PUBLISHED RESEARCH 2009

Stem cells have been isolated from many tissues and organs, including dental tissue. Five types of dental stem cells have been established: dental pulp stem cells, stem cells from exfoliated deciduous teeth, stem cells from apical papilla, periodontal ligament stem cells, and dental follicle progenitor cells. The main characteristics of dental stem cells are their potential for multilineage differentiation and self-renewal capacity. Dental stem cells can differentiate into odontoblasts, adipocytes, neuronal-like cells, glial cells, osteoblasts, chondrocytes, melanocytes, myotubes, and endothelial cells. Possible application of these cells in various fields of medicine makes them good candidates for future research as a new, powerful tool for therapy. Although the possible use of these cells in therapeutic purposes and tooth tissue engineering is still in the beginning stages, the results are promising. The efforts made in the research of dental stem cells have clarified many mechanisms underlying the biological processes in which these cells are involved. This review will focus on the new findings in the field of dental stem cell research and on their potential use in the therapy of various disorders.

To date, 5 different human dental stem/progenitor cells have been isolated and characterized: dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAP), and dental follicle progenitor cells (DFPCs). These postnatal populations have mesenchymal-stem-cell-like (MSC) qualities, including the capacity for self-renewal and multilineage differentiation potential. MSCs derived from bone marrow (BMMSCs) are capable of giving rise to various lineages of cells, such as osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic cells. The dental-tissue-derived stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells. However, they also have the ability to give rise to other cell lineages similar to, but different in potency from, that of BMMSCs. This article will review the isolation and characterization of the properties of different dental MSC-like populations in comparison with those of other MSCs, such as BMMSCs. Important issues in stem cell biology, such as stem cell niche, homing, and immunoregulation, will also be discussed.

Tooth derived cells are readily accessible and provide an easy and minimally invasive way to obtain and store stem cells for future use. Banking ones own tooth-derived stem cells is a reasonable and simple alternative to harvesting stem cells from other tissues. Obtaining stem cells from human exfoliated deciduous teeth (SHED) is simple and convenient, with little or no trauma. Every child loses primary teeth, which creates the perfect opportunity to recover and store this convenient source of stem cells--should they be needed to treat future injuries or ailments and presents a far better alternative to simply discarding the teeth or storing them as mementos from the past. Furthermore, using ones own stem cells poses few, if any, risks for developing immune reactions or rejection following transplantation and also eliminates the potential of contracting disease from donor cells. Stem cells can also be recovered from developing wisdom teeth and permanent teeth. Individuals have different opportunities at different stages of their life to bank these valuable cells. It is best to recover stem cells when a child is young and healthy and the cells are strong and proliferative. The purpose of this review is to discuss the present scenario as well as the technical details of tooth banking as related to SHED cells.

OBJECTIVES: To investigate the biological effects of hypoxia on the proliferation ability of human dental pulp cells(HDPCs), detect the relative amount of the messenger RNA of hypoxia induced factor-1a(HIF-1a), stromal cell-derived factor-1a(SDF-1a) and CXCR4 in HDPCs under hypoxia condition, and explore the chemotactic effect of exogenous recombinant human SDF-1a(rhSDF-1a) on hypoxic HDPCs.
METHODS: Cultured HDPCs were exposed to normoxia(20%O2)or hypoxia(1% O2) for 6, 12, 18 and 24 hours, then the proliferation of HDPCs was assessed by MTT assay. After being exposed to hypoxia for 6,12,18 and 24 hours, the cell samples were collected and the relative amount of the messenger RNA of HIF-1a, CXCR4 and SDF-1a were assayed by real-time quantitative PCR. The chemotactic effect of 100ng/ml rhSDF-1a on HDPCs either cultured in normoxia or hypoxia for 18 hours was measured by the in vitro chemotaxis assay.
RESULTS: MTT assay showed increased OD value in all hypoxic groups during the 24hs' cultivation (P<0.05). Comparing with the normoxic group, Real-time quantitative PCR showed that the expression level of CXCR4 increased in the 18h's groups (P<0.05), while the expression level of SDF-1a decreased in the 6h's, 18h's and 24h's groups (P<0.05). The chemotaxis assay showed that hypoxia enhanced the migration ability of HDPCs, and 100ng/ml rhSDF-1a migrated more hypoxic HDPCs than normoxic HDPCs (P<0.05).
CONCLUSIONS: During the 24hs' cultivation in hypoxic environment, HDPCs proliferated at a faster rate. Comparing with the normoxic group, the expression level of CXCR4 increased and the expression level of SDF-1a decreased. Furthermore, hypoxia enhanced the migration ability of HDPCs and recombinant human SDF-1a could migrate hypoxic HDPCs efficiently. These results suggest that hypoxia may recruit HDPCs to sites of injury through activating the SDF-1-CXCR4 pathway.

