Evaluation of in vivo lithium chloride effects as a GSK3-ß inhibitor on human adipose derived stem cells differentiation into oligodendrocytes and re-myelination in an animal model of multiple sclerosis.

BACKGROUND: The application of neuroprotective agents in combination with stem cells is considered a potential effective treatment for multiple sclerosis (MS). Therefore, the effects of lithium chloride as a neuroprotective agent and a GSK3-ß inhibitor were evaluated in combination with human adipose derived stem cells on re-myelination, oligodendrocyte differentiation, and functional recovery. METHODS: After inducing a mouse model of MS and proving it by the hanging wire test, the mice were randomly assigned to five experimental groups: Cup, Sham, Li, hADSC, and Li + hADSC. Additionally, a control group with normal feeding was considered. Finally, toluidine blue staining was carried out to estimate the level of myelination. Furthermore, immunofluorescent staining was used to evaluate the mean of OLIG2 and MOG positive cells. The mRNA levels of ß-Catenin, myelin and oligodendrocyte specific genes were determined via the Real-Time PCR. RESULTS: The results of the hanging wire test and toluidine blue staining showed a significant increase in myelin density and improvements in motor function in groups, which received lithium and stem cells, particularly in the Li + hADSC group compared with the untreated groups (P < 0.01). Moreover, immunostaining results indicated that the mean percentages of MOG and OLIG2 positive cells were significantly higher in the Li + hADSC group than in the other groups (P < 0.01). Finally, gene expression studies indicated that the use of lithium could increase the expression of ß-Catenin, myelin and oligodendrocyte specific genes. CONCLUSION: The use of Lithium Chloride can increase stem cells differentiation into oligodendrocytes and improve re-myelination in MS.

In vitro evaluation of the pogostone effects on the expression of PTEN and DACT1 tumor suppressor genes, cell cycle, and apoptosis in ovarian cancer cell line.

Background and purpose: Ovarian cancer is one of the most dangerous cancers among women. Pogostone has anticancer effects and is rich in polyphenol compounds. In the present study, we investigated the effects of pogostone on ovarian cancer cell lines (OVCAR-3). Experimental approach: OVCAR-3 cells were treated with pogostone at IC50(90 µg/mL) for 24 and 48 h. Cell viability and apoptotic rate in the cells were measured using MTT assay and flow cytometry. Real-time PCR was used to determine the expression of genes involved in the cell cycle and apoptosis. The expression of caspase-3 (CASP3) protein was evaluated by the CASP3 assay. Findings/Results: Treatment of OVCAR-3 cells with pogostone increased the expression levels of phosphatase and tensin homologue deleted on chromosome ten (PTEN) and Dapper antagonist of catenin-1 (DACT1) tumor suppressor genes, as well as the apoptotic genes CASPs3, 8, and 9. Moreover, the ratio of the expressed BCL2 associated X (BAX)/BCl2 genes, as pro- and anti-apoptotic genes, was increased. The expression levels of the genes related to the cell cycle progression including cyclin D1 (CCND1) and cyclin- dependent kinase 4 (CDK4) were inhibited. The data obtained from flow cytometry indicated that pogostone induced cell apoptosis in 24 and 48 pogostone groups. The CASP3 colorimetric assay revealed that pogostone increased the expression of CASP3 protein in the treated groups. Conclusion and implication: Pogostone, by inducing the expression of PTEN and DACT1 tumor suppressor genes and regulation of downstream genes may decrease cell proliferation and increase the rate of apoptosis in OVCAR-3.

Neurogenic induction of human dental pulp derived stem cells by hanging drop technique, basic fibroblast growth factor, and SHH factors.

