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Human dental pulp-derived stem cells promote locomotor recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms
Kiyoshi Sakai, Akihito Yamamoto, Kohki Matsubara, Shoko Nakamura, Mami Naruse, Mari Yamagata, Kazuma Sakamoto, Ryoji Tauchi, Norimitsu Wakao, Shiro Imagama, Hideharu Hibi, Kenji Kadomatsu, Naoki Ishiguro and Minoru Ueda
2011 December 1
[Link: JCI]

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Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.

Differentiation of induced pluripotent stem cells into dental mesenchymal cells.
Otsu K, Kishigami R, Oikawa-Sasaki A, Fukumoto S, Yamada A, Fujiwara N, Ishizeki K, Harada H.
2011 November 15
[Link: PubMed]

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Similar to embryonic stem cells, induced pluripotent stem (iPS) cells can differentiate into various cell types upon appropriate induction, and thus, may be valuable cell sources for regenerative medicine. However, iPS cells have not been reported to differentiate into odontogenic cells for tooth regeneration. Here we demonstrated that neural crest-like cells (NCLC) derived from mouse iPS cells have the potential to differentiate into odontogenic mesenchymal cells. We developed an efficient culture protocol to induce the differentiation of mouse iPS cells into NCLC. We confirmed that the cells exhibited NC cell markers by immunocytochemistry, flow cytometry, and real-time RT-PCR. Furthermore, in the recombination culture of NCLC and mouse dental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration as well as tooth development studies.

Dental pulp stem cells (DPSCs) are an adult stem cell population with high proliferative potential and the ability to differentiate in many cell types, and this has led scientists to consider these cells to be an alternative source of postnatal stem cells comparable to mesenchymal stem cells from bone marrow.
Giorgio Mori, Giacomina Brunetti, Angela Oranger, Claudia Carbone, Andrea Ballini, Lorenzo Lo Muzio, Silvia Colucci, Claudio Mori, Felice Roberto Grassi, Maria Grano
2011 November 14
[Link: Wiley Online Library]

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Dental pulp stem cells (DPSCs) are an adult stem cell population with high proliferative potential and the ability to differentiate in many cell types, and this has led scientists to consider these cells to be an alternative source of postnatal stem cells comparable to mesenchymal stem cells from bone marrow. In this work, we studied the osteoblastic phenotype developed by DPSCs cultured in osteogenic medium. In particular, we analyzed the expression of the typical osteoblast markers such as alkaline phosphatase, collagen type I, osteocalcin, osteopontin, as well as mineralized matrix production. Furthermore, the gene expression during DPSC differentiation into osteoblastic cells was studied by microarray technology. Using microarray and reverse transcriptase–polymerase chain reaction (RT-PCR) analysis, we found that IGFBP-5, JunB, and NURR1 genes are upregulated during the differentiation of DPSCs. These data indicate that opportunely differentiated DPSCs show a correct osteoblastic phenotype. Therefore, during the osteoblastic differentiation process, IGFBP-5, JunB, and NURR1 gene expression is significantly increased.

Human dental pulp stem cells demonstrate better neural and epithelial stem cell properties than bone marrow-derived mesenchymal stem cells.
Karaöz E, Demircan PC, Sağlam O, Aksoy A, Kaymaz F, Duruksu G.
2011 August 31
[Link: PubMed]

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Dental pulp stem cells (hDP-SCs) were primarily derived from pulp tissues of primary incisors, exfoliated deciduous and permanent third molar teeth. To understand the characteristics of hDP-SCs from impacted third molar, proliferation capacities, gene expression profiles, phenotypic, ultrastructural, and differentiation characteristics were analyzed in comparison with human bone marrow-derived mesenchymal stem cells (hBM-MSCs), extensively. hDP-SCs showed more developed and metabolically active cells. Contrary to hBM-MSCs, hDP-SCs strongly expressed both cytokeratin (CK)-18 and -19, which could involve in odontoblast differentiation and dentine repair. The intrinsic neuro-glia characteristics of hDP-MSCs were demonstrated by the expression of several specific transcripts and proteins of neural stem cell and neurons. These cells not only differentiate into adipogenic, osteogenic, and chondrogenic lineage, but also share some special characteristics of expressing some neural stem cell and epithelial markers. Under defined conditions, hDP-SCs are able to differentiate into both neural and vascular endothelial cells in vitro. Dental pulp might provide an alternative source for human MSCs. hDP-SCs with a promising differentiation capacity could be easily isolated, and possible clinical use could be developed for neurodegenerative and oral diseases in the future.