OBJECTIVES: Stromal stem cells display extensive proliferative capacity of multilineage differentiation and offer a large therapeutic potential in the field of regenerative medicine. The stromal compartment of mesenchymal tissues is considered to harbor stem cells. The present study is a comparison of differentiation towards endodermal lineage properties of mesenchymal cell cultures from milk tooth pulp and third molar pulp.
METHODS: Cell cultures were isolated from milk tooth and third molar pulp and were grown in DMEM supplemented with 10 % FBS. Cells were characterized for expressing stem cell markers CD117, CD44H, Oct3/4 by immunofluorescency and flow-cytometry. After 3 to 5 passages we added to the media 20 ng/ml hepatocyte growth factor (HGF) for 5 days for hepatic commitment. For hepatic differentiation the cells were cultured in DMEM, 20 ng/ml HGF, 10 nM dexamethasone, insulin-transferrin-selenium X, 10 ng/ml oncostatin and 2% FBS for 15 days.
RESULTS: Both mesenchymal cell lines were proven to be positive for pluripotent cell markers CD117, CD44H, Oct3/4. After hepatic induction both cell types changed from spindle shaped, fibroblast like to polygonal, parenchimal-like morphology. The alpha feto-protein and albumin expression were found during the differentiating process by immunofluorescency and ELISA. Mesenchymal cells were expanded in vitro and maintained in an undifferentiated state for more than 50 population doublings. Thus the cells differentiated into cells with morphological, phenotypic, and functional characteristics of hepatocytes.
CONCLUSIONS: The present results demonstrated the ability of both wisdom and milk tooth pulp mesenchymal cell cultures to differentiate to endodermal type of cells, normally not presented in tooth's pulp. These cells also acquired functional characteristics of hepatocytes: they secreted alpha feto-protein. Dental pulp mesenchymal cells obtained from each patient, requiring liver transplantation may therefore be ideal for in vivo therapies for these patients.

BACKGROUND AIMS: We have isolated human neuronal stem cells from exfoliated third molars (wisdom teeth) using a simple and efficient method. The cultured neuronal stem cells (designated tNSC) expressed embryonic and adult stem cell markers, markers for chemotatic factor and its corresponding ligand, as well as neuron proteins. The tNSC expressed genes of Nurr1, NF-M and nestin. They were used to treat middle cerebral artery occlusion (MCAO) surgery-inflicted Sprague-Dawley (SD) rats to assess their therapeutic potential for stroke therapy.
METHODS: For each tNSC cell line, a normal human impacted wisdom tooth was collected from a donor with consent. The tooth was cleaned thoroughly with normal saline. The molar was vigorously shaken or vortexed for 30 min in a 50-mL conical tube with 15-20mL normal saline. The mixture of dental pulp was collected by centrifugation and cultured in a 25-cm(2) tissue culture flask with 4-5mL Medium 199 supplemented with 5-10% fetal calf serum. The tNSC harvested from tissue culture, at a concentration of 1-2x10(5), were suspended in 3 microL saline solution and injected into the right dorsolateral striatum of experimental animals inflicted with MCAO.
RESULTS: Behavioral measurements of the tNSC-treated SD rats showed a significant recovery from neurologic dysfunction after MCAO treatment. In contrast, a sham group of SD rats failed to recover from the surgery. Immunohistochemistry analysis of brain sections of the tNSC-treated SD rats showed survival of the transplanted cells.
CONCLUSIONS: These results suggest that adult neuronal stem cells may be procured from third molars, and tNSC thus cultivated have potential for treatment of stroke-inflicted rats.

The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-l-lysine coated surfaces and pretreated for 48h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol.

Dental pulp is a promising source of mesenchymal stem cells with the potential for cell-mediated therapies and tissue engineering applications. We recently reported that isolation of dental pulp-derived stem cells (DPSC) is feasible for at least 120h after tooth extraction, and that cryopreservation of early passage cultured DPSC leads to high-efficiency recovery post-thaw. This study investigated additional processing and cryobiological characteristics of DPSC, ending with development of procedures for banking. First, we aimed to optimize cryopreservation of established DPSC cultures, with regards to optimizing the cryoprotective agent (CPA), the CPA concentration, the concentration of cells frozen, and storage temperatures. Secondly, we focused on determining cryopreservation characteristics of enzymatically digested tissue as a cell suspension. Lastly, we evaluated the growth, surface markers and differentiation properties of DPSC obtained from intact teeth and undigested, whole dental tissue frozen and thawed using the optimized procedures. In these experiments it was determined that Me(2)SO at a concentration between 1 and 1.5M was the ideal cryopreservative of the three studied. It was also determined that DPSC viability after cryopreservation is not limited by the concentration of cells frozen, at least up to 2x10(6) cells/mL. It was further established that DPSC can be stored at -85 degrees C or -196 degrees C for at least six months without loss of functionality. The optimal results with the least manipulation were achieved by isolating and cryopreserving the tooth pulp tissues, with digestion and culture performed post-thaw. A recovery of cells from >85% of the tissues frozen was achieved and cells isolated post-thaw from tissue processed and frozen with a serum free, defined cryopreservation medium maintained morphological and developmental competence and demonstrated MSC-hallmark trilineage differentiation under the appropriate culture conditions.

