Regulation of CCl4-induced liver cirrhosis by hepatically differentiated human dental pulp stem cells.

Liver transplantation is the most effective treatment for treating liver cirrhosis. However, a limited number of donors, graft rejection, and other complications can undermine transplant success. It is considered that cell transplantation is an alternative approach of liver transplantation. We previously developed a protocol for hepatic differentiation of cluster of differentiation 117+ stem cells isolated from human exfoliated deciduous tooth pulp (SHEDs) under hydrogen sulfide exposure. These cells showed excellent hepatic function. Here, we investigated whether hepatocyte-like cell transplantation is effective for treating carbon tetrachloride (CCl4)-induced liver cirrhosis. SHEDs were hepatically differentiated, which was confirmed via immunological analyses and albumin concentration determination in the medium. Rats were intraperitoneally injected with CCl4 for and the differentiated cells were injected into rat spleen. Histopathological and immunohistochemical analyses were performed. Liver functions were serologically and pathologically determined. Quantitative real-time-polymerase chain reaction was implemented to clarify the treatment procedure of liver cirrhosis. In vitro-differentiated hepatocyte-like cells were positive for all examined hepatic markers. SHED-derived hepatocyte transplantation eliminated liver fibrosis and restored liver structure in rats. Liver immunohistochemical analyses showed the presence of human-specific hepatic markers, i.e., a large amount of human hepatic cells were very active in the liver and spleen. Serological tests revealed significant liver function recovery in the transplantation group. Expression of genes promoting fibrosis increased after cirrhosis induction but was suppressed after transplantation. Our results suggest that xenotransplantation of hepatocyte-like cells of human origin can treat cirrhosis. Moreover, cell-based therapy of chronic liver conditions may be an effective option.

Regeneration of insulin-producing islets from dental pulp stem cells using a 3D culture system.

AIM: In this study, we aimed to establish the differentiation protocol of dental pulp stem cells (DPSCs) into pancreatic islets using a 3D structure. MATERIALS & METHODS: DPSCs were differentiated in a 3D culture system using a stepwise protocol. Expression of ß-cell markers, glucose-stimulated insulin secretion, and PI3K/AKT and WNT pathways were compared between monolayer-cultured pancreatic cells and islets. RESULTS: Islet formation increased insulin and C-peptide production, and enhanced the expression of pancreatic markers. Glucose-dependent secretion of insulin was increased by islets. Pancreatic endocrine markers, transcriptional factors, and the PI3K/AKT and WNT pathways were also upregulated. CONCLUSION: Pancreatic islets were generated from DPSCs in a 3D culture system. This system could provide novel strategies for controlling diabetes through regenerative medicine.

Hydrogen sulfide enhances pancreatic ß-cell differentiation from human tooth under normal and glucotoxic conditions.

AIM: Glucotoxicity obstructs pancreatic differentiation from adult stem cells. The aim was to develop a novel protocol for differentiation of dental pulp stem cells (DPSCs) into pancreatic ß cells and determine the effect of H2S on glucotoxicity. MATERIALS & METHODS: DPSCs were differentiated with media containing 5.5 or 25.0 mM glucose, exposed to 1 ng/ml H2S. Glucotoxicity, expression of ß-cell markers, INS, PDX1 and GLUT2, and PI3K/AKT pathway were assessed. RESULTS: H2S exposure increased insulin and C-peptide, and protected DPSC-derived pancreatic ß-like cells from glucotoxicity and upregulated INS, PDX1 and GLUT2, and genes of PI3K/AKT pathway. CONCLUSION: H2S improved effects of glucotoxicity on ß-like cells via PI3K/AKT pathway. The protocol for pancreatic ß-cell differentiation might have applications in regenerative medicine rather than swine pancreas transplantation.

Novel management of acute or secondary biliary liver conditions using hepatically differentiated human dental pulp cells.

The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.

