Cell Journal (Yakhteh)

115-123

Authors: Ali Ehsanpour, Najmaldin Saki, Marziyeh Bagheri, Masumeh Maleki Behzad, Saeid Abroun

Microvesicles (MVs) are the smallest subclass of the extracellular vesicles (EVs) spontaneously secreted by the external budding from the cell membranes in physiologic and pathologic conditions. The MVs derived from leukemic cells (LCs) can be detected by the expression of specific cluster of differentiation (CD) markers indicating their cellular origin while they can transfer different agents such as microRNAs, cytokines, and chemokines. The secretion of these agents from MVs can affect the vital processes of LCs such as cell cycle, proliferation, differentiation, and apoptosis. According to the effects of MVs components on the vital processes of LCs, it has been postulated that a change in the expression of MVs might be involved in the progression and prognosis of leukemia. However, further studies are needed to confirm the association between the presence of MVs and their components with the prognosis of leukemia. It seems that the identification of the prognostic values and the application of them for the detection of MVs in leukemia can provide new therapeutic targets for monitoring the status of patients with leukemia.

135-142

Authors: Fatemeh Firouzabadi, Shahrbanoo Oryan, Mohammad Hasan Sheikhha, Seyed Mehdi Kalantar, Ameneh Javed

Objective Today, in clinical trials, we suffer from the lack of effective methods with minimal side effects to deliver medication. Thus, efforts to identify better conditions for delivery of biomedical drugs seem necessary. The purpose of this study was to design a new liposomal formula for transportation of microRNA in osteosarcoma. Materials and Methods In this experimental study, several liposomal formulations were synthesized. Physical and chemical parameters, including size, zeta potential, polydispersity index, long-term stability of the liposomal-microRNA complex and the amount of miR-143 loading in liposome based nano-vesicles were optimized using different techniques. Similarly, the effect of free and encapsulated microRNA toxicity were investigated and compared in a human bone osteosarcoma cell line, named SaOs-2. Results In this study, we could produce a novel and optimized formulation of cationic PEGylated liposomal microRNA for gene delivery. The present synthesized microRNA lipoplex system was non-agglomerated. The system remained stable after four months and miR-143 leakage was not observed by performing gel electrophoresis. The microRNA lipoplex could enhance conduction of the loaded miR-143, and it also showed good biocompatibility to the healthy cells. Conclusion The PEGylated microRNA lipoplex system had a high potential for the systematic migration of miR-143 and it could improve intracellular stability of the released microRNA.

150-160

Authors: Mohammad Ali Khalilifar, Mohamadreza Baghaban Eslaminejad, Mohammad Ghasemzadeh, Samaneh Hosseini, Hossein Baharvand

Objective Systematic studies indicate a growing number of clinical studies that use mesenchymal stem cells (MSCs) for the treatment of cartilage lesions. The current experimental and preclinical study aims to comparatively evaluate the potential of MSCs from a variety of tissues for the treatment of cartilage defect in rabbit’s knee which has not previously been reported. Materials and Methods In this experimental study, MSCs isolated from bone marrow (BMMSCs), adipose (AMSCs), and ears (EMSCs) of rabbits and expanded under in vitro culture. The growth rate and differentiation ability of MSCs into chondrocyte and the formation of cartilage pellet were investigated by drawing the growth curve and real-time polymerase chain reaction (RT-PCR), respectively. Then, the critical cartilage defect was created on the articular cartilage (AC) of the rabbit distal femur, and MSCs in collagen carrier were transplanted. The studied groups were as the control (only defect), sham (defect with scaffold), BMMSCs in the scaffold, EMSCs in the scaffold, and EMSCs in the scaffold with cartilage pellets. Histological and the gene expression analysis were performed following the transplantation. Results Based on our comparative in vitro investigation, AMSCs possessed the highest growth rate, as well as the lowest chondrogenic differentiation potential. In this context, MSCs of the ear showed a significantly higher growth rate and cartilage differentiation potential than those of bone marrow tissue (P<0.05). According to our in vivo assessments, BMMSC- and EMSC-seeded scaffolds efficiently improved the cartilage defect 4 weeks post-transplantation, while no improvement was observed in the group contained the cartilage pellets. Conclusion It seems that the ear contains MSCs that promote cartilage regeneration as much as the conventional MSCs from the bone marrow. Considering a high proliferation rate and easy harvesting of MSCs of the ear, this finding could be of value for the regenerative medicine.

169-178

Authors: Dian Dayer, Mohammad Reza Tabandeh, Eskandar Moghimipour, Mahmoud Hashemi Tabar, Ata Allah Ghadiri, Elham Allah Bakhshi, Mahmoud Orazizadeh, Mohammad Ali Ghaffari

Objective We proposed a novel differentiation method for the efficient differentiation of adipose-derived mesenchymal stem cells (ADMSCs) into functional insulin-producing cells (IPCs) based on MafA overexpression. Materials and Methods In this experimental study, a eukaryotic expression vector containing MafA [MafA/pcDNA3.1(+)] was constructed and purified. ADMSCs were differentiated into IPCs. ADMSCs were assigned in two groups including control (C), and the MafA overexpressed (MafA+) groups. The ADMSCs were transfected by MafA/pcDNA 3.1(+) at day 10 of the differentiation. Differentiated cells were analyzed for the expression of multiple β cell specific genes (Nkx2.2, Ngn3, Isl-1, Pdx1, MafA, Nkx6.1, and Insulin) using real-time polymerase chain reaction (PCR). The insulin secretion potency of the differentiated cells in response to glucose exposure was also determined using an enzyme-linked immunosorbent assay (ELISA) method and Dithizone (DTZ) staining. The IPCs from the control manipulated group, and un-differentiated ADMSCs group were transplanted to streptozotocin (STZ)-diabetic rats. Rats were monitored for blood glucose and insulin concentration. Results The results revealed that ADMSCs were successfully differentiated into IPCs through the 14 day differentiation protocol. The expression of β-cell specific genes in MafA+ IPCs was higher than in control cells. Glucose-induced insulin secretion after the exposure of IPCs to glucose was higher in MafA+ group than the control group. The STZ- diabetic rats showed an ability to secrete insulin and apparent hyperglycemic condition adjustment after transplantation of the control IPCs. The mean insulin concentration of diabetic rats that were transplanted by manipulated IPCs was significantly higher than ADMSCs-transplanted rats; however, no effect was observed in the concentration of blood glucose. Conclusion The overexpression of MafA can be used as a novel promising approach for the efficient production of IPCs from ADMSCs in vitro. However, the future therapeutic use of the MafA+ IPCs in diabetic animals needs further investigations.

