Exploring the Correlation between Expression of EMT Transcription Factors and CXCR4 Expression

Background: Metastasis is one the most leading cause of death from cancer. The chemokine receptor of CXCR4 has an important role in cell migration and cancer metastasis. Additionally, metastasis is always associated with the process of epithelial-mesenchymal transition (EMT). In this study the correlation between expression of CXCR4 and EMT-TFs has been examined.Methods: The expression of CXCR4 in knocked out SUM159 cell line for EMT-TFs of slug, snail, twist and ZEB1 were examined.Results: The results revealed that the expression of CXCR4 decreased significantly in twist and ZEB1 knocked out cells, however in other groups no change was observed. Decreased expression of CXCR4 indicated that ZEB1 and twist may be one of regulators of CXCR4 expression.Conclusions: ChIP assay should be performed in future experiments to see the definite role of ZEB1 and twist as transcription factors for CXCR4

Exploring the Correlation between Expression of EMT Transcription Factors and CXCR4 Expression

Background: Metastasis is one the most leading cause of death from cancer. The chemokine receptor of CXCR4 has an important role in cell migration and cancer metastasis. Additionally, metastasis is always associated with the process of epithelial-mesenchymal transition (EMT). In this study the correlation between expression of CXCR4 and EMT-TFs has been examined.Methods: The expression of CXCR4 in knocked out SUM159 cell line for EMT-TFs of slug, snail, twist and ZEB1 were examined.Results: The results revealed that the expression of CXCR4 decreased significantly in twist and ZEB1 knocked out cells, however in other groups no change was observed. Decreased expression of CXCR4 indicated that ZEB1 and twist may be one of regulators of CXCR4 expression.Conclusions: ChIP assay should be performed in future experiments to see the definite role of ZEB1 and twist as transcription factors for CXCR4

Effects of Two Calcium Silicate Cements on Transforming Growth Factor-β1 Secretion from Human Dental Pulp Stem Cells

Introduction: The aims of this in vitro study were to evaluate the effects of two calcium silicate based cements, Calcum-enriched Mixture (CEM) and Biodentine  on proliferation of human dental pulp stem cells (hDPSCs) and the effects of proposed cements on the secretion of Transforming Growth Factor β1 (TGF-β1). Methods and materials: The cell cultures of human Dental Pulp Stem Cells (hDPSCs) at passage 3-5 were treated with various dilutions (1/1, 1/2, 1/4, 1/8, 1/16, and 1/32) of CEM and Biodentine extracts to assess the cell proliferation using 3-(4, 5-dimethylthiazol-2-Y1)-2, 5-diphenyltetrazolium brovide (MTT) assay after 48 and 72 h. The amount of TGF-β 1 secretion were estimated after 72 h using an enzyme-linked immunosorbent assay. Data were analyzed using the one-way analysis of variance (ANOVA) followed by the Dunnett’s test at the level of significance set at 0.05. Result: CEM showed the highest rates of cell proliferation compared to Biodentine after 72 h (P<0.05). A greater amount of TGF-β1 was secreted by hDPSCs treated with Biodentine compared to CEM (P<0.05). These differences were statistically significant (P<0.05). Conclusion: In this in vitro study hDPSCs showed more proliferation capacity with CEM rather than Biodentine and TGF-β1 secretion rate in Biodentine was higher.Keyword: Biodentine; Calcium-Enriched Mixture; Human Dental Pulp Stem Cells; Proliferation; Transforming Growth Factor-β1

The Effects of Extracellular Matrices and Co-Culture Systems on Cultured Limbal Stem Cells

Objective: To study the effects of different matrices and co-culture systemson cultured limbal stem cells (LSCs).Materials and Methods: Limbal explants were co-cultured with limbalfibroblasts (LF) and/or mouse embryonic fibroblasts (MEF) on filter insertscoated with amniotic membrane (AM), matrigel (MAT) and collagen type I(COL).Results: This study revealed that AM facilitated the cell migration andexpansion significantly in comparison with other matrices. However, thegene expression profile of stemness markers of LSCs showed no significantdifferences among the experimental groups. The data indicated that at least intwo-dimensional culture systems, the mentioned matrices have no significanteffect on switching the expression of genes involved in differentiation process.In addition, the results of the two co-culture systems in case of differentfeeders, including MEF and LF were similar in growth rate and also preservingstemness quality of cultured limbal cells.Conclusion: To exclude the pollution of transplantable cultivated cells withprobable mouse viruses, LF with human origin is recommended as feeder.Hence, limbal explants grown on AM in co-culture with LF will promise a quickand safe model for preparing undifferentiated epithelial sheets suitable fortransplantation

Strategies to Improve Homing of Stem Cells to achieve better Efficacy in Stem Cell Therapy

Stem/progenitor cell based therapeutic approach in our daily routine clinical practice, has been elusive dream in medical sciences and improvement of stem cell homing as one of major challenges in cell therapy programs, has been considered a promising milestone. It has been proved that stem/progenitor cells exhibit a homing response to injured tissues/organs, mediated by interactions of chemokine receptors expressed on the cells and chemokines secreted by the injured tissue. For improvement of directed homing of the cells, many techniques have been developed either to engineer stem/progenitor cells with higher amount of chemokine receptors (stem cell-based strategies) or to modulate the target tissues to release higher level of the corresponding chemokines (target tissue-based strategies). Treatment with chemical compounds, preconditioning of the cells with hypoxia, cytokine and growth factor priming of the cells, genetic modifications, coating of cell surface with antibodies, glycoengineering, coating of stem cells with homing ligands by streptavidin linkers are some of strategies to increase the ability of stem cells to respond to the migratory stimuli. On the other side to modulate target sites to be more attractive for stem cells, some strategies like direct injection of chemokines, direct transfection of the target tissue with chemokine encoding genes, injection of ectopic chemokine expressing cells, application of scaffolds, electrical fields and low level laser have been introduced. These extensive investigations have provided significant potentials to enhance targeted stem/progenitor cells homing. Meanwhile there are still some limitations to apply these findings in clinics. To overcome these limitations, further studies should be aimed, unveiling the molecular and cellular mechanisms underlying endogenous cell trafficking during physiological and pathological events like embryogenesis, inflammation, wound healing, or cancer metastasis. Keywords: Cell therapy, Homing, Stem cells