Regeneration of insulin-producing pancreatic cells using a volatile bioactive compound and human teeth.

Transplantation of insulin (INS)-secreting cells differentiated in vitro from stem cells promises a safer and easier treatment of severe diabetes mellitus. A volatile bioactive compound, hydrogen sulfide (H2S), promotes cell differentiation; human tooth-pulp stem cells undergo hepatic differentiation. The aim of this study is to develop a novel protocol using H2S to enhance pancreatic differentiation from the CD117(+) cell fraction of human tooth pulp. During the differentiation, the cells were exposed to 0.1 ng ml(-1) H2S. Immunocytochemistry, RT-PCR, determination of INS c-peptide content and flow cytometry of pancreatically related markers were carried out. Expression of WNT and the PI3K/AKT signaling pathway were also determined by PCR array. After differentiation, INS, glucagon (GCG), somatostatin (SST) and pancreatic polypeptide (PPY) were positive when examined by immunofluorescence. INS and GCG were also determined flow-cytometrically. Only the cells expressing INS increased after H2S exposure. The number of cells expressing GCG was significantly decreased. Genes involved in canonical WNT and the WNT/calcium pathways were highly expressed after H2S exposure. H2S accelerated INS synthesis and secretion by regenerated INS-producing cells from human teeth. All signaling pathway functions of the PI3K-AKT pathway were extremely activated by H2S exposure. The matured INS-producing cells originating in human teeth were increased by H2S in order to control blood-glucose level.

[Halitosis management by the general dental practitioner- results of an International Consensus Workshop*]. / Halitosismanagement für die Zahnarztpraxis. Ergebnisse eines internationalen Konsunsus-Workshops*.

Clinical investigations on patients suffering from halitosis clearly reveal that in the vast majority of cases the source for an offensive breath odor can be found within the oral cavity (90%). Based on these studies, the main sources for intra-oral halitosis where tongue coating, gingivitis/periodontitis and a combination of the two. Thus, it is perfectly logical that general dental practitioners (GDPs) should be able to manage intra-oral halitosis under the conditions found in a normal dental practice. However, GDPs who are interested in diagnosing and treating halitosis are challenged to incorporate scientifically based strategies for use in their clinics. Therefore, the present paper summarizes the results of a consensus workshop of international authorities held with the aim to reach a consensus on general guidelines on how to assess and diagnose patients’ breath odor concerns and general guidelines on regimens for the treatment of halitosis.

Pancreatic differentiation of human dental pulp CD117⁺ stem cells.

AIM: Adult stem cells cannot proliferate to produce enough cells for human transplantation with keeping stem cell characteristics shown in the primary culture. We established a novel culture protocol using human dental pulp stem cells (DPSCs) that can produce quantities sufficient for human transplantation. The present study assessed differentiation of DPSCs toward a pancreatic lineage in serum-free conditions, which is essential for safe transplantation. MATERIALS & METHODS: CD117⁺ stem cells were separated from human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth; SHED) and adult DPSCs. The cells were characterized with real-time reverse-transcription PCR for a panel of embryonal lineage markers. RESULTS: 82 out of 84 markers were expressed in different levels in SHED or DPSCs. After pancreatic differentiation in vitro, we found expression of pancreatic-specific endocrine markers insulin, glucagon, somatostatin and pancreatic polypeptide, and exocrine marker amylase-2a in both cultures. We also found reprogramming in both cell cultures mimicking the embryonal stages of development of the pancreas. Transcription factors PDX1, HHEX, MNX1, NEUROG3, PAX4, PAX6 and NKX6-1, crucial markers for the pancreatic development, were all activated. Expression of these factors strongly implies that the cells differentiated toward a distinguished pancreatic lineage. CONCLUSION: Our results show that CD117⁺ SHED and DPSCs are capable of differentiation toward all functional endocrine and exocrine subsets of pancreatic cells in serum-free conditions. SHED and DPSCs may therefore have great potential for future cell therapy of pancreatic disorders.

