerve damage due to traumatic injuries. Moreover, DPSCs have also differentiated into islet-like cells, giving a new alternative route for diabetes research, and when injected into hearts resulted in increased function and vasculature due to paracrine effects, which is a valuable option for treating ischaemic diseases. In addition, DPSCs and SHED were shown to differentiate into hepatocyte lineage, indicating another potential cell sources for treating liver disease. Importantly, they are derived from an easily accessible, abundant source. Although the amount of cells that can be derived from one donor or tooth may be small, their proliferative capacity may allow us produce an adequate number of cells for therapeutic uses. Still, dental stem cell research is currently in early stages and it is far from the clinics. Preclinical evaluation in animal models needs to be performed in order to verify the capability of dental stem cells to regenerate functional tissues or restore the function of diseased/injured tissues. 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