BACKGROUND: The progressive destruction of nerve cells in nervous system will induce neurodegenerative diseases. Recently, cell-based therapies have attracted the attention of researchers in the treatment of these abnormal conditions. Thus, the aim of this study was to provide a simple and efficient way to differentiate human dental pulp stem cells into neural cell-like to achieve a homogeneous population of these cells for transplantation in neurodegenerative diseases. MATERIALS AND METHODS: In this basic research, human dental pulp stem cells were isolated and characterized by immunocytochemistry and flow cytometry techniques. In the following, the cells were cultured using hanging drop as three-dimensional (3D) and tissue culture plate as 2D techniques. Subsequently, cultured cells were differentiated into neuron cell-like in the presence of FGF and Sonic hedgehog (SHH) factors. Finally, the percentage of cells expressing Neu N and ß tubulin III markers was determined using immunocytochemistry technique. Finally, all data were analyzed using the SPSS software. RESULTS: Flow cytometry and immunocytochemistry results indicated that human dental pulp-derived stem cells were CD90, CD106-positive, but were negative for CD34, CD45 markers (P ≤ 0.001). In addition, the mean percentage of ß tubulin positive cells in different groups did not differ significantly from each other (P ≥ 0.05). Nevertheless, the mean percentage of Neu N-positive cells was significantly higher in differentiated cells with embryoid bodies' source, especially in the presence of SHH than other groups (P ≤ 0.05). CONCLUSION: It is concluded that due to the wide range of SHH functions and the facilitation of intercellular connections in the hanging droop method, it is recommended that the use of hanging drop method and SHH factor can be effective in increasing the efficiency of cell differentiation.

RET Protein Expression in Colorectal Cancer; An Immunohistochemical Assessment.

BACKGROUND: RET (rearranged during transfection) is a transmembrane receptor tyrosine kinase and a receptor for the GDNF-family ligands. It plays the role of a tumor suppressor in colorectal cancer. Therefore, it is expected that RET gene becomes downregulated in colorectal cancer (CRC). In this study, we evaluated immuno-histochemical expression of RET in CRC and assessed its correlation with some of the clinicopathological features to study the prognostic value in CRC. MATERIALS AND METHODS: In total, 60 cases of colorectal cancer (CRC) from the patients who underwent surgical gastroenterology operations were randomly selected. The samples included one tumor-rich section per case and one adjacent tumor-free section as the normal control for that case. Then, immunohistochemistry (ICH) was performed for RET on all the samples and the expression of RET was analyzed. Furthermore, the correlation of RET with clinicopathological features including age, gender, location of the tumor, grade, and stage was evaluated. RESULTS: The expression of RET caused significant downregulation in cancer samples compared to the normal control ones (P = 0.002). This downregulation increased in correlation to both grade and metastasis to lymph nodes (P = 0.03 & 0.02 respectively). However, no correlation was found between the expression of RET and gender as well as location of the tumor. CONCLUSION: RET may be considered as a protein marker in CRC detection and prognosis.

Astaxanthin Reduces Demyelination and Oligodendrocytes Death in A Rat Model of Multiple Sclerosis.

OBJECTIVE: Astaxanthin (AST) is a carotenoid with anti-oxidative, anti-inflammatory, and anti-apoptotic properties. It has also been reported that AST exerts protective effects against neurodegenerative diseases and reduces oxidative stress-induced the central nervous system (CNS) injury. In this study, we aimed to evaluate the protective potential of AST in inhibiting demyelination and oligodendrocyte death in a rat model of multiple sclerosis (MS). MATERIALS AND METHODS: In this experimental study, forty Wistar rats were randomly assigned to four experimental groups: control group (with normal feeding), cuprizone (CPZ group) that daily received 0.6% CPZ for 4 weeks, sham group that daily received 0.6% CPZ plus dimethyl sulfoxid (DMSO) for 4 weeks, and AST group that daily received 0.6% CPZ and after 12 hours were treated with AST (3 mg/kg), for 4 weeks. Muscle strength was evaluated by the behavioral basket test at the end of every week for 4 weeks. Luxol Fast Blue (LFB) staining was utilized for the identification of myelination and demyelination. Myelin density was evaluated by the ImageJ software. The expression of A2B5 (oligodendrocyte precursor protein) and myelin oligodendrocyte protein (MOG) were assessed by immunohistochemistry (IHC) and the expression of myelin basic protein (MBP), MOG, and platelet-derived growth factor-alpha (PDGFR-α) genes was examined by the real-time polymerase chain reaction (RT-PCR) technique. RESULTS: The administration of AST reduced the oligodendrocyte damage and myelin sheath disruption in a rat model of MS. The basket behavioral test showed the improvement of muscle strength in the AST group compared with CPZ and sham groups. Besides, the results of real-time PCR and IHC indicated the beneficial effects of AST in declining demyelination and oligodendrocyte death in a rat model of MS. CONCLUSION: AST reduces damages to the myelin sheath and oligodendrocyte death in a rat model of MS.