186-193

Authors: Hossein Azizi, Hatef Ghasemi Hamidabadi, Thomas Skutella

Objective Spermatogonial stem cells (SSCs) provide the cellular basis for sperm production transforming the male’s genetic information to the next generation. We aimed to examine the effect of different feeder layer on proliferation of SSCs. Materials and Methods In this experimental study, we compared the in vitro effects of the co-culture of mouse SSCs with mouse embryonic fibroblasts (MEFs), sandos inbred mice (SIM) embryo-derived thioguanine- and ouabain- resistant (STO) feeders, and neonate and adult testicular stroma cell (TSC) feeders on the efficiency of mouse SSC proliferation and colony formation. Cells were cultivated on top of MEFs, STO, and neonate and adult TSCs feeder layers for 30 days. The number and diameter of colonies and also the number of cells were evaluated during day 7, 15, 25, and 30 of culture. The mRNA expression of germ cells and somatic cells were analyzed. Results In our study, we observed a significant difference in the proliferation rates and colony size of SSCs among the groups, especially for MEFs (P<0.05). SSCs can proliferate on MEFS, but not on STO, neonate or adult TSCs. Using immunocytochemistry by KI67 the proliferative activities of SSC colonies on MEFs were confirmed. The results of Fluidigm real-time polymerase chain reaction (RT-PCR) showed a high expression of the germ cell genes the promyelocytic leukemia zinc finger protein (PLZF), deleted in azoospermia-like (DAZL), octamer-binding transcription factor 4 (OCT4), and DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4 or VASA) in SSCs, and a low expression of these genes in the feeder layers. Furthermore, we observed a higher expression of vimentin and integrin-B1 in feeder layers than in SSCs (P<0.05). Conclusion Based on the optimal effect of MEFs for better colonization of SSCs, these feeder cells seem to be appropriate candidates for SSC cultures prior to transplantation. Therefore, it is suggested using these feeder cells for SSC cultivation.

204-209

Authors: Tahereh Zare, Reza Fardid, Samaneh Naderi

Objective The use of nanoscale particles, for instance silver nanoparticles (Ag NPs) has considerably increased recently. Since Ag NPs can be transmuted into silver ions; the toxicity and genotoxicity of these NPs along with other external factors such as ultraviolet type C (UVC) irradiation must be evaluated. In the present study, the aim was to investigate the genotoxic effects Ag NPs and UVC co-exposure on human lymphoblastoid TK6 cells. Materials and Methods In this experimental study, Ag NPs (~20 nm) were purchased from US Research Nanomaterials Inc. and H2AX gene expression was evaluated using quantitative real time polymerase chain reaction (qRT-PCR), 1 and 24 hours post Ag NPs and UVC treatment. Results Results showed that treatment of TK6 cells with different Ag NP concentrations without exposure to UVC can reduce H2AX gene expression, but treatment of these cells with Ag NPs in combination UVC irradiation can reduce viability that leads to a synergistic increase in the amount of H2AX gene expression. Conclusion According to our findings, Ag NPs can act to sensitize cells to UVC radiation when used for cancer treatment. So, combination of Ag NPs and UVC irradiation could be used in radiotherapy.

220-228

Authors: Soheila Pourkhodadad, Shahrbanoo Oryan, Gholamreza Kaka, Seyed Homayoon Sadraei

Objective Traumatic spinal cord injury (SCI) is considered one of the most devastating injuries leading to neuronal disruption. Olfactory ensheathing cells (OECs) and minocycline have been shown to promote locomotor function after spinal cord injury. In this study, we have tested the efficacy of combined treatment with minocycline and OECs after contusive spinal cord injury. Materials and Methods In this experimental study, adult female Wistar rats were randomly divided into five groups. Rats received an intraperitoneal injection of minocycline immediately after SCI, and then 24 hours after the injury. Transplantations were performed 7 days after the injury. Functional recovery was evaluated using the Basso, Beattie and Bresnahan scale (BBB). After that, the animals were sacrificed, and T11 segment of the spinal cord was removed after 5 weeks, and then used for histopathological, immunohistochemical, and biochemical assessments. Western blot analysis was applied to determine the protein expression of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL1β) and caspase3. Results The results of this study showed that the combination of OECs graft and minocycline reduced the functional deficits and diminished cavitation and astrogliosis in spinal tissue. The analysis of protein expression by western blotting revealed that minocycline treatment along with OECs transplantation further decreased the level of IL-1β, TNF-α, caspase-3, and the oxidative stress as compared with when minocycline or OECs transplantation was used alone. Conclusion The combinatory treatment with OECs graft and minocycline induced a more effective response to the repair of spinal cord injury, and it is considered a therapeutic potential for the treatment of SCI.

7-13

Authors: Mustafa Numan BUCAK, Mustafa Bodu, Nuri Başpınar, Şükrü Güngör, Pınar İli, Begimay Acibaeva, Tohid Rezaei Topraggaleh, Şükrü Dursun

Objective The purpose of the present study was to assess the effects of ellagic acid and ebselen on sperm and oxidative stress parameters during liquid preservation of ram semen. Materials and Methods In this experimental study, sixty ejaculates from six mature Merino rams were used. In experiment 1, the ejaculates were diluted in base extender contained ellagic acid at 0 (control), 0.5, 1, and 2 mM. In experiment 2, ebselen at 0 (control), 10, 20, and 40 μM were added to the extender. Sperm motility, viability, mitochondrial membrane potential, DNA integrity, lipid peroxidation (LPO), the antioxidant potential (AOP), and total glutathione (tGSH) were evaluated at 0, 24, 48, and 72 hours of preservation. Results Supplementation of ellagic acid at 1 and 2 mM resulted in higher sperm motility and viability at 0 hours of storage. Ellagic acid at 2 mM led to higher motility and viability compared to controls after 0, 24, and 48 hours of preservation and increased AOP after 24 and 72 hours. Higher tGSH was at 1 mM ellagic acid, compared to control after 72 hours. Addition of ebselen at a concentration of 40 μM increased motility at 24 and 48 hours and 10 μM produced the same effect after 48 and 72 hours of storage as well as higher viability, compared to the controls after 0 hours of storage. Sperm DNA integrity was significantly improved after 24, 48, and 72 hours with the addition of ebselen at 10 μM, and after 72 hours at 40 μM. Addition of 40 mM ebselen also reduced the LPO levels after 24 hours of storage compared to the controls. Conclusion The results showed that supplementation of ellagic acid and ebselen in semen extender has a potential effect on sperm and oxidative stress parameters during liquid preservation of ram semen.