Biological approaches toward dental pulp regeneration by tissue engineering.
Sun HH, Jin T, Yu Q, Chen FM.
2011 April 5
[Link: PubMed]

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Root canal therapy has been the predominant approach in endodontic treatment, wherein the entire pulp is cleaned out and replaced with a gutta-percha filling. However, living pulp is critical for the maintenance of tooth homeostasis and essential for tooth longevity. An ideal form of therapy, therefore, might consist of regenerative approaches in which diseased/necrotic pulp tissues are removed and replaced with regenerated pulp tissues to revitalize the teeth. Dental pulp regeneration presents one of the most challenging issues in regenerative dentistry due to the poor intrinsic ability of pulp tissues for self-healing and regrowth. With the advent of modern tissue engineering and the discovery of dental stem cells, biological therapies have paved the way to utilize stem cells, delivered or internally recruited, to generate dental pulp tissues, where growth factors and a series of dentine extracellular matrix molecules are key mediators that regulate the complex cascade of regeneration events to be faithfully fulfilled.

Human neural crest-derived postnatal cells exhibit remarkable embryonic attributes either in vitro or in vivo
d'Aquino R, Tirino V, Desiderio V, Studer M, De Angelis GC, Laino L, De Rosa A, Di Nucci D, Martino S, Paino F, Sampaolesi M, Papaccio G.
2011 Mar 22
[Link: PubMed]

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During human embryonic development, odontogenic tissues, deriving from the neural crest, remain undifferentiated until the adult age. This study was aimed at characterising the cells of the follicle enveloping the dental germ, due to its direct origin from neural crests. Sixty dental follicles were collected from patients aged 18 to 45 years. This research has clarified that dental follicles, if extracted in a very early stage, when dental roots did not start to be formed, contain a lineage of cells, characterised by a high degree of plasticity in comparison with other adult stem cell populations. In particular, we found that these cells share the following features with ES: (i) high levels of embryonic stem cell markers (CD90, TRA1-60, TRA1-81, OCT-4, CD133, and SSEA-4); (ii) mRNA transcripts for Nanog and Rex-1; (iii) broader potency, being able to differentiate in cell types of all three germ layer, including smooth and skeletal muscle, osteoblasts, neurons, glial cells, and adipocytes; (iv) high levels of telomerase activity; (v) ability to form embryoid bodies; (vi) ability, after injection in murine blastocysts, to be localised within the inner cell mass; (vii) no teratoma formation after injection; (viii) in vivo tissue formation after transplantation. Our results demonstrate that these cells represent a very easy accessible and extraordinary source of pluripotent cells and point out the fact that they own the cardinal feature of embryonic stem cells.

Comparative Analysis of Telomere Length, Telomerase and Reverse Transcriptase Activity in Human Dental Stem Cells.
Jeon BG, Kang EJ, Mohana Kumar B, Maeng GH, Ock SA, Kwack DO, Park BW, Rho GJ.
2011 March 8
[Link: PubMed]

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Stem cells from dental tissues have been isolated and established for tooth regenerative applications. However, basic characterization on their biological properties still needs to be investigated before employing them for effective clinical trials. In this study, we compared the telomere length, relative telomerase activity (RTA) and relative reverse transcriptase activity (RRA) as well as the surface antigen profile and mesenchymal differentiation ability in human dental papilla stem cells (DPaSCs) , dental pulp stem cells (DPuSCs) and dental follicle stem cells (DFSCs) with mesenchymal stem cells (MSCs) derived from bone marrow. Dental stem cells (DSCs) were strongly positive for cell surface markers, such as CD44 and CD90. However, slightly lower expression of CD105 was observed in DPaSCs and DPuSCs compared to DFSCs and MSCs. Following specific induction, DPaSCs, DFSCs and MSCs were successfully differentiated into adipocytes and osteocytes. However, DPuSCS, in particular, were able to differentiate into adipocytes but failed to induce into osteogenic differentiation. Further, all DSCs, MSCs, and MRC- 5 fibroblasts as control were investigated for telomere length by non- radioactive chemiluminescent assay, RTA by relative- quantitative telomerase repeat amplification protocol (RQ- TRAP), and RRA by PCR- based assay. Mean telomere lengths in DPaSCs, DPuSCs, DFSCs and MSCs was ~11 kb, and the values did not differ significantly (P<0.05) among the cells analyzed. RTA levels in DPaSCs were significantly (P<0.05) higher than in MSCs, DPuSCs, DFSCs, and MRC- 5 fibroblasts and among DSCs, DFSCs showed a significantly (P<0.05) lower RTA. Moreover, RRA levels were significantly (P<0.05) higher in DPaSCs, DPuSCs and MSCs than in DFSCs. Based on these observations, we conclude that among DSCs, DPaSCs possessed ideal characteristics on telomere length, telomerase activity and reverse transcriptase (RTase) activity, and may serve as suitable alternative candidates for regenerative medicine.