Evaluation Avocado Soybean Unsaponifiables Loaded in Poly (lactic-co-glycolic) Acid/Avocado Soybean Unsaponifiables-Fibrin Nanoparticles Scaffold (New Delivery System) is an Effective Factor for Tissue Engineering.

BACKGROUND: Growth factors and chemical stimulants have key role in cartilage tissue engineering, but these agents have unfavorable effects on cells. Avocado soybean unsaponifiables (ASU) has chondroprotective and anti-inflammatory effects. In this study, fibrin2nanoparticles (FNP)/ASU, as a new delivery system, with stem cells applied for cartilage tissue engineering in poly (lactic-co-glycolic) acid (PLGA) scaffold. MATERIALS AND METHODS: FNP/ASU prepared by freeze milling and freeze drying. NFP/ASU was characterized by dynamic light scattering (DLS). PLGA-NFP/ASU scaffold was fabricated and assessed by scanning electron microscope (SEM). Human adipose-derived stem cells (hADSCs) were seeded on scaffold and induced for chondrogenesis. After 14 days, cell viability and gene/protein expression evaluated. RESULTS: The results of DLS and SEM indicated that nanoparticles had high quality. The expression of type II collagen and SOX9 and aggrecan (ACAN) genes in differentiated cells in the presence of ASU was significantly increased compared with the control group (P and lt; 0.01), on the other hand, type I collagen expression was significantly decreased and western blot confirmed it. CONCLUSIONS: This study indicated FNP/ASU loaded in PLGA scaffold has excellent effect on chondrogenic differentiation of hADSCs and tissue engineering.

Anti-proliferative and anti-apoptotic effects of grape seed extract on chemo-resistant OVCAR-3 ovarian cancer cells.

BACKGROUND AND PURPOSE: Ovarian cancer is the deadliest cancer in women. The main challenge in the inhibition of ovarian cancer cells is chemo-resistance. Seeking to overcome this issue, several strategies have been suggested, including the administration of natural products. Grape seed extract (GSE) is a good source of polyphenols and its anticancer effects have been reported by many studies. In this study we aimed to evaluate the effects of GSE on OVCAR-3, a chemo-resistant ovarian cancer line. EXPERIMENTAL APPROACH: OVCAR-3 cells were treated with GSE (71 µg/mL) for 24 and 48 h. Cell viability and cell apoptosis were measured by MTT and flow cytometry. The real-time polymerase chain reaction was used to determine the expression of genes involved in the cell cycle (PTEN, DACT1, AKT, MTOR, GSK3B, C-MYC, CCND1, and CDK4) and apoptosis (BAX, BCl2, CASP3, 8 and 9). The expression of CASP3 protein was evaluated by the CASP3 assay. FINDINGS / RESULTS: The results showed that treatment of OVCAR-3 cells with GSE, increased the expression level of PTEN and DACT1 tumor suppressor genes, as well as apoptotic genes, CASP3, 8, and 9 (P < 0.001). Also, the induction of tumor suppressor genes expression was associated with an increase in the expression of BAX/BCL2 gene ratio as pro- and anti-apoptotic genes. The expression of the genes involved in the cell cycle, CCND1 and CDK4, was inhibited (P < 0.001). The results indicated that GSE induced cell apoptosis in a time-dependent manner (P < 0.001). Also, the GSE treatment resulted in the CASP3 protein expression (P < 0.001). CONCLUSION AND IMPLICATIONS: According to the results of this study, GSE may exert anti-tumorigenic effects on chemo-resistant OVCAR-3 ovarian cancer cells which might be mediated by the expression of tumor suppressor genes that interact with cell signaling pathways, cell cycle, and cell apoptosis. Hence, the consumption of GSE extract during chemotherapy may overcome part of chemo-resistance in ovarian cancer.

