Effect of testosterone on the mRNA expression of Wnt-2 and dickkopf1 (DKK1), collagen deposition and oxidative stress in the cardiac tissue in male rats.

Some studies suggest that misuse of androgenic-anabolic steroids may increase the risk of cardiovascular diseases in males. This study explored the effects of testosterone enanthate (TE) on the total antioxidant capacity (TAC) and malondialdehyde (MDA) levels as biomarkers of oxidative stress in the cardiac tissue of rats that were treated with TE. Also, we evaluated the levels of collagen deposition as a marker for cardiac fibrosis and the mRNA expression of the Wnt-2 and dickkopf1 (DKK1) as potential factors that may be involved in the increase of collagen deposition. In this study, 21 male Wistar rats were divided into three groups (n=7): CO: controls; T-T: normal rats that were treated with 25 mg/kg/day TE for 2 weeks and served as an androgen abuse model; V-T: these animals were treated with the sesame oil as a solvent of TE. At the end of treatment, the relative mRNA expression of Wnt-2 and DKK1 in the ventricular tissue was determined by q-RT-PCR. The degree of collagen deposition in the myocardial tissue was evaluated by Masson's trichrome staining. Results showed that the mRNA expression of DKK1 was down-regulated following excess androgen exposure (p=0.009) but Wnt-2 mRNA expression wasn't affected (p=0.069). Increased collagen deposition was observed in the T-T group (p=0.000). The levels of MDA and TAC in heart tissue weren't altered significantly (p>0.05). These results suggest that the raised collagen deposition by exogenous testosterone may be mediated, at least in part, by the reduction of expression of DKK1 mRNA. These findings may explain some structural alterations in the heart of some androgens abusers.

Biomedical applications of MnO2 nanomaterials as nanozyme-based theranostics.

Manganese dioxide (MnO2) nanoenzymes/nanozymes (MnO2-NEs) are 1-100 nm nanomaterials that mimic catalytic, oxidative, peroxidase, and superoxide dismutase activities. The oxidative-like activity of MnO2-NEs makes them suitable for developing effective and low-cost colorimetric detection assays of biomolecules. Interestingly, MnO2-NEs also demonstrate scavenging properties against reactive oxygen species (ROS) in various pathological conditions. In addition, due to the decomposition of MnO2-NEs in the tumor microenvironment (TME) and the production of Mn2+, they can act as a contrast agent for improving clinical imaging diagnostics. MnO2-NEs also can use as an in situ oxygen production system in TME, thereby overcoming hypoxic conditions and their consequences in the progression of cancer. Furthermore, MnO2-NEs as a shell and coating make the nanosystems smart and, therefore, in combination with other nanomaterials, the MnO2-NEs can be used as an intelligent nanocarrier for delivering drugs, photosensitizers, and sonosensitizers in vivo. Moreover, these capabilities make MnO2-NEs a promising candidate for the detection and treatment of different human diseases such as cancer, metabolic, infectious, and inflammatory pathological conditions. MnO2-NEs also have ROS-scavenging and anti-bacterial properties against Gram-positive and Gram-negative bacterial strains, which make them suitable for wound healing applications. Given the importance of nanomaterials and their potential applications in biomedicine, this review aimed to discuss the biochemical properties and the theranostic roles of MnO2-NEs and recent advances in their use in colorimetric detection assays of biomolecules, diagnostic imaging, drug delivery, and combinatorial therapy applications. Finally, the challenges of MnO2-NEs applications in biomedicine will be discussed.

Amino acid profile changes during enrichment of spheroid cells with cancer stem cell properties in MCF-7 and MDA-MB-231 cell lines.

BACKGROUND: Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis. AIMS: This study aimed to evaluate the changes in the amino acid profile in MCF-7 and MDA-MB-231 cells during spheroid formation to discover the specific metabolic properties in CSCs. METHODS: MCF-7 and MDA-MB-231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high-performance liquid chromatography (HPLC). RESULTS: The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF-7 and MDA-MB-231 spheroids (histidine and serine, respectively). CONCLUSION: Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation.

Thymoquinone-treated mouse mesenchymal stem cells-derived conditioned medium inhibits human breast cancer cells in vitro.