Chewing gum containing allyl isothiocyanate from mustard seed extract is effective in reducing volatile sulfur compounds responsible for oral malodor.

PURPOSE: To evaluate the in vivo effect of chewing gum containing allyl isothiocyanate alone, and in combination with zinc salts on reduction of the level of volatile sulfur compounds responsible for oral malodor. METHODS: 15 healthy volunteers between the ages of 20-50 chewed either an experimental gum or a placebo gum for 12 minutes. Their mouth air was analyzed for volatile sulfur compounds by a gas chromatograph at baseline, immediately after chewing, and at 60, 120 and 180 minutes after treatment. RESULTS: The study revealed that allyl isothiocyanate, a constituent of mustard seed extract, can effectively reduce the concentration of volatile sulfur compounds in mouth air. Chewing gum containing 0.1% zinc lactate and 0.01% of allyl isothiocyanate eliminated 89%, 55.5%, 48% and 24% of the total VSC concentration immediately after chewing and at 1, 2, and 3 hours after chewing, respectively.

Tongue thickness relates to nutritional status in the elderly.

Many elderly people under long-term care suffer from malnutrition caused by dysphagia, frequently leading to sarcopenia. Our hypothesis is that sarcopenia may compromise oral function, resulting in dysphagia. The objectives of this study were to evaluate sarcopenia of the lingual muscles by measuring the tongue thickness, and elucidate its relationship with nutritional status. We examined 104 elderly subjects (mean age = 80.3 ± 7.9 years). Anthropometric data, such as triceps skinfold thickness and midarm muscle area (AMA), were obtained. The tongue thickness of the central part was determined using ultrasonography. Measurement was performed twice and the mean value was obtained. The relationship between tongue thickness and nutritional status was analyzed by Pearson's correlation coefficient and Spearman's rank correlation coefficient. AMA and age were identified by multiple-regression analysis as factors influencing tongue thickness. The results of this study suggest that malnutrition may induce sarcopenia not only in the skeletal muscles but also in the tongue.

High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium.

INTRODUCTION: We have previously differentiated hepatocyte like cells from deciduous tooth pulp stem and extracted third molar pulp stem cells with a protocol that used fetal bovine serum, but it showed high contaminations of nondifferentiated cells. Both the lower purity of hepatically differentiated cells and usage of serum are obstacles for application of cell therapy or regenerative medicine. Objective of this study was to investigate the capacity for and purity of hepatocyte-like differentiation of CD117-positive dental pulp stem cells without serum. METHODS: Mesenchymal cells from human deciduous and extracted third molar pulp were isolated and expanded in vitro. We separated CD117-positive cells by using a magnetic-activated cell sorter. The cells were characterized immunofluorescently by using known stem cell markers. For hepatic differentiation, the media were supplemented with hepatic growth factor, insulin-transferrin-selenium-x, dexamethasone, and oncostatin M. Expression of hepatic markers alpha fetoprotein, albumin, hepatic nuclear factor-4 alpha, insulin-like growth factor-1, and carbamoyl phosphate synthetase was examined immunofluorescently after differentiation. The amount of differentiated cells was assessed by using flow cytometry. Glycogen storage and urea concentration in the medium were defined. RESULTS: Both cell cultures demonstrated a number of cells positive for all tested hepatic markers after differentiation, ie, albumin-positive cells were almost 90% of differentiated deciduous pulp cells. The concentration of urea in the media increased significantly after differentiation. Significant amount of cytoplasmic glycogen storage was found in the cells. CONCLUSIONS: Without serum both cell types differentiated into high-purity hepatocyte-like cells. These cells offer a source for hepatocyte lineage differentiation for transplantation in the future.

Tooth loss as risk factor for foreign-body asphyxiation in nursing-home patients.

