Establishment of in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of BrdU label-retaining dental pulp cells

We have proposed the new hypothesis that dental pulp stem cells play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with progenitors. This study aimed to establish an in vitro culture system for evaluating dentin–pulp complex regeneration with special reference to the differentiation capacity of slow-cycling long-term label-retaining cells (LRCs). Three intraperitoneal injections of BrdU were given to pregnant ICR mice to map LRCs in the mature tissues of born animals. The upper bilateral first molars of 3-week-old mice were extracted and divided into two pieces and cultured for 0, 1, 3, 5 and 7 days using the Trowel’s method. We succeeded in establishing an in vitro culture system for evaluating dentin–pulp complex regeneration, where most odontoblasts were occasionally degenerated and lost nestin immunoreactivity because of the separation of cell bodies from cellular processes in the dentin matrix by the beginning of in vitro culture. Numerous dense LRCs mainly resided in the center of the dental pulp associating with blood vessels throughout the experimental periods. On postoperative days 1–3, the periphery of the pulp tissue including the odontoblast layer showed degenerative features. By Day 7, nestin-positive odontoblast-like cells were arranged along the pulp–dentin border and dense LRCs were committed in the odontoblast-like cells. These results suggest that dense LRCs in the center of the dental pulp associating with blood vessels were supposed to be dental pulp stem/progenitor cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells.     

Responses of BrdU label-retaining dental pulp cells to allogenic tooth transplantation into mouse maxilla

Recently, we demonstrated that a pulse of BrdU given to prenatal animals reveals the existence of slow-cycling long-term label-retaining cells (LRCs), putative adult stem or progenitor cells, which reside in the dental pulp. This study aims to clarify responses of LRCs to allogenic tooth transplantation into mouse maxilla using prenatal BrdU-labeling, in situ hybridization for osteopontin and periostin, and immunohistochemistry for BrdU, nestin, and osteopontin. The upper-right first molars were allografted in the original socket between BrdU-labeled and non-labeled mice or between GFP transgenic and wild-type mice. Tooth transplantation caused degeneration of the odontoblast layer, resulting in the disappearance of nestin-positive reactions in the dental pulp. On postoperative days 5–7, tertiary dentin formation commenced next to the preexisting dentin where nestin-positive odontoblast-like cells were arranged in the successful cases. In BrdU-labeled transplanted teeth, dense LRCs were maintained in the center of the dental pulp beneath the odontoblast-like cells including LRCs, whereas LRCs disappeared in the area surrounding the bone-like tissue. In contrast, LRCs were not recognized in the pulp chamber of non-labeled transplants through the experimental period. Tooth transplantation using GFP mice demonstrated that the donor cells constituted the dental pulp of the transplant except for endothelial cells and some migrated cells, and the periodontal tissue was replaced by host-derived cells except for epithelial cell rests of Malassez. These results suggest that the maintenance of BrdU label-retaining dental pulp cells play a role in the regeneration of odontoblast-like cells in the process of pulpal healing following tooth transplantation.     

The relationship between cell proliferation and differentiation and mapping of putative dental pulp stem/progenitor cells during mouse molar development by chasing BrdU-labeling

Human dental pulp contains adult stem cells. Our recent study demonstrated the localization of putative dental pulp stem/progenitor cells in the rat developing molar by chasing 5-bromo-2’-deoxyuridine (BrdU)-labeling. However, there are no available data on the localization of putative dental pulp stem/progenitor cells in the mouse molar. This study focuses on the mapping of putative dental pulp stem/progenitor cells in addition to the relationship between cell proliferation and differentiation in the developing molar using BrdU-labeling. Numerous proliferating cells appeared in the tooth germ and the most active cell proliferation in the mesenchymal cells occurred in the prenatal stages, especially on embryonic Day 15 (E15). Cell proliferation in the pulp tissue dramatically decreased in number by postnatal Day 3 (P3) when nestin-positive odontoblasts were arranged in the cusped areas and disappeared after postnatal Week 1 (P1W). Root dental papilla included numerous proliferating cells during P5 to P2W. Three to four intraperitoneal injections of BrdU were given to pregnant ICR mice and revealed slow-cycling long-term label-retaining cells (LRCs) in the mature tissues of postnatal animals. Numerous dense LRCs postnatally decreased in number and reached a plateau after P1W when they mainly resided in the center of the dental pulp, associating with blood vessels. Furthermore, numerous dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 and CD146. Thus, dense LRCs in mature pulp tissues were believed to be dental pulp stem/progenitor cells harboring in the perivascular niche surrounding the endothelium.     