Breast cancer is now the most prevalent cancer in females, therefore, it is essential to identify factors affecting its initiation and progression. Mesenchymal stem cells (MSCs) have received considerable attention in stem cell-based therapies and drug delivery applications. Because the therapeutic potential of MSCs is primarily achieved by their paracrine effects, thus identifying and employing bioactive molecules that promote the paracrine activity of MSCs is crucial for their efficient use in cancer treatment. Thymoquinone (TQ) has many biomedical properties, including anti-inflammatory, anti-diabetic, anti-aging, anti-cancer, etc. In addition, it has been found that TQ affects the self-renewal and immunomodulatory properties of MSCs. The present study aimed to investigate the effect of TQ-treated mouse bone marrow-derived MSCs conditioned medium (TQ-MSC-CM) on the biological characteristics of breast cancer cell line MCF7. MSCs were cultured and treated with TQ for 24 h. The TQ-MSC-CM and MSC-CM were collected, and their effects were investigated on ROS production, mitochondrial membrane potential (MMP), cell death, cell cycle, and migration of MCF7 cells by DCFDA-cellular ROS assay, Rhodamine-123 MMP assay, Annexin-PI staining and Caspase-3/7 activity assays, PI-staining and flow-cytometry, and in vitro wound healing assay, respectively. Moreover, the effects of TQ-MSC-CM and MSC-CM were studied on Cdk4, Sox2, c-Met, and Bcl2 gene expression by real-time PCR. Results demonstrated that MSC-CM and TQ-MSC-CM did not have a significant effect on the apoptosis induction in MCF7 cells; however, they significantly stimulated necrosis in the cells. Although TQ-MSC-CM promoted ROS production in MCF7 cells, it decreased the MMP of the cells. TQ-MSC-CM also induced Bcl2 anti-apoptosis gene expression and Casp-3/7 activity in cells. In addition, although MSC-CM induced MCF7 cells to enter the cell cycle, TQ-MSC-CM inhibited its progression. TQ-MSC-CM also downregulated the Cdk4 and Sox2 gene expression. Furthermore, TQ-MSC-CM induced the migration potential of MCF7 in a c-Met-independent manner. Altogether, we conclude that TQ may induce programmed necrosis and inhibits the proliferation and migration of the breast cancer cells by affecting the paracrine activity of MSCs.

Thymoquinone promotes mouse mesenchymal stem cells migration in vitro and induces their immunogenicity in vivo.

Mesenchymal stem cells (MSCs) have unique potentials, including migration and immunomodulation. Identification of the factors that enhance these activities can improve clinical applications of MSCs. This study aimed to investigate total antioxidant capacity (TAC) and migration potential of mouse MSCs exposed to thymoquinone (TQ) in vitro, and to examine the effect of TQ-treated MSCs on the expression of mouse immune cell markers. The results of total antioxidant capacity and wound healing assays showed that TQ increased the rate of MSCs TAC and migration in a dose- and time-dependent manner. The maximum TAC and migration were detected at 600 and 250 ng/ml of TQ, respectively. Functionally, the real-time PCR data analysis indicated that TQ induced c-Met and Cxcr4 expression and therefore, there may be a correlation between upregulation of these genes and increased MSCs migration. TQ also enhanced the up and down regulating impact of MSCs on Rorγt and Plzf expression and the expression of Tcf4 in mouse immune cells, respectively. Overall, this study declares that TQ increases the TAC of MSCs. It also proposes that TQ may, through activation of c-MET and CXCR4 signalling pathways, promote MSCs migration. TQ may also augment MSCs immunogenicity through its influence on the expression of genes involved in commitment of mouse immune system cells in vivo.

In silico analysis of single-cell RNA sequencing data from 3 and 7 days old mouse spermatogonial stem cells to identify their differentially expressed genes and transcriptional regulators.