Foreign body asphyxiation causes severe medical conditions including pneumonia in the elderly requiring nursing care. The objective of this study was to elucidate the relationships between insufficient occlusal support due to tooth loss and the onset of asphyxiation accidents, and determine preventive measures for such accidents in nursing homes in Japan. The subjects were 437 elderly (110 men and 327 women) requiring nursing care. The frequency and risk factors for asphyxiation accidents and the food causing asphyxiation were examined in these subjects for 2.5 years, from June 2006 to December 2008. During the study period, 51 of the 437 subjects suffered asphyxiation. Self-feeding ability and loss of occlusal support were associated with a covariate-adjusted relative ratio for asphyxiation of 3.1 (95% confidence interval (CI)=1.50-6.44) and 1.7 (95% CI=1.12-2.74), respectively. To prevent asphyxiation in elderly people, it was found that maintaining or restoring occlusal support may be required. It was concluded that self-feeding ability and loss of occlusal support are significant risk factors for foreign-body asphyxiation among elderly people requiring nursing care.

Standardization of clinical protocols in oral malodor research.

The objective of this study is to standardize protocols for clinical research into oral malodor caused by volatile sulfur compounds (VSCs). To detect VSCs, a gas chromatograph (GC) using a flame photometric detector equipped with a bandpass filter (at 393 nm) is the gold standard (sensitivity: 5 × 10(-11) gS s(-1)). The baselines of VSC concentrations in mouth air varied considerably over a week. When the subjects refrained from eating, drinking and oral hygiene including mouth rinsing, the VSC concentrations remained constant until eating. Over a 6 h period after a meal, VSC concentrations decreased dramatically (p < 0.01). These results point to optimal times and conditions for sampling subjects. Several portable devices were compared with the measurements by the GCs. Portable GCs demonstrated capabilities similar to those of the GCs. We also applied the recommended protocols described below to clinical research testing the efficacy of ZnCl(2) products, and confirmed that using the recommended protocols in a randomized crossover design would provide very clear results. Proposed protocols include: (a) a short-term study rather than a long-term study is strongly recommended, since the VSC concentrations are constant in the short term; (b) a crossover study would be the best design to avoid the effects of individual specificities on each clinical intervention; (c) measurements of VSCs should preferably be carried out using either a GC or portable GCs.

Hydrogen sulfide increases hepatic differentiation in tooth-pulp stem cells.

The toxicity of hydrogen sulfide (H(2)S), an oral malodorous compound, is well reported. We have recently established an experimental model of hepatic differentiation from human tooth-pulp stem cells (HTPC) using serum-free medium. The objective of the present study is to determine the effect of H(2)S on hepatic differentiation. The CD117 positive cell fraction was obtained from deciduous HTPC using magnetic cell sorting. After 3-4 passages, cells were grown in Dulbecco's modified Eagle's medium supplemented with insulin-transferrin-selenium-x (ITS-x), embryotrophic factor (ETF) and hepatocyte growth factor (HGF) for hepatic commitment (five days). For hepatic differentiation the cells were cultured in Iscove's modified Dulbecco's medium supplemented with ITS-x, ETF, oncostatin, HGF and dexamethasone for 15 days in air containing 5% CO(2), with or without H(2)S at 0.05 ng ml(-1). Cells were assayed for the expression of hepatic markers α-fetoprotein, albumin or carbamoyl phosphate synthetase, and urea concentrations and glycogen synthesis were also determined. The panel of hepatic markers was expressed more in the test groups exposed to H(2)S than in the control groups. Urea and glycogen production were also increased, especially glycogen which was approximately five times greater compared to the control (p < 0.01). We concluded that H(2)S at physiological concentrations increased the ability of HTPC to undergo hepatogenic differentiation.

The role of p53 in an apoptotic process caused by an oral malodorous compound in periodontal tissues: a review.