27-34

Authors: Hye-Jung Park, Moon-Moo Kim

Objective Amentoflavone is the main component of Selaginella tamariscina widely known as an oriental traditional medicinal stuff that has been known to have a variety of medicinal effects such as the induction of apoptosis, anti- metastasis, and anti-inflammation. However, the effect of amentoflavone on autophagy has not been reported until now. The aim of this study was to investigate whether amentoflavone has a positive effect on the induction of autophagy related to cell aging. Materials and Methods In this experimental study, the aging of young cells was induced by the treatment with insulin- like growth factor-1 (IGF-1) at 50 ng/mL three times every two days. The effect of amentoflavone on the cell viability was evaluated in A549 and WI-38 cells using 3-(4,5-dimethyl-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay. The induction of autophagy was detected using autophagy detection kit. The expression of proteins related to autophagy and IGF-1 signaling pathway was examined by western blot analysis and immunofluorescence assay. Results First of all, it was found that amentoflavone induces the formation of autophagosome. In addition, it enhanced the expression level of Atg7 and increased the expression levels of Beclin1, Atg3, and LC3 associated with the induction of autophagy in immunofluorescence staining and western blot analyses. Moreover, amentoflavone inhibited the cell aging induced by IGF-1 and hydrogen peroxide. In particular, the levels of p53 and p-p21 proteins were increased in the presence of amentoflavone. Furthermore, amentoflavone increased the level of SIRT1 deacetylating p53. Conclusion Our results suggest that amentoflavone could play a positive role in the inhibition of various diseases associated with autophagy and the modulation of p53.

43-48

Authors: Zohre Soheily, Mohammad Soleimani, Keivan Majidzadeh-Ardebili

Objective Mycoplasmas are major contaminants of cell culture and affect in vitro biological and diagnostic tests. Mycoplasma detection is conducted using culture and molecular methods. These methods vary in terms of accuracy, reliably and sensitivity. Loop-mediated isothermal amplification (LAMP) is used to amplify target DNA in a highly specific and rapid manner. This study aimed to develop a LAMP method for rapid detection of Mycoplasma in culture samples. Materials and Methods In this descriptive laboratory study, for LAMP detection of Mycoplasma contaminations in cell culture, we used primers specifically designed for targeting the 16S rRNA conserved gene of Mycoplasma spp. For a positive control structure, 16S rRNA amplified based on PCR, was cloned in a plasmid vector and sequenced. The assay specificity was evaluated using Mycoplasma genomic DNA and a panel containing genomes of gram-positive and gram-negative organisms. Results In this study, the method developed for detection of Mycoplasma contamination of cell cultures was a rapid, sensitive and cost-effective LAMP approach. The results demonstrated that this method benefits from high specificity (100%) for amplification of Mycoplasma strains and high speed (multiplication within 60 minutes), while it does not require expensive laboratory equipment compared to those needed for polymerase chain reaction (PCR)-based detection. Conclusion Our study is the first report about application of LAMP assay based on 16S rRNA gene for detection of Mycoplasma strains; this technique could be considered a useful tool for rapid detection of contamination of cell culture.

57-61

Authors: Sara Ahmadi Badi, Shohreh Khatami, Shiva Irani, Seyed Davar Siadat

Objective Gastrointestinal (GI) tract, like other mucosal surface, is colonized with a microbial population known as gut microbiota. Outer membrane vesicles (OMVs) which are produced by gram negative bacteria could be sensed by Toll like receptors (TLRs). The interaction between gut microbiota and TLRs affects homeostasis and immune responses. In this study, we evaluated TLR2, TLR4 genes expression and cytokines concentration in Caco-2 cell line treated with Bacteroides fragilis (B. fragilis) and its OMVs. Materials and Methods In this experimental study, OMVs were extracted using sequential centrifugation and their physicochemical properties were evaluated as part of quality control assessment. Caco-2 cells were treated with B. fragilis and its OMVs (180 and 350 µg/ml). Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed to assess TLR2 and TLR4 mRNA expression levels. Pro-inflammatory (IFNᵧ) and anti-inflammatory (IL- 4 and IL-10) cytokines were evaluated by ELISA. Results B. fragilis significantly decreased TLR2 and slightly increased TLR4 mRNA levels in Caco-2 cell line. The TLR2 mRNA level was slightly increased at 180 and 350 µg/ml of OMVs. Conversely, the TLR4 mRNA level was decreased at 180 µg/ml of OMVs, while it was significantly increased at 350 µg/ml of OMVs. Furthermore, B. fragilis and its OMVs significantly increased and decreased IFNᵧ concentration, respectively. Anti-inflammatory cytokines were increased by B. fragilis and its OMVs. Conclusion B. fragilis and its OMVs have pivotal role in the cross talk between gut microbiota and the host especially in the modulation of the immune system. Based on the last studies on immunomodulatory effect of B. fragilis derived OMVs on immune cells and our results, we postulate that B. fragilis derived OMVs could be possible candidates for the reduction of immune responses.

70-77

Authors: Ahoura Nozari, Ehsan Aghaei-moghadam, Aliakbar Zeinaloo, Afagh Alavi, Saghar Ghasemi Firouzabadi, Shohre Minaee, Marzeieh Eskandari Hesari, Farkhondeh Behjati

Objective Tricuspid atresia (TA) is a rare life-threatening form of congenital heart defect (CHD). The genetic mechanisms underlying TA are not clearly understood. According to previous studies, the endocardial cushioning event, as the primary sign of cardiac valvulogenesis, is governed by several overlapping signaling pathways including Ras/ ERK pathway. RASA1, a regulator of cardiovascular development, is involved in this pathway and its haploinsufficiency (due to heterozygous mutations) has been identified as the underlying etiology of the autosomal dominant capillary malformation/arteriovenous malformation (CM/AVM). Materials and Methods In this prospective study, we used whole exome sequencing (WES) followed by serial bioinformatics filtering steps for two siblings with TA and early onset CM. Their parents were consanguineous which had a history of recurrent abortions. Patients were carefully assessed to exclude extra-cardiac anomalies. Results We identified a homozygous RASA1 germline mutation, c.1583A>G (p.Tyr528Cys) in the family. This mutation lies in the pleckstrin homology (PH) domain of the gene. The parents who were heterozygous for this variant displayed CM. Conclusion This is the first study reporting an adverse phenotypic outcome of a RASA1 homozygous mutation. Here, we propose that the phenotypic consequence of the homozygous RASA1 p.Tyr528Cys mutation is more serious than the heterozygous type. This could be responsible for the TA pathogenesis in our patients. We strongly suggest that parents with CM/AVM should be investigated for RASA1 heterozygous mutations. Prenatal diagnosis and fetal echocardiography should also be carried out in the event of pregnancy in heterozygous parents.

86-91

Authors: Fateme Karami, Iman Salahshourifar, Masoud Houshmand

Objective: APOB-related familial hypercholesterolemia (FH) is the most common hereditary hyperchlosterolemia with an autosomal dominant pattern. A number of APOB variants are the most important risk factors for hyperchlosterolemia. APOB is a large glycoprotein that plays an important role in the metabolism of lipoproteins in the human body. Small changes in the structure and function of APOB can cause major problems in lipid metabolism. Two forms of APOB are produced by an editing process of gene replication. APOB48 is required for the production of chylomicrons in the small intestine and APOB100 is essential in liver for the production of very low density lipoprotein (VLDL) and is also a ligand for LDL receptor (LDLR) that mediates LDL endocytosis. Materials and Methods: In this case-control study, rs693 (in exon 26 of APOB) and rs515135 (5 'end of APOB) single nucleotide polymorphisms (SNPs) were analyzed in 120 cases of familial hypercholesterolemia and 120 controls. Both SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) where PCR products were digested with specific restriction enzymes recognising each single nucleotide polymorphism. Results: This study was analyzed by odds-ratio (OR) and its 95% confidence interval (CI) to examine the association of the two SNPs with familial hypercholostermia susceptibility. Statistical analysis showed that both SNPs were in Hardy- Weinberg equilibrium. Conclusion: We found no significant relationship between rs515135 and familiar hypercholesterolemia. However, there was a significant association between the C allele of rs693 and high familial cholesterol levels. Furthermore, it seems the dominant model of T allele occurrence has a protective role in emergence of disease.