Spermatogonial stem cells (SSCs), which are at the basis of spermatogenesis process, are valuable cells with different applications in biotechnology and regenerative medicine. Understanding the molecular basis of SSC self-renewal and differentiation at various developmental stages of the male organism is crucial to find key factors in the SSCs fate and function. Therefore, this study was aimed to use single-cell RNA-sequencing dataset analysis for identification of differentially expressed genes (DEGs) and their regulators in 3 and 7 days old mouse-derived single SSCs (mSSCs). Results showed 68 upregulated and 203 downregulated genes in 7 days old mouse-derived SSCs compared to 3 days old mSSCs, which were associated with 1493 and 3077 biological processes, respectively. It also found that DAZL, FKBP6, PAIP2, DDX4, H3F3B, TEX15, XRN2, MAEL, and SOD1 are important factors with the higher gene expression pattern, which may be pivotal for mSSCs fate and function during development of germ cells. Moreover, NR3C1, RXRA, NCOA, ESR1, PML, ATF2, BMI1, POU5F1, and CHD1 were the main central regulators for the upregulated DEGs, while HNF1A, C/EBPα, and NFATC1 were the master regulators for the downregulated DEGs. In this regard, two significant protein complexes were found in the protein-protein interactions network for the upregulated DEGs regulators. Furthermore, 24 protein kinases detected upstream of the main central regulators of DEGs. In conclusion, this study presents DEGs and their transcriptional regulators that are crucial for inducing and regulating SSCs commitment during development, and for developing efficient protocols to identify and isolate SSCs for different applications.

The effect of resveratrol on oxidative stress in the liver and serum of a rat model of polycystic ovary syndrome: An experimental study.

BACKGROUND: Studies of oxidative status in polycystic ovarian syndrome (PCOS) patients are limited with inconsistent results. The effects of resveratrol as a natural antioxidant on oxidative status in PCOS aren't clear. OBJECTIVE: This study evaluated effects of resveratrol on oxidative stress in the liver and serum of the PCOS rats. MATERIALS AND METHODS: Fifteen female Wistar rats (3 wk old) were divided into 3 groups (n=5/each e): Control group, PCO-Control group, and PCO-Resveratrol group. For induction of polycystic ovary phenotype, testosterone enanthate 10 mg/kg was injected for 35 days subcutaneously. Then, resveratrol 10 mg/kg was injected intraperitoneally for 28 days to rats of the PCO-Resveratrol group. Ovarian sections were stained with hematoxylin/eosin. The serum glucose and insulin and the levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) in serum and liver were measured. RESULTS: Control animals showed normal ovarian morphology and PCO-Control animals exhibited cystic follicles. There were no significant differences in liver TAC between groups. The serum MDA (p=0.034), and homeostatic model assessment insulin resistance (HOMA-IR) (p=0.014) levels in PCO-Control rats were higher than the controls. The liver MDA in PCO-Control rats was more than that of controls (p=0.001). The HOMA-IR (p=0.008) and serum MDA (p=0.006) levels in PCO-Control rats were more than those of PCO-Resveratrol rats (p=0.008). In PCO-Resveratrol group, serum TAC was higher than that of PCO-Control group (p=0.022) and liver MDA was more than controls (p=0.01). CONCLUSION: Results indicated that the induction of PCOS in rats increased lipid peroxidation and insulin resistance and resveratrol improved these complications.

Thymoquinone influences the expression of genes involved in self-renewal and immunomodulatory potential of mouse bone marrow-derived mesenchymal stem cells in vitro.

Thymoquinone (TQ) is an active ingredient of some medicinal herbs. Despite extensive studies on the biological and pharmacological properties of TQ, its effect on the characteristics of stem cells remains to be clarified. Therefore, this study was aimed to investigate the effect of TQ on viability, proliferation and immunomodulatory potential of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) in vitro. The BM-MSCs were isolated from young NMRI mice. The cytotoxic effect of TQ on the BM-MSCs was evaluated using MTT assay. Then, the effect of TQ on the proliferation of BM-MSCs and the mRNA expression of genes involved in self-renewal and immunomodulatory potential of MSCs was assessed by the cell counting and real-time PCR assays. Results showed that TQ reduces the number of BM-MSCs in a dose- and time-dependent manner. In addition, the half-maximal inhibitory concentration values of TQ on the BM-MSCs were 8 µg/ml at 24h and 4 µg/ml at 48 and 72h after treatment. Furthermore, about 90% of the BM-MSCs were alive after treatment with concentrations ≤2 µg/ml of TQ for 24h. The results of cell counting assay indicated that TQ at concentrations of 1-2 µg/ml significantly enhanced the proliferation of BM-MSCs (P < 0.05). The gene expression analysis also showed that Tlr3, Tlr4, Ccl2, Ccl3, Sox2, and Rex1 are overexpressed (Fold change ≥1.5) in the TQ-treated BM-MSCs compared with the untreated samples. In conclusion, these findings propose that TQ may regulate self-renewal and immunomodulatory potential of MSCs. However, the exact mechanisms and the roles of this regulation are required to be elucidated in further study.

