Accepted Manuscript

Title: Neural Crest-Derived Dental Stem Cells–Where We Areand Where We Are Going

Author: Vera Mayo Yoh Sawatari C.-Y. Charles HuangFranklin Garcia-Godoy

PII: S0300-5712(14)00115-8DOI: http://dx.doi.org/doi:10.1016/j.jdent.2014.04.007Reference: JJOD 2281

To appear in: Journal of Dentistry

Received date: 30-1-2014Revised date: 11-4-2014Accepted date: 14-4-2014

Please cite this article as: Mayo V, Sawatari Y, Huang C-YC, Garcia-Godoy F, NeuralCrest-Derived Dental Stem Cells–Where We Are and Where We Are Going, Journalof Dentistry (2014), http://dx.doi.org/10.1016/j.jdent.2014.04.007

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Neural Crest-Derived Dental Stem Cells – Where We Are and Where We Are Going

Vera Mayo1, Yoh Sawatari2, C.-Y. Charles Huang1, Franklin Garcia-Godoy3

1Department of Biomedical Engineering, University of Miami, Coral Gables, FL 33146, USA

2 Division of Oral and Maxillofacial Surgery, University of Miami

3Department of Bioscience Research, College of Dentistry, University of Tennessee Health

Science Center, College of Dentistry, University of Tennessee

Corresponding Author C.-Y. Charles Huang, Ph.D. Department of Biomedical Engineering College of Engineering University of Miami P.O. Box 248294 Coral Gables, FL 33124-0621 USA Telephone: (305) 284-1320 Fax: (305) 284-6494 Email: c.huang@miami.edu

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Abstract

There are five types of post-natal human dental stem cells that have been identified,

isolated and characterized: dental pulp stem cells, stem cells from exfoliated deciduous teeth,

periodontal ligament stem cells, dental follicle precursor cells, and stem cells from apical papilla.

These populations present properties similar to those of mesenchymal stem cells, such as the

ability to self-renew and the potential for multilineage differentiation. While the dental stem cells

have greater capacity to give rise to odontogenic cells and regenerate dental pulp and periodontal

tissue, they also have the capacity to differentiate into chondrogenic, osteogenic, neurogenic,

myogenic, hepatogenic, adipogenic, and insulin-producing cells. In this way they have been

differentiated into all three germ line cells, proving that a population of pluripotent stem cells

exists in the dental tissues. Thus, dental stem cells have the potential to develop solutions to

different clinical problems such as dental implants, bone repair, neurodegenerative diseases,

heart failure and diabetes. Here, we review the information available on dental stem cells as well

as their potential application in dentistry, regenerative medicine and the development of other

therapeutic approaches.

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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 incisors1, 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

(Figure 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 pulp2. 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)3,4. In 2005, Morsczeck et al. identified dental follicle precursor cells (DFPCs)5, 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 al6.

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

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and perineural sheath regions, while Chen et al. localized PDLSCs in small clusters in the

perivascular region, where Sonoyama et al. also found SCAP 6-8.

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 remodeling. 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, 9.

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 10. Even though positional

information seems to determine cell fate 11, 12, 13, all of these cell populations have different

potentials, being susceptible to environmental and extracellular signals that play a key role in

their differentiation 10.

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

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Anderson 10. 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 14.

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 15.

The undifferentiated state and self-renewal capacity of early NCSCs has been preserved

by activating the bone morphogenic protein (BMP) and Wnt signaling pathways concurrently in

vitro 16; 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 17 and the modulation of their fates both in vitro

and in vivo 18, 19. In 2008, Teng et al. found that Foxd3 is required for the preservation of the

neural crest population in vivo, modulating Sox10 20. Its overexpression; however, upregulates

HNK-1 and Cad-7 in embryonic chicken neural tubes, which are markers of migratory neural

crest 21.

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 22. 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 23,24. This spatiotemporal property allows the NCSCs to react appropriately

according to specific cues 25.

Dental Pulp Stem Cells (DPSCs)

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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 2.

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) 26. 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 27. 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 signaling, metabolism, or

communication 28. 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 29.

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 cells27. Interestingly, DPSCs have preserved their self-renewal

capability and have formed dentin-like and pulp-like tissue as well as bone-like tissue, once

transplanted into animal models 30. When cultured in vitro, DPSCs can be differentiated into

odontoblasts with their characteristic accumulation of mineralized nodules and polarized cell

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bodies. In the last few years, investigators have used DPSCs for neural tissue 31 and corneal 32,33

regeneration, and the treatment of myocardial infarction 34 as well as cerebral ischemia 35.

DPSC colonies grow at different rates and they are composed of a heterogeneous group

of cells, with different morphologies and sizes 9. 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

dentin 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 36.

In a consequent study, the same group isolated human DPSCs through enzyme separation and

seeded them onto mechanically and chemically treated dentin 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 37.

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 dentin 2, 38. 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 dentin sialophosphoprotein (DSPP),

involved in the production of dentin, which resulted in the thickening of dentin 38. 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-

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expressing tissue grew in all three settings; however, it was more similar to connective tissue

than to dentin 39. 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 40. Additionally, another group

found that nanohydroxyapatite (nHA) enhances the differentiation of DPCSs into odontoblast-

like cells 41.