Allogenic tooth transplantation inhibits the maintenance of dental pulp stem/progenitor cells in mice

Our recent study suggested that allogenic tooth transplantation may affect the maintenance of dental pulp stem/progenitor cells. This study aims to elucidate the influence of allograft on the maintenance of dental pulp stem/progenitor cells following tooth replantation and allo- or auto-genic tooth transplantation in mice using BrdU chasing, immunohistochemistry for BrdU, nestin and Ki67, in situ hybridization for Dspp, transmission electron microscopy and TUNEL assay. Following extraction of the maxillary first molar in BrdU-labeled animals, the tooth was immediately repositioned in the original socket, or the roots were resected and immediately allo- or auto-grafted into the sublingual region in non-labeled or the same animals. In the control group, two types of BrdU label-retaining cells (LRCs) were distributed throughout the dental pulp: those with dense or those with granular reaction for BrdU. In the replants and autogenic transplants, dense LRCs remained in the center of dental pulp associating with the perivascular environment throughout the experimental period and possessed a proliferative capacity and maintained the differentiation capacity into the odontoblast-like cells or fibroblasts. In contrast, LRCs disappeared in the center of the pulp tissue by postoperative week 4 in the allografts. The disappearance of LRCs was attributed to the extensive apoptosis occurring significantly in LRCs except for the newly-differentiated odontoblast-like cells even in cases without immunological rejection. The results suggest that the host and recipient interaction in the allografts disturbs the maintenance of dense LRCs, presumably stem/progenitor cells, resulting in the disappearance of these cell types.     

Comparative analysis of ABCG2-expressing and label-retaining cells in mouse submandibular gland

The submandibular gland (SMG) is a tissue that can be regenerated in a tissue injury model and that has adult stem cells capable of self-renewal and differentiation into functional cells. We have analyzed the localization of label-retaining cells (LRCs), which are putative progenitor cells, by using the BrdU-labeling method. 5-Bromo-2′-deoxyuridine (BrdU) injection followed by a long chasing period permitted the identification of LRCs based on the slow-cycling characteristic. In order to confirm the accurate localization of LRCs, BrdU and SMG-specific markers, including aquaporin5, cytokeratin, and smooth muscle actin, were examined by double-immunofluoresence staining. We found that LRCs were distributed in the acinus, duct, myoepithelium, and connective tissue. Moreover, ABCG2 (a known stem cell marker) was used for the characterization of LRCs and the localization of cells as putative stem/progenitor cells. ABCG2-expressing cells were distributed in various regions of the SMG but did not co-localize with LRCs. We suggest that putative progenitor cells exist in various regions of the SMG and have diverse capacities to differentiate into specific cells. Yeun-Jung Kim and Hyuk-Jae Kwon contributed equally to this work. This work was supported by Korea Research Foundation Grant (KRF-2006–013-E00143).     

Identification of label-retaining cells in nasopharyngeal epithelia and nasopharyngeal carcinoma tissues