Expression Pattern of Oct-4, Sox2, and c-Myc in the Primary Culture of Human Dental Pulp Derived Cells.
Liu L, Wei X, Ling J, Wu L, Xiao Y.
2011 Feb 25
[Link: PubMed]

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Dental pulp cells (DPCs) have shown promising potential in dental tissue repair and regeneration. However, during in vitro culture, these cells undergo replicative senescence and result in significant alteration in cell proliferation and differentiation. Recently, the transcription factors of Oct-4, Sox2, c-Myc, and Klf4 have been reported to play a regulatory role in the stem cell self-renewal process, namely cell reprogramming. Therefore, it is interesting to know whether the replicative senescence during the culture of dental pulp cells is related to the diminishing of the expression of these transcription factors. In this study, we investigated the expression of the reprogramming markers Oct-4, Sox2, and c-Myc in the in vitro explant cultured dental pulp tissues and explant cultured dental pulp cells (DPCs) at various passages by immunofluorescence staining and real-time polymerase chain reaction analysis. Our results demonstrated that Oct-4, Sox2, and c-Myc translocated from nucleus in the first 2 passages to cytoplasm after the third passage in explant cultured DPCs. The mRNA expression of Oct-4, Sox2, and c-Myc elevated significantly over the first 2 passages, peaked at second passage (P < .05), and then decreased along the number of passages afterwards (P < .05). For the first time we demonstrated that the expression of reprogramming markers Oct-4, Sox2, and c-Myc was detectable in the early passaged DPCs, and the sequential loss of these markers in the nucleus during DPC cultures might be related to the cell fate of dental pulp derived cells during the long-term in vitro cultivation under current culture conditions.

Stem cells from human exfoliated deciduous teeth (SHED) enhance wound healing and the possibility of novel cell therapy.
Nishino Y, Yamada Y, Ebisawa K, Nakamura S, Okabe K, Umemura E, Hara K, Ueda M.
2011 Feb 22
[Link: PubMed]

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Abstract Background aims. In recent years, stem cells from human exfoliated deciduous teeth (SHED) have received attention as a novel stem cell source with multipotent potential. We examined the effect on wound-healing promotion with unique stem cells from deciduous teeth as a medical waste. Methods. An excisional wound-splinting mouse model was used and the effect of wound healing among SHED, human mesenchymal stromal cells (hMSCs), human fibroblasts (hFibro) and a control (phosphate-buffered saline; PBS) was evaluated by macroscopy, histology and enzyme-linked immunosorbent assay (ELISA), and the expression of hyaluronan (HA), which is related to wound healing, investigated. Results. SHED and hMSCs accelerated wound healing compared with hFibro and the control. There was a statistically significant difference in wound healing area among hFibro, hMSCs and SHED compared with the control after day 5. At days 7 and 14 after cell transplantation, the histologic observation showed that transplanted PKH26-positive cells were surrounded by human HA binding protein, especially in hMSCs and SHED. HA expression volume values were 1558.41 ± 60.33 (control), 2092.75 ± 42.56 (hFibro), 2342.07 ± 188.10 (hMSCs) and 2314.85 ± 164.91 (SHED) ng/mg, respectively, and significantly higher in hMSCs and SHED compared with hFibro and control at days 7 and 14 (P < 0.05). Conclusions. Our results show that SHED hMSCs have similar effects of wound-healing promotion as hFibro and controls. This implies that SHED might offer a unique stem cell resource and the possibility of novel cell therapies for wound healing in the future.

Differentiation of Dental Pulp Stem Cells Into Islet Like Aggregates
V. Govindasamy, V. S. Ronald, A. N. Abdullah, K. R. Ganesan Nathan, Z. A. C. Ab. Aziz, M. Abdullah, S. Musa, N. H. Abu Kasim, R. R. Bhonde
2011 Feb 18
[Link: SagePub]