99-102

Authors: Fatemeh Behrouznezhad, Fatemeh Ejeian, Mojtaba Emadi-Baygi, Parvaneh Nikpour, Mohammad Hossein Nasr-Esfahani

Neurodegenerative diseases have now become a major challenge, especially in aged societies. Most of the traditional strategies used for treatment of these diseases are untargeted and have little efficiency. Developments in stem cell investigations have given much attention to cell therapy as an alternative concept in the regeneration of neural tissues. Dental pulp stem cells (DPSCs) can be readily obtained by noninvasive procedures and have been shown to possess properties similar to well-known mesenchymal stem cells. Furthermore, based on their neural crest origin, DPSCs are considered to have a good potential to differentiate into neural cells. Zfp521 is a transcription factor that regulates expression of many genes, including ones involved in the neural differentiation process. Therefor based on neural crest origin of the cell and high expression of neural progenitor markers, we speculate that sole overexpression of Zfp521 protein can facilitate differentiation of dental stem cells to neural cells and researchers may find these cells suitable for therapeutic treatment of neurodegenerative diseases.

459-468

Authors: Niloufar Shayan Asl, Farhad Nejat, Parvaneh Mohammadi, Abdolhossein Nekoukar, Saeed Hesam, Marzieh Ebrahimi, Khosrow Jadidi

Objective Human amniotic membrane (HAM) is used as a supporter for limbal stem cell (LSC) expansion and corneal surgery. The aim of study is to use HAM extracts from healthy donors to enhance proliferation of LSCs in vitro and in vivo. Materials and Methods In this interventional experimental study, the effective and cytotoxic doses of the amniotic membrane extract eye drops (AMEED) was assessed by adding different concentrations of AMEED (0-2.0 mg/ml) to LSC cultures for 14 days. Subsequently, the expression levels of ATP-binding cassette sub-family G member 2 (ABCG2, a putative stem cell marker), cytokeratin 3 (K3, corneal maker), K12 and K19 (corneal-conjunctival cell makers) were assessed by real-time polymerase chain reaction (PCR). In the second step, the corneal epithelium of 10 rabbits was mechanically removed, and the right eye of each rabbit was treated with 1 mg/ml AMEED [every 2 hours (group 1) or every 6 hours (group 2)]. The left eyes only received an antibiotic. The corneal healing process, conjunctival infection, degree of eyelid oedema, degree of photophobia, and discharge scores were evaluated during daily assessments. Finally, corneal tissues were biopsied for pathologic evidences. Results In comparison to the positive control [10% foetal bovine serum (FBS)], 0.1-1 mg/ml AMEED induced LSC proliferation, upregulated ABCG2, and downregulated K3. There were no remarkable differences in the expression levels of K12 and K19 (P>0.05). Interestingly, in the rabbits treated with AMEED, the epithelium healing duration decreased from 4 days in the control group to 3 days in the two AMEED groups, with lower mean degrees of eyelid oedema, chemosis, and infection compared to the control group. No pathologic abnormalities were observed in either of the AMEED groups. Conclusion AMEED increases LSCs proliferation ex vivo and accelerates corneal epithelium healing in vivo without any adverse effects. It could be used as a supplement for LSC expansion in cell therapy.

477-482

Authors: Samaneh Karimi, Jafar Ai, Layasadat Khorsandi, Darioush Bijannejad, Ghasem Saki

Objective Type 1 diabetes is caused by destruction of beta cells of pancreas. Vildagliptin (VG), a dipeptidyl peptidase IV (DPP IV) inhibitor, is an anti-diabetic drug, which increases beta cell mass. In the present study, the effects of VG on generation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs) is investigated. Materials and Methods In this experimental study, ASCs were isolated and after characterization were exposed to differentiation media with or without VG. The presence of IPCs was confirmed by morphological analysis and gene expression (Pdx-1, Glut-2 and Insulin). Newport Green staining was used to determine insulin-positive cells. Insulin secretion under different concentrations of glucose was measured using radioimmunoassay method. Results In the presence of VG the morphology of differentiated cells was similar to the pancreatic islet cells. Expression of Pdx-1, Glut-2 and Insulin genes in VG-treated cells was significantly higher than the cells exposed to induction media only. Insulin release from VG-treated ASCs showed a nearly 3.6 fold (P<0.05) increase when exposed to a high- glucose medium in comparison to untreated ASCs. The percentage of insulin-positive cells in the VG-treated cells was approximately 2.9-fold higher than the untreated ASCs. Conclusion The present study has demonstrated that VG elevates differentiation of ASCs into IPCs. Improvement of this protocol may be used in cell therapy in diabetic patients.

496-504

Authors: Sadaf Vahdat, Sara Pahlavan, Nasser Aghdami, Behnaz Bakhshandeh, Hossein Baharvand

Objective Cardiovascular progenitor cells (CPCs) are introduced as one of the promising cell sources for preclinical studies and regenerative medicine. One of the earliest type of CPCs is cardiogenic mesoderm cells (CMCs), which have the capability to generate all types of cardiac lineage derivatives. In order to benefit from CMCs, development of an efficient culture strategy is required. We aim to explore an optimized culture condition that uses human embryonic stem cell (hESC)-derived CMCs. Materials and Methods In this experimental study, hESCs were expanded and induced toward cardiac lineage in a suspension culture. Mesoderm posterior 1-positive (MESP1+) CMCs were subjected to four different culture conditions: i. Suspension culture of CMC spheroids, ii. Adherent culture of CMC spheroids, iii. Adherent culture of single CMCs using gelatin, and iv. Adherent culture of single CMCs using Matrigel. Results Although, we observed no substantial changes in the percentage of MESP1+ cells in different culture conditions, there were significantly higher viability and total cell numbers in CMCs cultured on Matrigel (condition iv) compared to the other groups. CMCs cultivated on Matrigel maintained their progenitor cell signature, which included the tendency for cardiogenic differentiation. Conclusion These results showed the efficacy of an adherent culture on Matrigel for hESC-derived CMCs, which would facilitate their use for future applications.