Myricetin Exerts its Apoptotic Effects on MCF-7 Breast Cancer Cells through Evoking the BRCA1-GADD45 Pathway.

OBJECTIVE: Myricetin is a polyphenol flavonoid with nutraceutical values which is abundantly found as the main ingredient of various foods and beverages. It has been reported that the function of myricetin is to trigger apoptosis in several types of cancers. The present study intended to investigate the apoptotic effects of myricetin on MCF-7 breast cancer cells and to assess its possible mechanisms of action. MATERIALS AND METHODS: MCF-7 breast cancer cells were assigned to four groups: Control (cells in normal condition); myricetin (cells treated with the IC50 dosage of myricetin) in three different incubation times (24, 48, and 72 h). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V assay, flow cytometry, real-time polymerase chain reaction (PCR), and caspase-3 assay were used to estimate the apoptosis function of myricetin in breast cancer. RESULTS: The expression levels of apoptosis-related genes caspase-3, caspase-8, caspase-9, and the BAX /Bcl-2 ratio as well as the expression of p53, BRCA1, GADD45 genes were significantly increased following the treatment of MCF-7 breast cancer cells with myricetin. The annexin V assay demonstrated the significant expression of annexin which was also detected by flow cytometry. CONCLUSION: Myricetin efficiently induces apoptosis in MCF-7 breast cancer cells by evoking both extrinsic and intrinsic apoptotic pathways. Myricetin may exert its apoptotic effects on MCF-7 cells by inducing the BRCA1- GADD45 pathway.
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Myricetin Apoptotic Effects on T47D Breast Cancer Cells is a P53-Independent Approach.

OBJECTIVE: Using nutraceuticals in cancer therapy is a strategy contributing with other approaches to promote apoptosis in cancer cells. Myricetin is a polyphenol flavonoid that forms main ingredients of various type of foods and beverages. The inducing properties of myricetin in apoptosis is reported by several investigations. The present study aimed to assess apoptotic effects of myricetin on T47D breast cancer cells and to evaluate part of the mechanisms of action. MATERIALS AND METHODS: T47D breast cancer cells were assigned into five groups: control (cells in normal condition), myricetin (cells treated with myricetin IC50 concentration) in two different incubation times (24, 48 and 72 hours). MTT assay, annexin v assay, flow cytometry, caspase-3 assay and Real-time PCR were used to evaluate apoptosis in breast cancer cells. RESULTS: The expression rate of apoptotic genes caspase-3, caspase-8, caspase-9, the ratio of BAX /Bcl-2 as well as the expression of P53, BRCA1, GADD45 genes were increased significantly after treatment of T47D breast cancer cells with myricetin. Annexin v assay confirmed significant expression of annexin as were displyed by flow cytometry. CONCLUSION: Myricetin enhances apoptosis in T47D breast cancer cells by evoking both extrinsic and intrinsic apoptotic pathways. myricetin may practices its apoptotic properties on T47D cells through inducing BRCA1- GADD45 pathway.
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Evaluating the Radiosensitization Effect of Hydroxyapatite Nanoparticles on Human Breast Adenocarcinoma Cell Line and Fibroblast.