Evaluation of TNF-α and IL-6 mRNAs expressions in visceral and subcutaneous adipose tissues of polycystic ovarian rats and effects of resveratrol.

OBJECTIVES: Some studies suggest that chronic low-grade inflammation is involved in insulin resistance in polycystic ovary syndrome (PCOS). This study assessed possible involvement of alteration in expression of two pro-inflammatory factors, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in adipose tissues of PCOS rats in the impairment of insulin actions. Also, effects of resveratrol as an anti-inflammatory agent were investigated. MATERIALS AND METHODS: Fifteen female Wistar rats (21 days old) were divided into three groups (n=5): I) Control, II) PCO-model-saline: served as PCOS rats and to induce PCOS, received subcutaneously testosterone enanthate 1 mg/100 g body weight subcutaneously for 35 days, III) PCO-model-resveratrol, after receiving testosterone, received resveratrol 10 mg/kg intraperitoneally for 28 days. The expression of Tnf-α and Il-6 mRNAs in adipose tissues was determined by the qRT-PCR method. RESULTS: The Il-6 mRNA expression in the visceral adipose tissue of PCOS rats was increased in comparison to controls (P<0.05). Tnf-α and Il-6 mRNA expression in visceral and subcutaneous adipose tissues of polycystic ovarian rats was similar to controls. The expression of Tnf-α mRNA in subcutaneous adipose tissue and Tnf-α and Il-6 mRNAs in the visceral adipose tissue of the PCO-model-resveratrol group were lower than PCOS rats (P<0.05). CONCLUSION: Increased expression of Il-6 mRNA in the visceral adipose tissue of polycystic ovarian rats may be one cause of insulin resistance observed in them and resveratrol as an anti-inflammatory and anti-hyperglycemic agent may decrease the risk of diabetes by reduction of expression of pro-inflammatory cytokines TNF-α and IL-6 in PCOS patients.

Eugenol enhances proliferation and migration of mouse bone marrow-derived mesenchymal stem cells in vitro.

Mesenchymal stem cells (MSCs) have received considerable attention in regenerative medicine during the past decade. Eugenol is a natural and versatile vegetable molecule, which has a wide variety of therapeutic effects. Although different biological and pharmaceutical functions of Eugenol are well known, its effect on MSCs has not been studied yet. Therefore, this study was focused on investigating the effect of Eugenol on the proliferation and migration of bone marrow (BM)-derived MSCs in vitro. To do so, BM-MSCs were isolated from 4 to 8 weeks old NMRI mice. Cytotoxicity of Eugenol on MSCs was evaluated by MTT assay at 24, 48 and 72 h after treatment. In addition, its effect was assessed on the proliferation and migration of MSCs using wound healing assay in vitro and quantitative gene expression analysis for Oct4, Sox2, Cyclin-D1, Rex1, Tex10, Cxcr4, Vla4 and c-Met. Results showed that Eugenol reduced the number of MSCs in a dose- and time-dependent manner. The median inhibition concentration of Eugenol on MSCs was 400 µg/ml at 24 and 48 h and 200 µg/ml at 72 h after treatment. Moreover, about 90% viability of MSCs was detected at concentrations ≤12.5 µg/ml. The wound healing assay and gene expression analysis demonstrated that Eugenol promoted the migratory potential of MSCs through up-regulation of c-Met. Moreover, Eugenol has enhanced the proliferation of MSCs via over-expression of Sox2, Rex1 and Tex10. In conclusion, this study revealed that Eugenol enhances the proliferation and migration of MSCs, and thus this will be beneficial to the field of regenerative medicine.

Comparative analysis of single-cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells.

Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells.

External factors influencing mesenchymal stem cell fate in vitro.

Mesenchymal stem cells (MSCs) have extensive potentials, which make them attractive candidates for the developmental biology, drug discovery and regenerative medicine. However, the use of MSCs is limited by their scarceness in tissues and in culture conditions. They also exhibit various degrees of potency which subsequently influencing their applications. Nowadays, questions remain about how self-renewal and differentiation of MSCs can be controlled in vitro and in vivo, how they will behave and migrate to the right place and how they modulate the immune system. Therefore, identification of factors and culture conditions to affect the fate and function of MSCs may be effective to enhance their applications in clinical situations. Studies have indicated that the fate of MSCs in culture is influenced by various external factors, including the specific cell source, donor age, plating density, passage number and plastic surface quality. Some other factors such as cell culture media and their supplementary factors, O2 concentration, mechano-/electro-stimuli and three-dimensional scaffolds are also shown to be influential. This review addresses the current state of MSC research for describing and discussing the findings about external factors that influence the fate and function of MSCs. Additionally, the new discoveries and suggestions regarding their molecular mechanisms will be explained.

Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

Emerging physiological and pathological implications of tunneling nanotubes formation between cells.

Cell-to-cell communication is a critical requirement to coordinate behaviors of the cells in a community and thereby achieve tissue homeostasis and conservation of the multicellular organisms. Tunneling nanotubes (TNTs), as a cell-to-cell communication over long distance, allow for bi- or uni-directional transfer of cellular components between cells. Identification of inducing agents and the cell and molecular mechanism underling the formation of TNTs and their structural and functional features may lead to finding new important roles for these intercellular bridges in vivo and in vitro. During the last decade, research has shown TNTs have different structural and functional properties, varying between and within cell systems. In this review, we will focus on TNTs and their cell and molecular mechanism of formation. Moreover, the latest findings into their functional roles in physiological and pathological processes, such as signal transduction, micro and nano-particles delivery, immune responses, embryogenesis, cellular reprogramming, apoptosis, cancer, and neurodegenerative diseases initiation and progression and pathogens transfer, will be discussed.

Thymoquinone and its therapeutic potentials.

Herbal medicine has attracted great attention in the recent years and is increasingly used as alternatives to chemical drugs. Several lines of evidence support the positive impact of medicinal plants in the prevention and cure of a wide range of diseases. Thymoquinone (TQ) is the most abundant constituent of the volatile oil of Nigella sativa seeds and most properties of N sativa are mainly attributed to TQ. A number of pharmacological actions of TQ have been investigated including anti-oxidant, anti-inflammatory, immunomodulatory, anti-histaminic, anti-microbial and anti-tumor effects. It has also gastroprotective, hepatoprotective, nephroprotective and neuroprotective activities. In addition, positive effects of TQ in cardiovascular disorders, diabetes, reproductive disorders and respiratory ailments, as well as in the treatment of bone complications as well as fibrosis have been shown. In addition, a large body of data shows that TQ has very low adverse effects and no serious toxicity. More recently, a great deal of attention has been given to this dietary phytochemical with an increasing interest to investigate it in pre-clinical and clinical researches for assessing its health benefits. Here we report on and analyze numerous properties of the active ingredient of N. sativa seeds, TQ, in the context of its therapeutic potentials for a wide range of illnesses. We also summarize the drug's possible mechanisms of action. The evidence reported sugests that TQ should be developed as a novel drug in clinical trials.

The molecular signature and spermatogenesis potential of newborn chicken spermatogonial stem cells in vitro.

Although chicken spermatogonial stem cells (SSCs) have received considerable attention in recent years, only a few studies so far have focused on their derivation and characterization in vitro. Identification of specific molecular biomarkers and differentiation capacity of chicken SSCs would not only help us to understand cell and molecular biology of these cells, but also can contribute to their applications in biotechnology. In this regard, we found that colony-forming cells (SSCs) in newborn chicken testicular cell cultures were positive for alkaline phosphatase activity and also expressed specific markers including DAZL, STRA-8, CVH, PLZF, SPRY-1, GFRα1, GDNF, POU5F1, NANOG, GPR125, THY-1, c-KIT, and BCL6B, at mRNA level. Moreover, these cells expressed POU5F1 and GPR125 proteins as reliable intracellular and cell surface markers, respectively; whereas they were negative for SSEA-1. Furthermore, we showed that newborn chicken colony-forming cells had spermatogenesis potential and thus could be produced sperm-like cells in a three-dimensional matrix in vitro. In conclusion, this study reports novel insights into the molecular signature of newborn chicken SSCs in comparison with mammalian SSCs and for the first time we report a successful protocol for in vitro spermatogenesis and thus production of sperm-like cells from newborn chicken testicular cell cultures.

SOX2 expression in gastrointestinal cancers of Iranian patients.