513-520

Authors: Mahdi Mohaqiq, Mansoureh Movahedin, Zohreh Mazaheri, Naser AmirJanati

Objective In vitro transplantation (IVT) of spermatogonial stem cells (SSCs) is one of the most recent methods in transplantation in recent decades. In this study, IVT and SSCs homing on seminiferous tubules of host testis in organ culture have been studied. Materials and Methods In this experimental study, human SSCs were isolated and their identities were confirmed by tracking their promyelocytic leukemia zinc finger (PLZF) protein. These cells were transplanted to adult azoospermia mouse testes using two methods, namely, IVT and in vivo transplantation as transplantation groups, and testes without transplantation of cells were assigned in the control group. Then histomorphometric, immunohistochemical and molecular studies were done after 2 weeks. Results After two weeks, histomorphometric studies revealed that the number of subsided spermatogonial cells (SCs) and the percentage of tubules with subsided SCs in IVT and in vivo groups were significantly more than those in the control group (P<0.05). Immunohistochemical studies in the transplantation groups confirmed that the PLZF protein was expressed in the cells subsided on the seminiferous tubule. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) demonstrated that the PLZF gene expression was only positive in the transplantation groups, but it was not significantly different between the IVT group and the in vivo group (P>0.05). Conclusion Testicular tissue culture conditions after SSC transplantation can help these cells subside on the seminiferous tubule basement membrane.

527-536

Authors: Azam Dalman, Mehdi Totonchi, Mojtaba Rezazadeh Valojerdi

Objective In this study, we have examined human theca stem cells (hTSCs) in vitro differentiation capacity into human oocyte like cells (hOLCs). Materials and Methods In this interventional experiment study, hTSCs were isolated from the theca layer of small antral follicles (3-5 mm in size). Isolated hTSCs were expanded and cultured in differentiation medium, containing 5% human follicular fluid, for 50 days. Gene expressions of PRDM1, PRDM14, VASA, DAZL, OCT4, ZP1, 2, 3 GDF9, SCP3 and DMC1 were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on days 0, 18, and 25 after monoculture as well as one week after co-culture with human granulosa cells (hGCs). In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were immune-localized in oocyte-like structures. Results After 16-18 days, the color of the medium became acidic. After 25 days, the cells started to differentiate into round-shaped cells (20-25 µm diameter). One week after co-culturing with hGCs, the size of the round cells increased 60 to70 µm and convert to hOLCs. However, these growing cells expressed some primordial germ cell (PGC)- and germ cell genes (PRDM1, PRDM14, VASA, DAZL, and OCT4) as well as oocyte specific genes (ZP1, 2, 3 and GDF9), and meiotic-specific markers (SCP3 and DMC1). In addition, GDF9, OCT4, DAZL, VASA, and ZP3 proteins were present in hOLCs. Conclusion To sum up, hTSCs have the ability to differentiate into hOLCs. This introduced model paved the way for further in vitro studies of the exact mechanisms behind germ cell formation and differentiation. However, the functionality of hOLCs needs further investigation.

544-551

Authors: Husamaldeen Alsalim, Farnoosh Jafarpour, Faezeh Ghazvini Zadegan, Mohammad Hossein Nasr-Esfahani, Amir Niasari-Naslaji

Objective In the present study, we investigated the possible epigenotoxic effect of dimethyl sulfoxide (DMSO) on buffalo fibroblast cells and on reconstructed oocytes during buffalo-bovine interspecies somatic cell nuclear transfer (iSCNT) procedure and its effect on rate and quality of blastocyst which derived from these reconstructed oocytes. Materials and Methods In this experimental study, cell viability of buffalo fibroblasts was assessed after exposure to various concentration (0.5, 1, 2 and 4%) of DMSO using MTS assay. The epigenetic effect of DMSO was also assessed in terms of DNA methylation in treated cells by flowcytometry. Reconstructed oocytes of buffalo-bovine iSCNT exposed for 16 hours after activation to non-toxic concentration of DMSO (0.5%) to investigate the respective level of 5-methylcytosine, cleavage and blastocyst rates and gene expression (pluripotent genes: OCT4, NANOG, SOX2, and trophectodermal genes: CDX2 and TEAD4) of produced blastocysts. Results Supplementation of culture medium with 4% DMSO had substantial adverse effect on the cell viability after 24 hours. DMSO, at 2% concentration, affected cell viability after 48 hours and increased DNA methylation and mRNA expression of DNMT3A in fibroblast cells. Exposure of reconstructed oocytes to 0.5% DMSO for 16 hours post activation did not have significant effect on DNA methylation, nor on the developmental competency of reconstructed oocyte, however, it decreased the mRNA expression of NANOG in iSCNT blastocysts. Conclusion Depending on the dose, DMSO might have epigenotoxic effect on buffalo fibroblast cells and reconstructed oocytes and perturb the mRNA expression of NANOG in iSCNT blastocysts.

559-563

Authors: Zeynep Ermiş Karaali, Gonca Candan, Mehmet Burak Aktuğlu, Mustafa Velet, Arzu Ergen

Objective Innate immunity factors are associated with type 2 diabetes (T2DM) and its complications. Therefore, T2DM has been suggested to be an immune-dependent disease. Elevated fasting glucose level and higher concentrations of innate immunity soluble molecules are not only related with insulin resistance, but inflammation is also an important factor in beta cell dysfunction in T2DM. Toll-like receptor 2 (TLR-2), which has an important role in inducing innate immune cells, is thought to have suppressive roles on immune responses in T2DM. We therefore aimed to investigate the possible role of TLR-2 del -196-174 and Arg753Gln variants in T2DM pathogenesis. Materials and Methods This study was designed as a case-control study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to genotype the two variants in 100 T2DM patients and 98 age-matched controls. Results We found significantly higher frequencies of TLR-2 del -196-174 DD genotype (P=0.003), ID genotype (P=0.009) and D allele (P=0.001) in patients compared with controls. In addition, the II genotype (P=0.001) and the I allele (P=0.003) frequencies were elevated in healthy controls. We did not find any significant differences in frequency distribution for the Arg753Gln variant in study groups. Conclusion We suggest that carrying the D allele of the TLR-2 del -196-174 variant may be related as a risk factor for T2DM.

569-575

Authors: Fatemeh Bahreini, Ali Reza soltanian

Objective We sought to apply Shannon’s entropy to determine colorectal cancer genes in a microarray dataset. Materials and Methods In the retrospective study, 36 samples were analysed, 18 colorectal carcinoma and 18 paired normal tissue samples. After identification of the gene fold-changes, we used the entropy theory to identify an effective gene set. These genes were subsequently categorised into homogenous clusters. Results We assessed 36 tissue samples. The entropy theory was used to select a set of 29 genes from 3128 genes that had fold-changes greater than one, which provided the most information on colorectal cancer. This study shows that all genes fall into a cluster, except for the R08183 gene. Conclusion This study has identified several genes associated with colon cancer using the entropy method, which were not detected by custom methods. Therefore, we suggest that the entropy theory should be used to identify genes associated with cancers in a microarray dataset.