BACKGROUND: Nanohydroxyapatite (nHAP) exhibit anti-proliferative effects on various cancer cells. However, to date, there are only a few studies on the radiosensitization effect of nHAP. The present study aimed to investigate the possible enhancement of the radiosensitization effect of nHAP on human breast adenocarcinoma cancer (MCF-7) and fibroblast. METHODS: nHAP was extracted from fish scales using the thermal alkaline method and characterized at Babol University of Medical Sciences (Babol, Iran) in 2017. The anti-proliferative and the radiosensitization effects of nHAP were investigated by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyltetrazolium Bromide (MTT), clonogenic assay, and apoptosis assay. MCF-7 cells and fibroblasts were incubated with different concentrations of nHAP and at different periods. The MTT solution was added and the absorbance was measured at 570 nm. The MCF-7 cells were exposed to 0, 1.5, 3.5, and 5 Gy X-ray irradiation and incubated for 10-14 days. The data were compared using the one-way analysis of variance (ANOVA) followed by the post hoc tests (Tukey's method). RESULTS: The results showed that nHAP significantly inhibited the growth of MCF-7 cells compared with controls (P<0.001), but the difference was not statistically significant for fibroblasts (P=0.686 at 400 µg/mL at 72 hours). After 48 hours, the proliferation of MCF-7 cells and fibroblasts was inhibited by about 81% and 34% at 400 µg/mL concentration, respectively. The radiosensitization enhancement factor for MCF-7 cells and fibroblasts at a dose of 3.5 Gy and 100 µg/mL concentration were 1.87 and 1.3, respectively. CONCLUSION: nHAP can be considered as a breast cancer radiosensitization agent with limited damage to the surrounding healthy tissue.

The Effects of Fibrin-icariin Nanoparticle Loaded in Poly (lactic-co-glycolic) Acid Scaffold as a Localized Delivery System on Chondrogenesis of Human Adipose-derived Stem Cells.

BACKGROUND: Nowadays, cartilage tissue engineering is the best candidate for regeneration of cartilage defects. This study evaluates the effect of fibrin/icariin (ICA) nanoparticles (F/I NPs) on chondrogenesis of stem cells. MATERIALS AND METHODS: F/I NPs were characterized by Dynamic Light Scattering DLS. Poly (lactic-co-glycolic) acid (PLGA)-F/I NP scaffold was fabricated and assessed by scanning electron microscope. Human adipose-derived stem cells (hADSCs) were seeded on scaffold and induced for chondrogenesis. After 14 days, cell viability and gene expression were analyzed by the 3-(4, 5- dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. MTT assay and real-time polymerase chain reaction (RT-PCR). RESULTS: The size and surface charge of F/I NP were about 28-30 nm and - 17, respectively. The average of pore size of PLGA and PLGA-fibrin/ICA was 230 and 340 µm, respectively. Cell viability of differentiated cells in P/F group was higher than others significantly (P ≤ 0.05). Furthermore, quantitative RT-PCR analysis demonstrated that ICA upregulated cartilaginous-specific gene expression. Furthermore, the results of the expression of type I collagen revealed that ICA downregulated this gene significantly (P < 0.01). CONCLUSIONS: The results indicated that F/I NP could be a potential factor for chondrogenesis of stem cells and downregulation of fibrocartilage marker.

Assessment of Immuno-Histochemical Expression of MBD1 in Colorectal Adenocarcinoma and Its Correlations with Prognostic Factors.

BACKGROUND MBD1, the largest member of methyl binding domain family, has the most downregulated mRNA expression and upregulated methylation levels in advanced colorectal cancer (CRC). In this study, we evaluated the immune-histochemical expression of MBD1 in CRC and assessed its correlation with clinicopathological features to study its prognostic value in CRC. METHODS A total of 60 samples of CRC, from patients who underwent surgical gastroenterology operations, were randomly selected. The samples included one tumor-rich section per case and one adjacent tumor-free section as a normal control for that case. Then, immunohistochemistry (ICH) was performed for MBD1 protein on all samples and the expression of MBD1 was analyzed in cancerous and normal samples. In the next step, the correlation between MBD1 and clinicopathological features including age, sex, location of the tumor, grade, and stage were evaluated. RESULTS The expression of MBD1 protein had a significant downregulation in cancerous samples compared with normal control samples. This downregulation increased corresponding to both grade and stage of cancer. However, no correlation was seen between the expression of MBD1 and sex, age and location of the tumor. CONCLUSION MBD1 protein may be considered as a protein marker in the detection of CRC and its progression.

Estrogen Receptor Beta (ERß) May Act as Mediator in Apoptotic Induction of Grape Seed Extract (GSE).