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 42.

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 43.

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 44.

Stem Cells from Human Exfoliated Deciduous Teeth (SHED)

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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 3. The cells survived for over 10 days expressing

neural markers and they have been shown to express both neuronal and glial cell markers 45. 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. 46.

Like DPSCs, SHED express STRO-1 and CD146 as well as Oct4 and tumor recognition

antigens and stage-specific embryonic antigens 46. 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 47.

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In murine models, transplanted SHED formed dentin-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 dentin-pulp

complexes, indicating a difference in odontogenic potential 3. 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 dentin, 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 [3]. In this way, SHED have been found to mend calvarial defects in a murine

model that resulted in bone formation 48. Another interesting property of SHED is that instead of

displaying the regular fibroblastic morphology, they show multicytoplasmic processes 3. They

also have been shown to possess chondrogenic and myogenic potentials 46. 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 49.

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 50. According to Nakamura et al. SHED have better proliferative potential than

DPSCs and BMSCs due to their higher levels of TGF-ß2 and FGF2. The group suggests that this

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is why SHED are potentially the most promising population of cells to be used in tissue

engineering and regeneration 51. 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 52.

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 53. 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 54. The pluripotency of PDLSCs was confirmed by a recent study 55 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

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proliferative properties 56. 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 4. 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 57. 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 58. In

addition, PDLSCs have the capacity to differentiate into both osteoblasts and cementoblasts 59-62,

suggesting that the function PDLSCs is to maintain homeostasis and regenerate periodontal

tissue 61, 62.

PDLSCs have been used to regenerate a cementum-like structure alongside collagen

fibers 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 fibers attached to the cementum-like structures much like it happens with Sharpey’s

fibers 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 4. In 2008, a study showed that PDLSCs are capable of giving rise

to cementum-like cells, when implanted along with noncollagenous dentin proteins 63. In the past

few years, PDLSCs were also found to present osteogenic, chondrogenic, and adipogenic

features when cultured under the right conditions 57, 58, 64. 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 65.

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Dental Follicle Precursor Cells (DFPCs)

The dental follicle controls osteogenesis and osteoclastogenesis and differentiates into the

periodontium during the eruption process 66-69. 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 5. 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 5, 70-72. DFPCs express markers such as nestin and Notch-1 5, as

well as various neural cell markers and cementoblast markers after the respective inductions 73,

74.

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) 6. 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 6,75. SCAP have been shown to differentiated in vitro into neurogenic and

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chondrogenic tissue; and, like DPSCs, have been able to form dentin-like structures containing

odontoblast-like cells in immunocompromised animal models 6.

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 6, 76. Another interesting feature of these cells is that they express neural

markers even when not exposed to neurogenic stimulation 76. However, when stimulated, more

neural markers are expressed 75. When transplanted into immunocompromised mice in an

adequate carrier matrix such as HA, these cells are capable of forming a dentin/pulp-like

complex like the one previously described for DPSCs 9 and they have also been found to

regenerate alveolar bone and periodontal ligament in vivo 6. 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 9. As of now, the field of tooth regeneration still has many other

obstacles to overcome.

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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 77. Conversely, Prescott et

al. used the same model to implant a collagen scaffold seeded with DPSCs and supplemented

with dentin matrix protein (DMP-1). This study resulted in the regeneration of pulp-like tissue 78.

Furthermore, by expanding human SCAP and DPSCs ex-vivo, Huang et al. have been able to

form a uniform dentin-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 dentin-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

tissue79.

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 dentin

was regenerated in this case, no enamel or root was observed 80. 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 dentin and root formation along with

periodontium 81. 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.

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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 6. 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 82.

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 83. 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 hours, 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

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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 83.

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 84.

Diabetes Treatment

Recently, Govindasamy et al. obtained islet-like cell aggregates (ICAs) from DPSCs

derived from deciduous teeth 26. 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 26.

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) 85. Large amounts

of these cells were positive for the stem cell surface marker CD117 as well as endodermal,

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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

lineages85; however, SHED seem to be a better hepatic progenitor source as they are believed to

be a more immature stem cell population9 and have been shown to have neurogenic

differentiation capabilities 3.

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 34. This could be

a breakthrough in the use of DPSCs to treat ischemic diseases.

Bone Repair

As early as 2005, there have been reports of DPSCs causing osteogenesis both in vitro

and in vivo 42. SHED have also been proven to have the ability to generate bone tissue, by

inducing host cells to produce osteoblasts 3. Since PDSCs and SHED are more proliferative and

obtained less invasively than BMMSCs, they are a good alternative for use in bone regeneration

86. Interestingly, a study used SHED/ß-TCP to successfully reconstruct defects in the mandibles

of minipigs, achieving bone regeneration in a large animal model for the first time 87. Since every

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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 3, 88, while DPSCs are able to obtain

neuronal morphology and express gene and protein markers specific to neuronal cells, both in

vitro and in vivo 27, 89. Researchers were also able to generate a sodium current under the effects

of inductive media 90. 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 91. 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

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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 ischemic 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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Pulp: DPSCs, SHED

Gum

Bone

Periodontal Ligament: PDLSCs

Apical Papilla: SCAP 

Figure 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

Dental Follicle: DFPCs  

Unerupted tooth