Adult stem cells can be identified by label-retaining cell (LRC) approach based on their ability to retain nucleoside analog, such as bromodeoxyuridine (BrdU). We hypothesized that mouse nasopharynx contains a small population of epithelial stem/progenitor cells that may be detected by the LRC technique. To identify LRCs in mice nasopharyngeal epithelia, neonatal mice were intraperitoneally injected with BrdU twice daily for 3 consecutive days. After an 8-week chase, long-term BrdU-labeled LRCs (∼2% of cells) were detected in the adult mice nasopharyngeal epithelia by immunostaining with BrdU antibody and some of LRCs (∼12% of cells) were found to be recruited into the S phase of cell cycle with an additional radioactive thymidine-labeling technique, indicating that the stem cells also divide, most likely asymmetrically. To further investigate whether the LRCs existed in human nasopharyngeal carcinoma (NPC) tissues, three NPC cell lines (5-8F, 6-10B and TMNE) were labeled with BrdU in vitro and then individually engrafted into the back of nude mice, which developed tumors. Again, label-retaining stem cells were found in all the three kinds of NPC xenograft tumors (∼0.3% of cells), around 16% of which were also labeled with radioactive thymidine. Thus, this study has demonstrated for the first time the presence of epithelial LRCs in mouse nasopharyngx and human NPC tissues and these stem-like LRCs are not completely quiescent, as they will be recruited into the cell cycle to participate physiological or pathological process at any moment. More importantly, our data showed that NPC also contained stem cells, which are most likely the cause for NPC spread, metastasis and recurrence.     

Identification and characterization of label-retaining cells in mouse pancreas

Pancreatic stem cells (PSCs) may play an important role in maintaining and repairing pancreatic tissues. However, both the existence and localization of PSCs in adult mammalian pancreas still remain elusive. In order to locate the potential pancreatic progenitor/stem cells, we used the tracing label-retaining cells (LRCs) method and identified slow-cycling cells in mouse pancreas. Characterization of the LRCs revealed that the differentiation marker-negative LRCs were located not only within and around the islets but also around the acini and ducts. About 30% of the LRCs around the acini and ducts expressed c-Met, which is a putative pancreatic progenitor/stem cell marker. Moreover, the LRCs around the acini could be activated to form duct-like structures in response to pancreatic damage, and the involvement of these LRCs in the neogenesis of islets and focal areas could also be observed in acini. Our data suggest that the LRCs located around the acini and ducts may represent potential pancreatic progenitor/stem cells, and characterization of these cells may aid in further identification of the specific markers of pancreatic progenitor/stem cells.     

Identification of Tympanic Border Cells as Slow-Cycling Cells in the Cochlea

Mammalian cochlear sensory epithelial cells are believed to possess minimal regenerative potential because they halt proliferation during late stage of embryogenesis and never regenerate after birth. This means that sensorineural hearing loss caused by the death of cochlear sensory epithelial cells is a permanent condition. However, stem cells were recently identified in neonatal mice following dissociation of their inner ear organs. This suggests that regenerative therapy for sensorineural hearing loss may be possible. Unfortunately, dissociation distorts the microanatomy of the inner ear, making it difficult to determine the precise location of stem cells in unaltered specimens. To develop new therapeutic approaches based on sensory epithelial cell regeneration, the location of these stem cells must be elucidated. Stem cells normally proliferate at a slow rate in adult organs. In fact, so-called label-retaining cells, or slow-cycling cells, of the brain and skin are recognized as stem cells. In this study, using the exogenous proliferation marker, 5′-bromo-2′-deoxyuridine (BrdU) in combination with the endogenous proliferation marker Ki-67, we identified tympanic border cells. These cells, which are located beneath the basilar membrane in vivo, represent slow-cycling cells of the murine cochlea. Immunohistochemically, these cells stained positive for the immature cell marker Nestin. But it will be difficult to achieve regeneration of the cochlear function because these slow-cycling cells disappear in the mature murine cochlea.     

Induction of transforming growth factor-beta 1 on dentine pulp cells in different culture patterns