584-591

Authors: Fereshteh Rahimi, Somayeh fardindoost, Naser Ansari-Pour, Fatemeh Sepehri, Farideh Makiyan, Azizolah Shafiekhani, Ali Hossein Rezayan

Objective Substantial effort has been put into designing DNA-based biosensors, which are commonly used to detect presence of known sequences including the quantification of gene expression. Porous silicon (PSi), as a nanostructured base, has been commonly used in the fabrication of optimally transducing biosensors. Given that the function of any PSi-based biosensor is highly dependent on its nanomorphology, we systematically optimized a PSi biosensor based on reflectometric interference spectroscopy (RIS) detecting the high penetrance breast cancer susceptibility gene, BRCA1. Materials and Methods In this experimental study, PSi pore sizes on the PSi surface were controlled for optimum filling with DNA oligonucleotides and surface roughness was optimized for obtaining higher resolution RIS patterns. In addition, the influence of two different organic electrolyte mixtures on the formation and morphology of the pores, based on various current densities and etching times on doped p-type silicon, were examined. Moreover, we introduce two cleaning processes which can efficiently remove the undesirable outer parasitic layer created during PSi formation. Results of all the optimization steps were observed by field emission scanning electron microscopy (FE-SEM). Results DNA sensing reached its optimum when PSi was formed in a two-step process in the ethanol electrolyte accompanied by removal of the parasitic layer in NaOH solution. These optimal conditions, which result in pore sizes of approximately 20 nm as well as a low surface roughness, provide a considerable RIS shift upon complementary sequence hybridization, suggesting efficient detectability. Conclusion We demonstrate that the optimal conditions identified here makes PSi an attractive solid-phase DNA-based biosensing method and may be used to not only detect full complementary DNA sequences, but it may also be used for detecting point mutations such as single nucleotide substitutions and indels.

592-598

Authors: Seyedeh Parisa Chavoshi Tarzjani, Seyed Abolhassan Shahzadeh Fazeli, Mohammad Hossein Sanati, Seyed Massood Nabavi

Multiple sclerosis (MS) is a chronic disease of the central nervous system and one of the most common causes of neurological disability among those aged 20-40 years, particularly in women. Major histocompatibility complex (MHC) Class II genes are known to be involved in the development of MS. One of the important groups of this complex is the HSP gene family, especially HSP70, which is induced under stress conditions. The aim of the present case-control study was to determine the association between the heat shock protein 70 (HSP70) and risk of MS in Iranian patients by genotyping the rs1061581 gene polymorphism. A total of 50 relapsing-remitting MS (RRMS) patients and 50 healthy control subjects were considered for this study. Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) method. PCR-RFLP results of twenty-five randomly selected samples were confirmed by DNA sequencing. Genotypic and allelic distributions were compared between the case and control groups. We observed no significant difference in the distribution of rs1061581 genotype and allele frequencies between RRMS patients and controls. In addition, there was no association between the HSP70 gene polymorphism and the clinical variables in the case group. Our data indicate that HSP70, in particular rs1061581, is unlikely to be involved in the susceptibility to or the severity of RRMS in Iranian patients. Further large prospective studies are required to confirm these findings.

302-311

Authors: Suresh Palamadai krishnan

Breast cancer (BC) is a widely prevalent form of neoplasia in women with fairly alarming mortality statistics. This aspect may be attributed, in part, to the current spatial and temporal heterogeneity-based limitations in therapies with possible recurrence of this tumour at primary and/or secondary sites. Such an extensive phenotypic heterogeneity in breast cancer is unlikely to be adequately or completely comprehended by an immuno-histopathology-based classification alone. This finding has warranted research and development in the area of microarray-based methods (i.e. transcriptomic and proteomic chips) for an improved molecular classification of this complex and heterogeneous tumour. Further, since epigenetics can also be an important determinant in terms of diagnosis, prognosis and therapy, this review provides an insight into the molecular portrait of BC in genetic and epigenetic terms. Specifically, the roles of characteristic DNA and histone-based modifications as well as mi-RNA- based alterations have been discussed with specific examples. Also, their involvement in epithelial mesenchymal transition (EMT) processes in cancer stem cells (CSCs) has been outlined. Last but not least, the salient aspects and the advantages of ex vivo/in vitro 3D model systems in recapitulating several aspects of BC tumour (particularly the architecture as well as the apico-basal polarity) are mentioned. This review hopes to provide not only an improved and updated understanding of the epigenetics of breast cancer, but to also elaborate on tumour model development/refinement, biomarker evaluation, drug resistance and test of individual drugs or drug combinations and drug delivery systems.

318-325

Authors: Reza Ranjbaran, Mahin Nikogoftar Zarif, Sedigheh Sharifzadeh, Habibollah Golafshan, Ali Akbar Pourfathollah

Objective Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies. Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced -117 G→A substitution in HBG promoter in prevention of transcriptional silencing of the γ-globin. Materials and Methods In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively. Results Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation confirmed erythroid differentiation in HSCs and K562 cells. γ to β-globin gene switching was estimated to be on days 18-21 of HSC differentiation. Flow cytometry analysis showed that more than 70% of erythroid progenitor cells (EPCs) were transfected with the chimeraplast. The highest gene conversion efficiency was 7.2 and 11.1% in EPCs and K562 cells respectively. The induced mutation led to a 1.97-fold decrease in β/γ-globin gene expression in transfected EPCs at the experimental end point (day 28) whereas, due to the absence of β-globin gene expression following K562 differentiation, this rate was not evaluable. Conclusion Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit γ-globin gene silencing during erythroid differentiation.

333-339

Authors: Fatemeh Bahreini, Massoud Houshmand, Mohammad Hossein Modaresi, Seyed Mohammad Akrami

Objective Pompe disease (PD) is a progressive neuromuscular disorder that is caused by glucosidase acid alpha (GAA) deleterious mutations. Mitochondrial involvement is an important contributor to neuromuscular diseases. In this study the sequence of MT-ATP 6/8 and Cytochrome C oxidase I/II genes along with the expression levels of the former genes were compared in classic and non-classic patients. Materials and Methods In this case-control study, the sequence of MT-ATP 6/8 and Cytochrome C oxidase was analyzed by polymerase chain reaction (PCR)-Sanger sequencing and expression of MT-ATP genes were quantified by real time-PCR (RT-PCR) in 28 Pompe patients. The results were then compared with 100 controls. All sequences were compared with the revised Cambridge reference sequence as reference. Results Screening of MT-ATP6/8 resulted in the identification of three novel variants, namely T9117A, A8456C and A8524C. There was a significant decrease in MT-ATP6 expression between classic (i.e. adult) and control groups (P=0.030). Additionally, the MT-ATP8 expression was significantly decreased in classic (P=0.004) and non-classic (i.e. infant) patients (P=0.013). In total, 22 variants were observed in Cytochrome C oxidase, five of which were non- synonymous, one leading to a stop codon and another (C9227G) being a novel heteroplasmic variant. The A8302G in the lysine tRNA gene was found in two brothers in a pedigree, while a T7572C variant in the aspartate tRNA gene was observed in two brothers in another pedigree. Conclusion The extent of mitochondrial involvement in the classic group was more significant than in the non-classic form. Beside GAA deleterious mutations, it seems that mtDNA variants have a secondary effect on PD. Understanding, the role of mitochondria in the pathogenesis of Pompe may potentially be helpful in developing new therapeutic strategies.