It is well known that interactions between epithelial components and mesenchymal components are essential for tooth development. Therefore, it has been postulated that both types of stem cells might be involved in the regeneration of dental hard tissues. Recently, mesenchymal dental pulp stem cells that have odontogenic potential were identified from human dental pulp. However, the existence of epithelial cells has never been reported in human dental pulp. In the present study, we isolated and characterized epithelial cell-like cells from human deciduous dental pulp. They had characteristic epithelial morphology and expressed epithelial markers. Moreover, they expressed epithelial stem cell-related genes such as ABCG2, Bmi-1, DeltaNp63, and p75. Taken together, our findings suggest that epithelial stem cell-like cells might exist in human deciduous dental pulp and might play a role as an epithelial component for the repair or regeneration of teeth.

INTRODUCTION: Postnatal human dental pulp is a potentially promising source of progenitor cells. Sustaining and amplifying progenitor cell populations would be beneficial for basic science research with application in pulpal regeneration. Hypoxia has been observed to promote the undifferentiated cell state in various stem cell populations. The purpose of this study was to examine human dental pulp cells (DPCs) proliferation in normoxia and hypoxia.
METHODS: Dental pulp cells were obtained from third molars of adult patients and cultured in alpha modification of Eagle's medium culture medium with 10% fetal bovine serum. For cell proliferation, DPCs were divided into two groups: (1) DPCs incubated in normoxic conditions (20% oxygen tension) and (2) DPC incubated in hypoxic conditions (3% oxygen tension). Cell proliferation assays were performed every 2 to 3 days from day 3 to day 14 by trypsinization and quantification of cells with a hemacytometer. Fluorescence-activated cell sorting analysis was completed to investigate stem cell markers, CD133, and STRO-1.
RESULTS: DPCs proliferated significantly more in hypoxia than in normoxia (ie, two-fold throughout the experiment, p < 0.0001). The primitive stem cell marker, CD133, decreased in hypoxia, whereas the osteoprogenitor marker, STRO-1, increased by 8.5-fold.
CONCLUSIONS: This study suggested that hypoxia is an effective treatment to amplify numbers of progenitor cells from human dental pulp.

AIMS: Our aims were to isolate dental pulp stem cells, to cultivate them in various media and to investigate their basic biological properties and phenotype.
METHODS: 16 lines of dental pulp stem cells (DPSCs) were isolated from an impacted third molar. After enzymatic dissociation of dental pulp, DPSCs were cultivated in modified cultivation media for mesenchymal adult progenitor cells containing 2 % or 10 % fetal calf serum (FCS), or in modified 2 % FCS cultivation media supplemented with ITS. Cell viability and other biological properties were examined periodically using a Vi-Cell analyzer and Z2-Counter. DNA analysis and phenotyping were done using flow cytometry.
RESULTS: We were able to cultivate DPSCs in all tested cultivation media over 40 population doublings. Our results showed that DPSCs cultivated in medium supplemented with ITS had shorter average population doubling time (24.5, 15.55-35.12 hours) than DPSCs cultivated in 2 % FCS (55.43, 21.57-187.14 hours) or 10 % FCS (42.56, 11.86 - 101.3 hours). Cell diameter was not affected and varied from 15 to 16 microm. DPSCs viability in the 9(th) passage was over 90 %. Our phenotypical analysis was highly positivity for CD29, CD44, CD90 and HLA I, and negative for CD34, CD45, CD71, HLA II. DPSC lines cultivated in all media showed no signs of degeneration or spontaneous differentiation during the expansion process.
CONCLUSIONS: We showed that ITS supplement in the cultivation media greatly increased the proliferative activity of DPSCs. Other DPSC biological properties and phenotype were not affected.