Recent studies have documented that TGF-beta1 takes part in dental pulp tissue repair. Moreover, dental pulp cells have the potential to differentiate into odontoblast-like cells and produce reparative dentine in this process. However, the molecular mechanisms and potential interactions between TGF-beta1 and dental pulp cells are not clear due to the complexity of the pulp/dentine microenvironment. In this study, we investigated the induction of TGF-beta1 on the dental pulp cells in cell culture, tissue culture and three-dimensional culture patterns. These results demonstrated that TGF-beta1 significantly increased the proliferation of cells and activity of ALPase. Dental pulp cells cultured in the presence of TGF-beta1 formed mineralization nodules. In the organ culture, dental pulp cells treated with TGF-beta1 differentiated into odontoblast-like cells and formed a pulp-dentinal complex; and TGF-beta1 significantly induced synthesis of dentine relative proteins DSPP, DMP-1. The dental pulp cells share some characteristics of the odontoblast, such as a parallel arrangement with columnar form and a unilateral cell process. Together, these data indicate that TGF-beta1 can make dental pulp cells differentiated into odontoblast-like cells and form the pulp-dentinal complex. Moreover, these results suggest that TGF-beta1 is an important regulatory factor in odontoblast differentiation during tooth development and pulp repair.     

Analysis of the odontogenic and osteogenic potentials of dental pulp in vivo using a Col1a1-2.3-GFP transgene

Recently, transgenic mice that carry a Green Fluorescent Protein (GFP) reporter gene fused to 2.3 kb fragment of rat Col1a1 regulatory sequences (pOBCol2.3GFPemd) were generated. In the present study, we have examined the patterns of expression of Col1a1-2.3-GFP during odontoblast differentiation in this transgenic line. We report that Col1a1-2.3-GFP is expressed in newly differentiated odontoblasts secreting predentin and fully differentiated odontoblasts. The pattern of expression of Col1a1-2.3-GFP in odontoblasts is correlated with that of dentin sialophosphoprotein (DSPP). Col1a1-2.3-GFP is also expressed in the osteoblasts and osteocytes of alveolar bone. The pattern of expression of Col1a1-2.3-GFP in osteocytes is correlated with the expression of Dmp1. These observations indicate the 2.3 kb rat Col1a1 promoter fragment has sufficient strength and specificity to monitor the stage-specific changes during both odontoblast and osteoblast differentiation. We also used coronal pulp tissues isolated from postnatal pOBCol2.3GFPemd transgenic animals to follow their differentiation after transplantation under the kidney capsule. Our observations provide direct evidence that the dental pulp contains competent progenitor cells capable of differentiating into new generations of odontoblast-like cells which express high levels of Col1a1-2.3-GFP and DSPP and secrete tubular containing reparative dentin. We also report that the dental pulp is capable of giving rise to atubular bone-like tissue containing osteocytes expressing high levels of Col1a1-2.3-GFP and Dmp1. Our studies indicate that pOBCol2.3GFPemd transgenic animals provide a powerful tool for direct examination of the underlying mechanisms and the signaling pathways involved in dentin regeneration and repair, stem cell properties and heterogeneity of the dental pulp.     

Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population

Mammary epithelium can functionally regenerate upon transplantation. This renewal capacity has been classically ascribed to the function of a multipotent mammary gland stem cell population, which has been hypothesized to be a primary target in the etiology of breast cancer. Several complementary approaches were employed in this study to identify and enrich mammary epithelial cells that retain stem cell characteristics. Using long-term BrdU labeling, a population of label retaining cells (LRCs) that lack expression of differentiation markers has been identified. LRCs isolated from mammary primary cultures were enriched for stem cell antigen-1 (Sca-1) and Hoechst dye-effluxing "side population" properties. Sca-1(pos) cells in the mammary gland were localized to the luminal epithelia by using Sca-1(+/GFP) mice, were progesterone receptor-negative, and did not bind peanut lectin. Finally, the Sca-1(pos) population is enriched for functional stem/progenitor cells, as demonstrated by its increased regenerative potential compared with Sca-1(neg) cells when transplanted into the cleared mammary fat pads of host mice.     