PURPOSE: Gastrointestinal (GI) malignancies are among the 5 most common cancers in Iran, and their high associated mortality rates are attributable to late diagnosis and poor treatment options. SOX2, a transcription factor necessary for maintenance and induction of pluripotency and self-renewal, has been identified as a lineage-survival oncogene in several cancers. In the present study, we examined SOX2 expression in esophageal squamous cell carcinoma (ESCC), gastric adenocarcinoma and colon squamous cell carcinoma (SCC), as well as normal GI tissues, in Iranian patients. METHODS: To elucidate the role of SOX2 in GI carcinogenesis, formalin-fixed tissues were analyzed using immunohistochemistry (IHC), while frozen ESCC samples were studied by quantitative reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: IHC studies indicated presence of SOX2+ cells in a subset of cancerous and normal tissues of stomach and colon, while no significant difference was observed between groups, and no correlation was found between SOX2 expression and tumors grades. Nevertheless, studying ESCC samples with IHC and qRT-PCR revealed overexpression of SOX2 in comparison with normal adjacent tissues. CONCLUSIONS: The present results are in line with other studies and indicate SOX2 up-regulation in ESCC; however, due to our small sample size and contradictory reports, more research is needed to determine the importance of SOX2 in GI cancers.

Construction and quantitative evaluation of a dual specific promoter system for monitoring the expression status of Stra8 and c-kit genes.

Applications of genetic constructs with multiple promoters, which are fused with reporter genes and simultaneous monitoring of various events in cells, have gained special attention in recent years. Lentiviral vectors, with their distinctive characteristics, have been considered to monitor the developmental changes of cells in vitro. In this study, we constructed a novel lentiviral vector (FUM-M), containing two germ cell-specific promoters (Stra8 and c-kit), fused with ZsGreen and DsRed2 reporter genes, and evaluated its efficiency in different cells following treatments with retinoic acid and DMSO. Several cell lines (P19, GC-1 spg and HEK293T) were transduced with this vector, and functional capabilities of the promoters were verified by flow cytometry and quantitative RT-PCR. Our results indicate that FUM-M shows dynamic behavior in the presence and absence of extrinsic factors. A correlation was also observed between the function of promoters, present in the lentiviral construct and the endogenous level of the Stra8 and c-kit mRNAs in the cells. In conclusion, we recommend this strategy, which needs further optimization of the constructs, as a beneficial and practical way to screen chemical inducers involved in cellular differentiation toward germ-like cells.

A simple method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are expected to participate in male infertility therapy, endangered species preservation, and transgenic animal technology by their unique unipotency to differentiate into spermatozoa. The main challenges, however, remain to be addressed including the appropriate conditions to reach good number of these cells and how to derive, culture, and maintain them in vitro. In the present study, the testicular tissues were isolated from 1-d-old male chickens to establish primary cell cultures. This culture led to development of distinguished colonies which were further characterized by alkaline phosphatase (AP) activity assay and gene expression analysis. They were shown to be positive for AP activity and expressed two main transcription factors of OCT4 and STRA8 as indicated by reverse transcription-polymerase chain reaction. These were indications of carrying characteristics of SSCs by these colonies. The cultures were also exposed to different concentrations of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) growth factors to seek optimum colony-forming conditions. Colony-forming activity assay indicated that they were able to propagate in vitro with an increased self-renewal property when cultured in the presence of 15 ng/mL of GDNF, 20 ng/mL of bFGF, and 15 ng/mL of LIF. The present work provides an easy and practical method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells and introduces appropriate cell culture conditions to improve and maintain their self-renewal property based on supplying the necessary growth factors.

Transdifferentiation: a cell and molecular reprogramming process.

Evidence has emerged recently indicating that differentiation is not entirely a one-way process, and that it is possible to convert one cell type to another, both in vitro and in vivo. This phenomenon is called transdifferentiation, and is generally defined as the stable switch of one cell type to another. Transdifferentiation plays critical roles during development and in regeneration pathways in nature. Although this phenomenon occurs rarely in nature, recent studies have been focused on transdifferentiation and the reprogramming ability of cells to produce specific cells with new phenotypes for use in cell therapy and regenerative medicine. Thus, understanding the principles and the mechanism of this process is important for producing desired cell types. Here some well-documented examples of transdifferentiation, and their significance in development and regeneration are reviewed. In addition, transdifferentiation pathways are considered and their potential molecular mechanisms, especially the role of master switch genes, are considered. Finally, the significance of transdifferentiation in regenerative medicine is discussed.