Regenerating human tooth ex vivo and biological repair of dental caries are hampered by non-viable odontogenic stem cells that can regenerate different tooth components. Odontoma is a developmental dental anomaly that may contain putative post-natal stem cells with the ability to differentiate and regenerate in vivo new dental structures that may include enamel, dentin, cementum and pulp tissues. We evaluated odontoma tissues from 14 patients and further isolated and characterized human odontoma-derived mesenchymal cells (HODCs) with neural stem cell and hard tissue regenerative properties from a group of complex odontoma tissues from 1 of 14 patients. Complex odontoma was more common (9 of 14) than compound type and females (9 of 14) were more affected than males in our set of patients. HODCs were highly proliferative like dental pulp stem cells (DPSCs) but demonstrated stronger neural immunophenotype than both DPSCs and mandible bone marrow stromal cells (BMSCs) by expressing higher levels of nestin, Sox 2 and betaIII-tubulin. When transplanted with hydroxyapatite/tricalcium phosphate into immunocompromised mice, HODCs differentiated and regenerated calcified hard tissues in vivo that were morphologically and quantitatively comparable to those generated by DPSCs and BMSCs. When transplanted with polycaprolactone (biodegradable carrier), HODCs differentiated to form new predentin on the surface of a dentin platform. Newly formed predentin contained numerous distinct dentinal tubules and an apparent dentin-pulp arrangement. HODCs represent unique odontogenic progenitors that readily commit to formation of dental hard tissues.

PURPOSE: Postnatal stem cells have been isolated from various tissues, including bone marrow, neural tissue, skin, retina, and dental epithelium. Recently, adult stem cells have been isolated from human dental pulp. Postnatal stem cells have been isolated from a variety of tissues. Previously, it was generally accepted that the differentiation potential of postnatal stem cells was lineage restricted.
MATERIALS AND METHODS: Normal impacted third molars were collected from adults and normal exfoliated deciduous teeth (SHED; stem cells from human exfoliated deciduous teeth) by single-colony selection and magnetic activated cell sorting.
RESULTS: BMP-2 treatment groups produced alkaline phosphatase in the cells and also produced and secreted osteocalcin in the culture medium, and were capable of inducing an upregulated expression of Osteocalcin or Sox9, Col 2, and Col X by reverse transcriptase polymerase chain reaction (RT-PCR). For adipogenic differentiation, there is potential for SHED and dental pulp stem cells (DPSC) to express 2 adipocyte-specific transcripts, PPARgamma2 and LPL, in vitro, as do bone marrow mesenchymal stem cells by RT-PCR.
CONCLUSION: This study demonstrated that pluripotential cells isolated from the pulp of human teeth expanded in vitro and differentiated into osteoblasts, chondrocytes, and adipocytes. DPSC and SHED are not only derived from a very accessible tissue resource but also capable of providing enough cells for potential clinical applications.

INTRODUCTION: It is now accepted that progenitor/stem cells reside within the post-natal dental pulp. Studies have identified several niches of multipotent mesenchymal progenitor cells, known as dental pulp stem cells, which have a high proliferative potential for self-renewal. These progenitor stem cells are now recognized as being vital to the dentine regeneration process following injury. Understanding the nature of these progenitor/stem cell populations in the pulp is important in determining their potentialities and development of isolation or recruitment strategies for use in regeneration and tissue engineering. Characterization of these cells, and determination of their potentialities in terms of specificity of regenerative response, may help direct new clinical treatment modalities. Such novel treatments may involve controlled direct recruitment of the cells in situ and possible seeding of stem cells at sites of injury for regeneration or use of the stem cells with appropriate scaffolds for tissue engineering solutions. Such approaches may provide an innovative and novel biologically based new generation of clinical materials and/or treatments for dental disease. AIM: This study aimed to review the body of knowledge relating to stem cells and to consider the possibility of these cell populations, and related technology, in future clinical applications.

Myocardial infarction is a major public health problem that causes significant mortality despite recent advances in its prevention and treatment. Therefore, approaches based on adult stem cells represent a promising alternative to conventional therapies for this life-threatening condition. Mesenchymal stem cells (MSCs) are self-renewing pluripotent cells that have been isolated from multiple tissues and differentiate to various cell types. Here we have analyzed the capacity of MSCs from human bone marrow (BMSC), adipose tissue (ATSC), and dental pulp (DPSC) to differentiate to cells with a cardiac phenotype. Differentiation of MSCs was induced by long-term co-culture with neonatal rat cardiomyocytes (CMs). Shortly after the establishment of MSC-CM co-cultures, expression of connexin 43 and the cardiac-specific markers troponin I, beta-myosin heavy chain, atrial natriuretic peptide, and alpha-sarcomeric actinin was detected in BMSCs, ATSCs, and DPSCs. Expression of differentiation markers increased over time in the co-cultures, reaching the highest levels at 4 weeks. Translocation of the transcription factors NKX2.5 and GATA4 to the nucleus was observed in all three cultures of MSCs during the differentiation process; moreover, nuclear localization of NKX2.5 and GATA4 correlated with expression of alpha-sarcomeric actinin. These changes were accompanied by an increase in myofibril organization in the resulting CM-like cells as analyzed by electron microscopy. Thus, our results provide novel information regarding the differentiation of tissue-specific MSCs to cardiomyocytes and support the potential use of MSCs in cell-based cardiac therapies.