348-354

Authors: Malihe Nakhaeifard, Maryam Haji ghasem kashani, Iran Goudarzi, Arezou Rezaei

Objective Adipose derived stem cells (ASCs) secrete numerous neurotrophic factors and cytokines in conditioned medium (CM), which protect neurons by its antioxidative and trophic effects. This research assesses the neuroprotective effect of ASC- CM on neurotrophins genes expressions and tyrosine hydroxylase positive (TH+) cell density in male Wistar rats lesioned by 6-hydroxydopamine (6-OHDA). Materials and Methods In this experimental study, the groups consisted of lesioned and sham rats with unilateral injections of 20 µg of 6-OHDA neurotoxin and phosphate buffered saline (PBS) into the striatum, respectively. Another groups received intravenous injections of 3×106 cells (ASCs group), 500 µl of CM (ASC-CM group) or medium [α-minimal essential medium (α-MEM) group)]. All rats underwent evaluations with the rotarod and apomorphine-induced rotation tests at 2, 4, 6, and 8 weeks post-injection. At 8 weeks we sacrificed some of the animals for real-time polymerase chain reaction (PCR) analysis, and evaluation of TH+cell counts. Results We observed a significant decrease in contralateral turns to the lesions in the ASCs and ASC-CM groups compared to the neurotoxin lesioned or α-MEM groups at 8 weeks post transplantation. Cell and CM- injected rats showed a significant increase of staying on the rotarod compared to the lesion or α-MEM groups. Cell and CM-treated rats showed significant increases in the NGF and NT3 genes, respectively, compared with the lesion group. Both treated groups showed significant increases in BDNF gene expression and TH+ cell density. Conclusion The results suggested that ASCs and ASC-CM protected dopaminergic neurons through the expressions of neurotrophin genes.

361-368

Authors: Azam Samadian, Mahdi Hesaraki, Sepideh Mollamohamadi, Behrouz Asgari, Mehdi Totonchi, Hossein Baharvand

Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. Conclusion Our investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.

377-387

Authors: Zahra Azhdari Tafti, Mehdi Mahmoodi, Mohamas Reza Hajizadeh, Vahid Ezzatizadeh, Hossein Baharvand, Massoud Vosough, Abbas Piryae

Objective Recent advances in cell therapy have encouraged researchers to provide an alternative for treatment and restoration of damaged liver through using hepatocytes. However, these cells quickly lose their functional capabilities in vitro. Here, we aim to use the secretome of mesenchymal stromal cells (MSCs) to improve in vitro maintenance conditions for hepatocytes. Materials and Methods In this experimental study, following serum deprivation, human adipose tissue-derived MSCs (hAT-MSCs) were cultured for 24 hours under normoxic (N) and hypoxic (H) conditions. Their conditioned media (CM) were subsequently collected and labeled as N-CM (normoxia) and H-CM (hypoxia). Murine hepatocytes were isolated by perfusion of mouse liver with collagenase, and were cultured in hepatocyte basal (William’s) medium supplemented with 4% N-CM or H-CM. Untreated William’s and hepatocyte-specific media (HepZYM) were used as controls. Finally, we evaluated the survival and proliferation rates, as well as functionality and hepatocyte-specific gene expressions of the cells. Results We observed a significant increase in viability of hepatocytes in the presence of N-CM and H-CM compared to HepZYM on day 5, as indicated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) assay. Indocyanine green (ICG) uptake of hepatocytes in the H-CM and HepZYM groups on days 3 and 5 also suggested that H-CM maintained the hepatocytes at about the same level as the hepatocyte-specific medium. The HepZYM group had significantly higher levels of albumin (Alb) and urea secretion compared to the other groups (P<0.0001). However, there were no significant differences in cytochrome activity and cytochrome gene expression profiles among these groups. Finally, we found a slightly, but not significantly higher concentration of vascular endothelial growth factor (VEGF) in the H-CM group compared to the N-CM group (P=0.063). Conclusion The enrichment of William’s basal medium with 4% hAT-MSC-H-CM improved some physiologic parameters in a primary hepatocyte culture.

396-402

Authors: Nassim Ghorbanmehr, Mojdeh Salehnia, Mahboobeh Amoushahi

Objective The aim of present study is to determine the effects of supplementation of oocyte maturation medium with sodium selenite (SS) on oocyte mitochondrial DNA (mtDNA) copy number and reactive oxygen species (ROS) levels. Materials and Methods In this experimental study germinal vesicle (GV), metaphase I (MI), and metaphase II (MII) stage oocytes were recovered from 6-8 week old female mice after superovulation. Some of the GV oocytes were cultured and matured in the presence and absence of SS. Then in vivo and in vitro matured (IVM) oocytes were subjected to mitochondria staining by MitoTracker green, ROS analysis, and mtDNA copy number determination using absolute real-time polymerase chain reaction (PCR). Results The maturation rate of GV oocytes to the MII stage significantly increased in the SS supplemented group (79.25%) compared to the control group (72.46%, P<0.05). The intensity of mitochondrial staining was not different among the studied groups, whereas the mitochondria distribution in the cytoplasm of the IVM oocytes showed some aggregation pattern. The in vivo obtained MII oocytes had lower ROS levels and higher mtDNA copy numbers than IVM-MII oocytes (P<0.05). The SS supplemented group had significantly lower ROS levels and higher mtDNA copy numbers than the non-treated group (P<0.05). Conclusion SS increased oocyte mtDNA copy number by decreasing oxidative stress. SS had an association with better oocyte developmental competence.

412-421

Authors: Mozafar Khazaei, Mona Pazhouhi, Saber Khazaei

Objective Glioblastoma multiforme is the most malignant form of brain tumors. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. Here we have investigated the effects of T. pratense extract on glioblastoma multiforme cell line (U87MG). Materials and Methods In this experimental study the effect of T. pratense extract on cell viability was investigated using trypan blue staining, MTT assay, and lactate dehydrogenase activity measurement. Apoptosis and autophagy cell death were detected by fluorescent staining. Nitric oxide (No) production was measured using Griess reaction. Expression levels of some apoptotic and autophagic-related genes were detected using real-time polymerase chain reaction (PCR). The combination effects of T. pratense extract and temozolomide (TMZ) were evaluated by calculating the combination index and dose reduction index values. Results After treatment with T. pratense extract, the cell viability was significantly reduced in a time- and dose- dependent manner (P<0.05). Apoptosis and autophagy of U87MG cells were significantly increased (P<0.05). Also, T. pratense extract significantly decreased NO production (P<0.05) by U87MG cells. Combination of TMZ and T. pratense extract had a synergistic cytotoxic effect. Conclusion T. pratense showed anti-cancer properties via induction of apoptosis and autophagy cell death.