Oral malodor is caused by volatile sulfur compounds (VSCs) composed mainly of hydrogen sulfide (H(2)S) and methyl mercaptan. In particular, H(2)S is an important compound, since it is a major component of physiologic halitosis. The toxicity of VSCs is similar to that of hydrogen cyanide, and is well investigated. The role of VSCs in reducing collagen in human gingival fibroblasts is one of the main sources of their toxicity to human oral tissues. It has been reported recently that H(2)S may cause apoptosis in several periodontal tissues. In human gingival fibroblasts, H(2)S inhibits not only cytochrome c oxidase activity but also superoxide dismutase activity. The levels of reactive oxygen species are markedly increased, which causes the release of cytochrome c into the cytoplasm, resulting in caspase-9 activation; finally, the executor caspase, caspase-3, is activated. This pathway is commonly observed in cells from all periodontal tissues. Moreover, p53, an apoptotic factor, and phosphorlylated p53, which is the activated form, are increased by H(2)S in keratinocyte stem cells and osteoblasts. H(2)S also increases the expression of Bax, a primary response gene playing an important role in p53-mediated apoptosis, but maintains a lower expression of Bcl-2, an anti-apoptotic factor, in osteoblasts. It is concluded that the Bax apoptotic pathway and the mitochondrial pathway are activated by H(2)S.

Combined effects of hydrogen sulfide and lipopolysaccharide on osteoclast differentiation in rats.

BACKGROUND: Lipopolysaccharide (LPS) stimulates osteoclast differentiation through toll-like receptors (TLRs) 2 and 4, and hydrogen sulfide (H(2)S) induces osteoclast differentiation. If H(2)S activates TLRs, H(2)S may enhance the effects of LPS on osteoclast differentiation. The purpose of the present study is to examine the combined effects of sodium hydrogen sulfide (NaHS, an H(2)S donor drug) and LPS on osteoclast differentiation and TLR expression in rat periodontal tissue. METHODS: Twenty-eight male Wistar rats (8 weeks old) were divided into four groups (n = 7 per group): a control (no treatment) group and three experimental groups (NaHS group, LPS group, and a combination [NaHS + LPS] group). At 1 day after topical application of NaHS and/or Porphyromonas gingivalis LPS into the gingival sulcus of first molars, the number of tartrate-resistant acid phosphate (TRAP)-positive osteoclasts in the periodontal tissue was counted. Expression of TLR2 and TLR4 mRNAs and proteins in the gingival was also assessed. RESULTS: The number of TRAP-positive osteoclasts was significantly higher in the combination group than in any other group (P <0.01). The combination group had 11.0-fold higher TLR4 mRNA levels than the control group. TLR4 protein levels were also higher in the combination group than in the NaHS or LPS group. However, the TLR2 mRNA and protein levels were not significantly different in the combination group and the LPS group. CONCLUSION: In rat periodontal tissue, NaHS and LPS had an additive effect on osteoclast differentiation through activation of the TLR4 pathway but not the TLR2 pathway.

Hydrogen sulfide causes apoptosis in human pulp stem cells.

INTRODUCTION: Untreated dental caries will eventually lead to pulpal inflammation. Although much is known regarding the interaction of microbial antigens and the immunologic defense systems of pulp, many aspects of the pathogenesis of pulpitis are not fully understood. The relationship between human pulp stem cells (HPSCs) and the pathogenesis of pulpitis remains among the poorly understood areas. Many of the invading bacteria are known to produce considerable amounts of hydrogen sulfide (H(2)S), which causes apoptosis in some tissues. The aims of this study were to determine whether H(2)S causes apoptosis in HPSCs and to examine its signaling pathway. METHODS: Stem cells were isolated from human dental pulp and incubated with 50 ng/mL H(2)S for 48 hours. To detect apoptosis, the cells were analyzed by using flow cytometry. The mitochondrial signaling pathway was examined by determining mitochondrial membrane depolarization. Activation of the key apoptotic enzymes caspase-9, caspase-8, and caspase-3 was assessed by using enzyme-linked immunosorbent assay. Release of cytochrome C from mitochondria was also determined. RESULTS: The number of apoptotic cells increased significantly with H(2)S treatment from 1.6% to 16.3% (P < .01). Significant increases were also measured in the amounts of caspase-9 and caspase-3 and in cytochrome C release (all P < .01) and in mitochondrial membrane depolarization (P < .05), whereas caspase-8 activity was not found. CONCLUSIONS: H(2)S causes apoptosis in HPSCs by activating the mitochondrial pathway. It is suggested that H(2)S might be one of the factors modifying the pathogenesis of pulpitis by causing loss of viability of HPSCs through apoptosis.

Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial.

We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H(2)S >1.5 or CH(3)SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH(3)SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00-0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47-1.00] ng/10 ml; P = 0.011). The median concentration of CH(3)SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log(10) copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log(10) copies/10 µl; P = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

Feeding therapy for children with food refusal.

Disabled children suffer not only from their primary disease, but also from other complications, including food refusal. The purpose of this study was to elucidate the relationship between these conditions and food refusal in disabled children. The effectiveness of feeding therapy in treating food refusal was also examined. The study subjects were 67 disabled children (35 boys and 32 girls; mean age at initial examination: 6.5 years, SD: 6.0 years) who attended the Nippon Dental University Hospital between April 2004 and August 2008. Of them, the 13 subjects who were diagnosed as those who refused food received feeding therapy combined with desensitization therapy for hypersensitivity. Approximately 20% of the subjects showed food refusal symptoms. Primary disease, respiratory impairment and gastroesophageal reflux were not causes of food refusal in this population. There was a significant relationship between food refusal and hypersensitivity (p = 0.021). After receiving feeding therapy, six of the seven subjects with hypersensitivity but without dysphagia at initial examination recovered from food refusal. Food refusal did not significantly correlate with tube feeding. Hypersensitivity and/or tube feeding may induce food refusal. For subjects with these conditions, feeding therapy combined with desensitization therapy is effective in achieving recovery from food refusal.

Expression of multiple stem cell markers in dental pulp cells cultured in serum-free media.

INTRODUCTION: Stem cell lines are usually grown in medium containing animal products. Fetal bovine serum (FBS) is an important additive for cell growth; however, the allergenic potential and the possibility of contamination when we use a medium containing serum would be a barrier to transplantation and consequently to the introduction of cell therapy methods into clinical applications. METHODS: Dental mesenchymal cells were isolated and expanded in vitro and maintained in 4 different serum-free media (SFMs): SFM#1 (ITS-X, embryotrophic factor [ETF]); SFM#2 (ITS-X); SFM#3 (ETF); and SFM#4 (ETF, sodium pyruvate, ascorbic acid, fibroblast growth factor [FGF-a], acidic). Viability, proliferative, and immunocytochemical tests for the cells were performed by using 4 stem cell markers (CD44H, CK19, nestin, and P63) for ectoderm, mesoderm, and endoderm. RESULTS: Viability tests showed a significant difference between the control and SFMs in both deciduous tooth pulp cells (DTPCs) and wisdom tooth pulp cells (WTPCs). However, all SFMs demonstrated 84%-90% viability, whereas the control showed 90%-93%. In both DTPCs and WTPCs, SFM#1 had the highest proliferation rate among the 4 SFMs. Immunocytochemistry stained positive stem cell markers most intensely in cells cultured with SFM#1. Furthermore, all stem cell markers for ectoderm, mesoderm, and endoderm were expressed in the cells cultured with SFM#1. CONCLUSIONS: SFM#1 showed an acceptable survival rate, the highest proliferation rate, and the strongest expression of all the stem cell markers. SFM#1 proved to be a suitable medium for the culture of human dental pulp stem cells and to preserve pluripotency in differentiation.