BACKGROUND: Grape seed extract is a complex mixture of polyphenols. Its anti-tumor effects have been reported by several studies. Estrogen receptors (ERs) are commonly considered as important markers for breast cancer. The present study aimed to evaluate the apoptotic effects of GSE on MCF7 breast cancer cells and assessed the expression of ERß during treatment of cells with GSE. MATERIAL AND METHODS: The half maximal inhibitory concentration (IC50) of GSE in MCF7 breast cancer cells were calculated by treating cells with serial dilution of GSE for 48 hours and cell viability evaluated using MTT assay. Then cells assigned to three groups: control (no treatment), DMSO (cells treated with 0.05% of DMSO) and GSE group (cells treated with of GSE for 48 hours). The apoptosis assay was performed by detecting Annexin V protein by flow cytometry. The gene expression of ERß and caspase-3 was evaluated by Real-Time PCR. RESULTS: Cells in GSE group treated with GSE IC50 concentration for 48 hours. Annexin V staining assay, represented early apoptosis detected by flow cytometry analysis showed significantly higher expression (p<0.01) than control and DMSO groups. Moreover, results of Real-Time PCR showed a significant expression in ERß and caspase-3 genes in GSE group compared to control and DMSO groups (Fold change = 2.3 and 3.5, respectively). CONCLUSION: GSE may induce apoptosis in MCF7 human breast cancer cells by activation of ERß gene.

BIO (6-bromoindirubin-3'-oxime) GSK3 inhibitor induces dopaminergic differentiation of human immortalized RenVm cells.

Parkinson's disease (PD) is one of the most neurodegenerative disorders which can lead to severe neural disability and neurological defects. Cell-based therapy using fully differentiated cells is a new method for the treatment of this abnormal condition. In the present study, we investigated the effects of 6-bromoindirubin-3'-oxime (BIO) on dopaminergic differentiation of human immortalized RenVm cells in order to obtain a set of fully differentiated cells for transplantation in Parkinson's disease. To this end, the immortalized RenVm cells were induced to dopaminergic differentiation using a neuro basal medium supplemented with N2 and different concentrations (75, 150, 300, 600, and 1200 nM) of BIO for 4, 8, and 12 days. The efficiency of dopaminergic differentiation was determined using immunocytochemistry for tyrosine hydroxylase expressions. In addition, the expression of a ß-catenin marker was measured using the western blot technique. The results of immunocytochemistry revealed that the mean percentage of Tuj1- and TH-positive sells in 150- and 300-nM-BIO-treated groups was significantly increased compared to that of other groups (p ≤ 0.01). In addition, the expression of the ß-catenin marker was higher in these groups as compared with that of other groups. Overall, BIO through its effect on the Wnt-Frizzled signaling pathway can promote dopaminergic differentiation of RenVm cells in a dose-dependent manner.

Lithium Chloride can Induce Differentiation of Human Immortalized RenVm Cells into Dopaminergic Neurons.

BACKGROUND: Stem cell-based therapy is a novel strategy for the treatment of neurodegenerative diseases. The transplantation of fully differentiated cells instead of stem cells in order to decrease serious adverse complications of stem cell therapy is a new idea. In this study, the effect of lithium chloride on dopaminergic differentiation of human immortalized RenVm cells was investigated in order to access a population of fully differentiated cells for transplantation in Parkinson disease. METHODS: The immortalized RenVm cells were induced to dopaminergic differentiation using a neurobasal medium supplemented with N2 and different concentrations (1, 3, 6 mM) of Lithium Chloride (LiCl) for 4, 8 and 12 days. The efficiency of dopaminergic differentiation was evaluated using immunocytochemistry and western blot techniques for tyrosine hydroxylase and ß-catenin marker expression. RESULTS: Our results indicated that LiCl can promote dopaminergic differentiation of RenVm cells in a dose-dependent manner. CONCLUSION: It can be concluded that LiCl is able to facilitate dopaminergic differentiation of cultured cells by affecting Wnt-frizzled signaling pathway.

Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells.

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.