Long-term label retaining cells localize to distinct regions within the female reproductive epithelium

The uterus is an extremely plastic organ that undergoes cyclical remodeling including endometrial regeneration during the menstrual cycle. Endometrial remodeling and regeneration also occur during pregnancy and following parturition, particularly in hemochorial implanting species. The mechanisms of endometrial regeneration are not well understood. Endometrial stem/progenitor cells are proposed to contribute to endometrial regeneration in both humans and mice. BrdU label retention has been used to identify potential stem/progenitor cells in mouse endometrium. However, methods are not available to isolate BrdU label-retaining cells (LRC) for functional analyses. Therefore, we employed a transgenic mouse model to identify H2B-GFP LRCs throughout the female reproductive tract with particular interest on the endometrium. We hypothesized that the female reproductive tract contains a population of long-term LRCs that persist even following pregnancy and endometrial regeneration. Endometrial cells were labeled (pulsed) either transplacentally/translactationally or peripubertally. When mice were pulsed transplacentally/translactationally, the label was not retained in the uterus. However, LRCs were concentrated to the distal oviduct and endocervical transition zone (TZ) following natural (i.e., pregnancy/parturition induced) and mechanically induced endometrial regeneration. LRCs in the distal oviduct and endocervical TZ expressed stem cell markers and did not express ERα or PGR, implying the undifferentiated phenotype of these cells. Oviduct and endocervical TZ LRCs did not proliferate during endometrial re-epithelialization, suggesting that they do not contribute to the endometrium in a stem/progenitor cell capacity. In contrast, when mice were pulsed peripubertally long-term LRCs were identified in the endometrial glandular compartment in mice as far out as 9 months post-pulse. These findings suggest that epithelial tissue of the female reproductive tract contains 3 distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.     

Differentiation and regenerative capacities of human odontoma-derived mesenchymal cells

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 βIII-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.     

Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano

Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.     

Dentinogenic potential of human adult dental pulp cells during the extended primary culture

Despite the frequent use of primary dental pulp cells in dental regenerative research, few systematic studies of stemness for osteogenic and dentinogenic differentiation of human adult pulp cells have been reported. To investigate the stemness of human adult dental pulp cells, pulp tissues were obtained from extracted third molars and used as a source of pulp cells. In FACS analysis and immunophenotyping, the general mesenchymal stem cell markers CD44, CD90, and CD146 were highly expressed in early passages of the pulp cell culture. The stem cell population was dramatically decreased in an expansion culture of human dental pulp cells. When pulp cells were treated with additives such as β-glycerophosphate, ascorbic acid, and dexamethasone, nodule formation was facilitated and mineralization occurred within 2 weeks. Expression of osteogenic markers such as alkaline phosphatase, osteocalcin, and osteonectin was relatively low in undifferentiated cells, but increased significantly under differentiation conditions in whole passages. Dentinogenic markers such as dentin sialophosphoprotein and dentin matrix protein-1 appeared to decrease in their expression with increasing passage number; however, peak levels of expression occurred at around passage 5. These data suggested that stem cells with differentiation potential might exist in the dental pulp primary culture, and that their phenotypes were changed during expansion culture over 8-9 passages. Under these conditions, a dentinogenic population of pulp cells occurred in limited early passages, whereas osteogenic cells occurred throughout the whole passage range.     

Isolation of nuclei from label-retaining cells and measurement of their turnover rates in rat colon

We describe here a new technique for isolating nuclei from long-term label-retaining cells (LRCs), a subpopulation enriched with stem cells from colon, and for measuring their proliferation rates in vivo. A double-label approach was developed, combining the use of bromodeoxyuridine (BrdU) and ²H₂O. Male Fisher 344 rats were administered BrdU in drinking water continuously for 2–8 wk. BrdU was then discontinued (BrdU washout), and animals (n = 33) were switched to ²H₂O in drinking water and killed after 2, 4, and 8 wk. Nuclei from BrdU-positive cells (LRCs) were collected by flow cytometry. The percentages of LRCs were 7 and 3.8% after 4 and 8 wk of BrdU washout, respectively. Turnover rates of LRCs were measured on the basis of deuterium incorporation from ²H₂O into DNA of LRC nuclei, as determined by mass spectrometry. The proliferation rate of the LRCs collected was 0.33–0.90% per day (half-life of 77–210 days). Significant contamination from other potentially long-lived colon cells was excluded. In conclusion, this double-labeling method allows both physical isolation of nuclei from colon epithelial LRCs and measurement of their in vivo proliferation rates. Use of this approach may allow better understanding of mechanisms by which agents induce or protect against colon carcinogenesis. carcinogenesis; deuterated water; long-term label-retaining cells; stable isotopes     