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The post-natal dental pulp tissue contains a population of multipotent mesenchymal progenitor cells known as dental pulp stromal/stem cells (DPSCs), with high proliferative potential for self-renewal. In this investigation, we explored the potential of DPSCs to differentiate into pancreatic cell lineage resembling islet-like cell aggregates (ICAs). We isolated, propagated, and characterized DPSCs and demonstrated that these could be differentiated into adipogenic, chondrogenic, and osteogenic lineage upon exposure to an appropriate cocktail of differentiating agents. Using a three-step protocol reported previously by our group, we succeeded in obtaining ICAs from DPSCs. The identity of ICAs was confirmed as islets by dithiozone-positive staining, as well as by expression of C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. There were several-fold up-regulations of these transcription factors proportional to days of differentiation as compared with undifferentiated DPSCs. Day 10 ICAs released insulin and C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Our results demonstrated for the first time that DPSCs could be differentiated into pancreatic cell lineage and offer an unconventional and non-controversial source of human tissue that could be used for autologous stem cell therapy in diabetes.
In conclusion, CD31-/CD146- SP cells promoted migration and differentiation of the endogenous neuronal progenitor cells (NPC) and induced vasculogenesis, and ameliorated ischemic brain injury after TMCAO.

Dental pulp stem cells in regenerative dentistry.
Casagrande L, Cordeiro MM, Nör SA, Nör JE.
2011 Jan 27
[Link: PubMed]

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Stem cells constitute the source of differentiated cells for the generation of tissues during development, and for regeneration of tissues that are diseased or injured postnatally. In recent years, stem cell research has grown exponentially owing to the recognition that stem cell-based therapies have the potential to improve the life of patients with conditions that span from Alzheimer's disease to cardiac ischemia to bone or tooth loss. Growing evidence demonstrates that stem cells are primarily found in niches and that certain tissues contain more stem cells than others. Among these tissues, the dental pulp is considered a rich source of mesenchymal stem cells that are suitable for tissue engineering applications. It is known that dental pulp stem cells have the potential to differentiate into several cell types, including odontoblasts, neural progenitors, osteoblasts, chondrocytes, and adipocytes. The dental pulp stem cells are highly proliferative. This characteristic facilitates ex vivo expansion and enhances the translational potential of these cells. Notably, the dental pulp is arguably the most accessible source of postnatal stem cells. Collectively, the multipotency, high proliferation rates, and accessibility make the dental pulp an attractive source of mesenchymal stem cells for tissue regeneration. This review discusses fundamental concepts of stem cell biology and tissue engineering within the context of regenerative dentistry.

Dental Pulp Derived CD31-/CD146- Side Population Stem/Progenitor Cells Enhance Recovery of Focal Cerebral Ischemia in Rats.
Sugiyama M, Iohara K, Wakita H, Hattori H, Ueda M, Matsushita K, Nakashima M.
Nagoya University Granduate School of Medicine
2011 Jan 12
[Link: Pub Med]

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Regenerative therapy using stem cells is a promising approach for the treatment of stroke.
Recently, we reported that CD31-/CD146- side population (SP) cells from porcine dental pulp exhibit highly vasculogenic potential in hindlimb ischemia.
In this study, we investigated the influence of CD31-/CD146- SP cells after transient middle cerebral artery occlusion (TMCAO).
Adult male Sprague-Dawley rats were subjected to 2 hours of TMCAO. Twenty-four hours after TMCAO, CD31-/CD146- SP cells were transplanted into the brain.
Motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined with immunochemical markers, and the levels of neurotrophic factors were assayed with real-time RT-PCR.
In the cell transplantation group, the number of doublecortin-positive cells increased twofold, and the number of NeuN-positive cells increased eightfold, as compared with the control PBS group.
The vascular endothelial growth factor (VEGF) level in the ischemic brain with transplanted cells was 28 times higher than that in the normal brain.

In conclusion, CD31-/CD146- SP cells promoted migration and differentiation of the endogenous neuronal progenitor cells (NPC) and induced vasculogenesis, and ameliorated ischemic brain injury after TMCAO.

Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo.
Király M, Kádár K, Horváthy DB, Nardai P, Rácz GZ, Lacza Z, Varga G, Gerber G.
Department of Oral Biology, Semmelweis University
2011 Jan 08
[Link: Pub Med]