Berberine suppresses migration of MCF-7 breast cancer cells through down-regulation of chemokine receptors

Objective(s): Berberine is one of the main alkaloids and it has been proven to have different pharmacological effects including inhibition of cell cycle and progression of apoptosis in various cancerous cells; however, its effects on cancer metastasis are not well known. Cancer cells obtain the ability to change their chemokine system and convert into metastatic cells. In this study, we examined the effect of berberine on breast cancer cell migration and its probable interaction with the chemokine system in cancer cells. Materials and Methods: The MCF-7 breast cancer cell line was cultured, and then, treated with berberine (10, 20, 40 and 80 μg/ml) for 24 hr. MTT assay was used in order to determine the cytotoxic effect of berberine on MCF-7 breast cancer cells. Wound healing assay was applied to determine the inhibitory effect of berberine on cell migration. Moreover, real-time quantitative PCR analysis of selected chemokine receptors was performed to determine the probable molecular mechanism underlying the effect of berberine on breast cancer cell migration. Results: The results of wound healing assay revealed that berberine decreases cell migration. Moreover, we found that the mRNA levels of some chemokine receptors were reduced after berberine treatment, and this may be the underlying mechanism for decreased cell migration.  Conclusion: Our results indicate that berberine might be a potential preventive biofactor for human breast cancer metastasis by targeting chemokine receptor genes

Chemically primed bone-marrow derived mesenchymal stem cells show enhanced expression of chemokine receptors contributed to their migration capability

Objective(s):The limited homing potential of bone-marrow-derived mesenchymal stem cells (BM-MSC) is the key obstacle in MSC-based therapy. It is believed that chemokines and chemokine receptor interactions play key roles in cellular processes associated with migration. Meanwhile, MSCs express a low level of distinct chemokine receptors and they even lose these receptors on their surface after a few passages which influence their therapeutic applications negatively. This study investigated whether treatment of BM-MSCs with hypoxia-mimicking agents would increase expression of some chemokine receptors and cell migration. Materials and Methods: BM-MSCs were treated at passage 2 for our gene expression profiling. All qPCR experiments were performed by SYBR Green method in CFX-96 Bio-Rad Real-Time PCR. The Boyden chamber assay was utilized to investigate BM-MSC homing. Results:Possible approaches to increasing the expression level of chemokine receptors by different hypoxia-mimicking agents such as valproic acid (VPA), CoCl2, and desferrioxamine (DFX) are described. Results show DFX efficiently up-regulate the CXCR7 and CXCR4 gene expression while VPA increase only the CXCR7 gene expression and no significant change in expression level of CXCR4 and the CXCR7 gene was detectable by CoCl2 treatment. Chemotaxis assay results show that pre-treatment with DFX, VPA, and Cocl2 enhances significantly the migration ability of BM-MSCs compared with the untreated control group and DFX treatment accelerates MSCs homing significantly with a higher rate than VPA and Cocl2 treatments. Conclusion: Our data supports the notion that pretreatment of MSC with VPA and DFX improves the efficiency of MSC therapy by triggering homing regulatory signaling pathways

Pretreatment of Mesenchymal Stem Cells and Stromal-derived Factor-1α Delivery from Chitosan-based Injectable Hydrogels for Better Cell Guidance and Retention

Clinical applications of mesenchymal stem cells (MSCs) rely on their capacity to home and engraft in the appropriate target tissues for a long time. Homing and engraftment capacity of these stem cells depend on the expression of Chemokines and their receptors. Ex vivo expanded MSCs exhibit homing potential when grafted to injury tissue but their homing efficiency has been observed very poor because of modifications in homing receptor expression and/or functions during culture and/or preparation steps. Hence, this study was designed to investigate the expression of surface CXCR4 by flow cytometric analysis (FACS) and in vitro modified Boyden chamber assay in adipose-derive MSCs (ASCs) stimulated with a hypoxia mimicking agents such as desferrioxamine mesilate (DFX), cobalt chloride (CoCl2), lithium chloride (LiCl), valproic acid (VPA) and hypoxia. Intracellular CXCR4 were also evaluated by conventional and real-time PCR. Then we evaluated the homing ability of DFX-pretreated human DiI-labeled ASCs in vivo, 2 weeks after intravenous (IV), local infusion towards subcutaneously implanted chitosan-glycerophophate-hydroxyethyl cellulose (CH-GP-HEC) injectable hydrogels releasing SDF1 in dorsum of Wistar Rats. Presence of human ASCs in the CH-GP-HEC injectable, spleen, and lung were analyzed histologically by fluorescent microscope, and also quantified by PCR for human specific CXCR4 gene, 2 weeks after transplantation in recipients' Rats. Results showed that short-term (24 hours) pretreatment to ASCs with the hypoxia mimicking agents up-regulate the CXCR4, increase in vitro migration capacity toward 100ng/ml SDF-1 (