Progress in the use of dental pulp stem cells in regenerative medicine

The field of tissue engineering is emerging as a multidisciplinary area with promising potential for regenerating new tissues and organs. This approach requires the involvement of three essential components: stem cells, scaffolds and growth factors. To date, dental pulp stem cells have received special attention because they represent a readily accessible source of stem cells. Their high plasticity and multipotential capacity to differentiate into a large array of tissues can be explained by its neural crest origin, which supports applications beyond the scope of oral tissues. Many isolation, culture and cryopreservation protocols have been proposed that are known to affect cell phenotype, proliferation rate and differentiation capacity. The clinical applications of therapies based on dental pulp stem cells demand the development of new biomaterials suitable for regenerative purposes that can act as scaffolds to handle, carry and implant stem cells into patients. Currently, the development of xeno-free culture media is emerging as a means of standardization to improve safe and reproducibility. The present review aims to describe the current knowledge of dental pulp stem cells, considering in depth the key aspects related to the characterization, establishment, maintenance and cryopreservation of primary cultures and their involvement in the multilineage differentiation potential. The main clinical applications for these stem cells and their combination with several biomaterials is also covered.     

Pulpal responses to cavity preparation in aged rat molars

The dentin-pulp complex is capable of repair after tooth injuries including dental procedures. However, few data are available concerning aged changes in pulpal reactions to such injuries. The present study aimed to clarify the capability of defense in aged pulp by investigating the responses of odontoblasts and cells positive for class II major histocompatibility complex (MHC) to cavity preparation in aged rat molars (300–360 days) and by comparing the results with those in young adult rats (100 days). In untreated control teeth, immunoreactivity for intense heat-shock protein (HSP)-25 and nestin was found in odontoblasts, whereas class-II-MHC-positive cells were densely distributed in the periphery of the pulp. Cavity preparation caused two types of pulpal reactions based on the different extent of damage in the aged rats. In the case of severe damage, destruction of the odontoblast layer was conspicuous at the affected site. By 12 h after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border but subsequently disappeared together with HSP-25-immunopositive cells, and finally newly differentiated odontoblast-like cells took the place of the degenerated odontoblasts and acquired immunoreactivity for HSP-25 and nestin by postoperative day 3. In the case of mild damage, no remarkable changes occurred in odontoblasts after operation, and some survived through the experimental stages. These findings indicate that aged pulp tissue still possesses a defense capacity, and that a variety of reactions can occur depending on the difference in the status of dentinal tubules and/or odontoblast processes in individuals.This work was supported in part by a grant from MEXT to promote 2001-multidisciplinary research project (in 2001–2005), KAKENHI (B) to H.O. (no. 16390523), and Daiwa Securities Health Foundation, Japan.     

Human dental pulp stem cells: Applications in future regenerative medicine

Stem cells are pluripotent cells, having a property of differentiating into various types of cells of human body. Several studies have developed mesenchymal stem cells (MSCs) from various human tissues, peripheral blood and body fluids. These cells are then characterized by cellular and molecular markers to understand their specific phenotypes. Dental pulp stem cells (DPSCs) are having a MSCs phenotype and they are differentiated into neuron, cardiomyocytes, chondrocytes, osteoblasts, liver cells and β cells of islet of pancreas. Thus, DPSCs have shown great potentiality to use in regenerative medicine for treatment of various human diseases including dental related problems. These cells can also be developed into induced pluripotent stem cells by incorporation of pluripotency markers and use for regenerative therapies of various diseases. The DPSCs are derived from various dental tissues such as human exfoliated deciduous teeth, apical papilla, periodontal ligament and dental follicle tissue. This review will overview the information about isolation, cellular and molecular characterization and differentiation of DPSCs into various types of human cells and thus these cells have important applications in regenerative therapies for various diseases. This review will be most useful for postgraduate dental students as well as scientists working in the field of oral pathology and oral medicine.