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Pluripotency and their neural crest origin make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement.
Despite recent encouraging results in this field, little is known about the integration of transplanted DPSC derived neuronal pecursors into the central nervous system.
To address this issue, NFneuronally predifferentiated DPSCs, labeled with a vital cell dye Vybrant DiD were introduced into postnatal rat brain. DPSCs were transplanted into the cerebrospinal fluid of 3-day-old male Wistar rats.
Cortical lesion was induced by touching a cold (-60°C) metal stamp to the calvaria over the forelimb motor cortex. Four weeks later cell localization was detected by fluorescent microscopy and neuronal cell markers were studied by immunohistochemistry.
To investigate electrophysiological properties of engrafted, fluorescently labeled DPSCs, 300μm-thick horizontal brain slices were prepared and the presence of voltage-dependent sodium and potassium channels were recorded by patch clamping.
Predifferentiated donor DPSCs injected into the cerebrospinal fluid of newborn rats migrated as single cells into a variety of brain regions. Most of the cells were localized in the normal neural progenitor zones of the brain, the subventricular zone (SVZ), subgranular zone (SGZ) and subcallosal zone (SCZ).
Immunohistochemical analysis revealed that transplanted DPSCs expressed the early neuronal marker N-tubulin, the neuronal specific intermediate filament protein NF-M, the postmitotic neuronal marker NeuN, and glial GFAP. Moreover, the cells displayed TTX sensitive voltage dependent (VD) sodium currents (I(Na)) and TEA sensitive delayed rectifier potassium currents (K(DR)). Four weeks after injury, fluorescently labeled cells were detected in the lesioned cortex. Neurospecific marker expression was increased in DPSCs found in the area of the cortical lesions compared to that in fluorescent cells of uninjured brain. TTX sensitive VD sodium currents and TEA sensitive K(DR) significantly increased in labeled cells of the cortically injured area.

In conclusion, our data demonstrate that engrafted DPSC-derived cells integrate into the host brain and show neuronal properties not only by expressing neuron-specific markers but also by exhibiting voltage dependent sodium and potassium channels. This proof of concept study reveals that predifferentiated hDPSCs may serve as useful sources of neuro- and gliogenesis in vivo, especially when the brain is injured.

Human dental pulp stem cells protect mouse dopaminergic neurons against MPP+ or rotenone.
Nesti C, Pardini C, Barachini S, D'Alessandro D, Siciliano G, Murri L, Petrini M, Vaglini F.
RRMR/CUCCS (Rete Regionale di Medicina Rigenerativa/Center for the Clinical Use of Stem Cells), Italy
2011 Jan 07
[Link: Pub Med]

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive death of substantia nigra dopaminergic neurons that results in a regional loss of striatal dopamine (DA) levels.
Dental pulp contains ex vivo-expandable cells called dental pulp stem cells (DPSCs), with the capacity to differentiate into multiple cell lineages.
More interestingly, due to their embryonic origin, DPSCs express neurotrophic factors such as brain-derived neurotrophic factor, nerve growth factor and glial cell-derived neurotrophic factor.
The aim of the present study was to investigate the neuroprotective effects of DPSCs against MPP+ (2.5, 5, and 10 μM) and rotenone (0.25, 0.5 and 1 μM) in an in vitro model of PD, using an indirect co-culture system with mesencephalic cell cultures.
When mesencephalic cultures were challenged with MPP+ or rotenone, in the presence of DPSCs a statistically significant protective effect was observed at all the tested doses in terms of DA uptake. DPSCs protective effect on DA neurons was also confirmed by immunocytochemistry: an increased number of spared tyrosine hydroxylase (TH)+ cells was observed in co-culture conditions compared to controls, and neurons showed longer processes in comparison with mesencephalic cells grown without DPSCs.

In conclusion, the co-culture with DPSCs significantly attenuated MPP+ or rotenone-induced toxicity in primary cultures of mesencephalic neurons. Considering that the direct contact between the two cell types was prevented, it can be speculated that neuroprotection could be due to soluble factors such as BDNF and NGF, released by DPSCs. Blocking BDNF and NGF with neutralizing antibodies, the neuroprotecting effect of DPSCs was completely abolished. Therefore DPSCs can be viewed as possible candidates for studies on cell-based therapy in neurodegenerative disorders.

Stem cell-based biological tooth repair and regeneration
Ana Angelova Volponi, Yvonne Pang and Paul T. Sharpe
Advanced Centre for Biochemical Engineering, University College London, London, UK
Web 2011 Jan 05
[Link: Pub Med]

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Teeth exhibit limited repair in response to damage, and dental pulp stem cells probably provide a source of cells to replace those damaged and to facilitate repair.
Stem cells in other parts of the tooth, such as the periodontal ligament and growing roots, play more dynamic roles in tooth function and development.
Dental stem cells can be obtained with ease, making them an attractive source of autologous stem cells for use in restoring vital pulp tissue removed because of infection, in regeneration of periodontal ligament lost in periodontal disease, and for generation of complete or partial tooth structures to form biological implants.
As dental stem cells share properties with mesenchymal stem cells, there is also considerable interest in their wider potential to treat disorders involving mesenchymal (or indeed non-mesenchymal) cell derivatives, such as in Parkinson's disease.

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