427-434

Authors: neda Ranjbar Kohan, Saeed Nazifi, Mohamad Reza Tabandeh, Maryam Ansari Lari

Objective L-carnitine (LC) has been shown to protect cardiac metabolism in diabetes patients with cardiovascular diseases (CVDs). Apelin, a newly discovered adipocytokines, is an important regulator of cardiac muscle function; however, the role of the level of expression of Apelin axis in improvement of cardiac function by LC in diabetic patients, is not clear. In the present study, obese insulin-resistant rats were used to determine the effect of LC, when given orally with a high-calorie diet, on Apelin and Apelin receptor (Apj) expression in cardiac muscle. Materials and Methods In this experimental study, rats were fed with high-fat/high-carbohydrate diet for five weeks and subsequently were injected with streptozotocin 30 mg/kg for induction of obesity and insulin resistance. After confirming the induction of diabetes (serum glucose above 7.5 mmol/L), the animals received LC 300 mg/kg in drinking water for 28 days. On days 0, 14 and 28 after treatment, cardiac Apelin and Apj gene expression was evaluated by real time polymerase chain reaction (PCR) analysis. Serum levels of insulin, Apelin, glucose, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and the homeostasis model assessment of insulin resistance (HOMA-IR) were also measured using commercial kits. Results Cardiac Apelin and Apj expression and serum Apelin were increased in obese rats, while LC supplementation decreased the serum levels of Apelin and down-regulated Apelin and Apj expression in cardiac muscle. These changes were associated with reduced insulin resistance markers and serum inflammatory factors and improved lipid profile. Conclusion We concluded that LC supplementation could attenuate the over-expression of Apelin axis in heart of diabetic rats, a novel mechanism by which LC improves cardiovascular complications in diabetic patients.

443-448

Authors: Mahdi Lotfipanah, Fereydoon Azadeh, Mehdi Totonchi, Reza Omani-Samani

Objective Stem cells that have unlimited proliferation potential as well as differentiation potency are considered to be a promising future treatment method for incurable diseases. The aim of the present study is to evaluate the future trend of stem cell researches from researchers’ viewpoints. Materials and Methods This was a cross-sectional descriptive study on researchers involved in stem cell research at Royan Institute. We designed a questionnaire using a qualitative study based on expert opinion and a literature review. Content validity was performed using three rounds of the Delphi method with experts. Face validity was undertaken by a Persian literature expert and a graphics designer. The questionnaire was distributed among 150 researchers involved in stem cell studies in Royan Institute biology laboratories. Results We collected 138 completed questionnaires. The mean age of participants was 31.13 ± 5.8 years; most (60.9%) were females. Participants (76.1%) considered the budget to be the most important issue in stem cell research, 79.7% needed financial support from the government, and 77.5% felt that charities could contribute substantially to stem cell research. A total of 90.6% of participants stated that stem cells should lead to commercial usage which could support future researches (86.2%). The aim of stem cell research was stipulated as increasing health status of the society according to 92.8% of the participants. At present, among cell types, importance was attached to cord blood and adult stem cells. Researchers emphasized the importance of mesenchymal stem cells (MSCs) rather than hematopoietic stem cells (HSCs, 57.73%). The prime priorities were given to cancer so that stem cell research could be directed to sphere stem cell research whereas the least preference was given to skin research. Conclusion Regenerative medicine is considered the future of stem cell research with emphasis on application of these cells, especially in cancer treatment.

138-149

Authors: Hamid Chobineh, Mahsa Kazemi, Mohammad Ali Sadighi Gilani, Tahereh Heydari, Saeed Shokri, Mahshid Bazrafkan, Gholamreza Hassanzadeh

Objective: This study evaluated the effects of exogenous testosterone molecule-1 (CADM1) pathological defect during early and chronic periods of spinal cord injury (SCI). Materials and Methods: In this experimental study, testosterone was administered immediately or after one week of SCI induction. Along with quantification of CADM1 gene expression and its immunoreactivity, we evaluated sperm parameters and serum testosterone level post-SCI. Results: Different grades of abnormalities in sperm parameters and testis architecture were observed along with significant reductions in the level of CADM1 expression and its immunoreactivity in the seminiferous tubules of both acute and chronic SCI groups. Exogenous testosterone, by compensating the serum testosterone level. reduced the percentage of apoptotic and both short head and abnormal sperm froms in the caudal epididymis. Importantly, the beneficial effects of immediate administration of testosterone were prominent. Increases in the level of CADM1 transcription and its immunoreactivity in the testis of SCI mice treated with testosterone were accompanied by improvement of sperm motility as well as testicular Johnsen’s and Miller’s criteria. Conclusion: Since immediate testosterone treatment improved the immunoreactivity and transcription level of CADM1, the observed beneficial effect of exogenouse testosterone can be attributed to its effect on CADM1 dynamics.

157-167

Authors: Mohamadreza Afarinesh, Gila Behzadi

Objective: The aim of the present study is to investigate the effects of chronic whisker deprivation on possible alterations to the development of nitrergic neurons in the whisker part of the somatosensory (wS1) and motor (wM1) cortices in offspring with congenital hypothyroidism (CH). Materials and Methods: In the experimental study, CH was induced by adding propylthiouracil to the rats drinking water from embryonic day 16 to postnatal day (PND) 60. In whisker-deprived (WD) pups, all the whiskers were trimmed from PND 1 to 60. Nitrergic interneurons in the wS1/M1 cortices were detected by NADPH-diaphorase histochemistry staining technique in the control (Ctl), Ctl+WD, Hypo and Hypo+WD groups. Results: In both wS1 and wM1 cortices the number of nitrergic neurons was significantly reduced in the Hypo and Hypo+WD groups compared to Ctl and Ctl+WD groups, respectively (P<0.05) while bilateral whisker deprivation had no remarkable effect. The mean soma diameter size of NADPH-d labeled neurons in the Ctl+WD and Hypo+WD groups was decreased compared to the Ctl and Hypo groups, respectively. A similar patterns of decreased NADPH-d labeled neurons in the wS1/M1 cortices occur in the processes of nitrergic neurons in both congenital hypothyroidism and whisker deprivation. Conclusion: Our results suggest that both congenital hypothyroidism and whisker deprivation may disturb normal development of the wS1 and wM1 cortical circuits in which nitrergic neurons are involved.

177-182

Authors: Azadeh Seyedjoodaki, Fereshteh Alsahebfosoul, Nahid Eskandari, Vahid Shaygannejad, Mansoor Salehi, Mohammad Kazemi, Mostafa Manian, Omid Mirmosayyeb, Mohmmad Taghi Kardi

Inflammation of the immune system and the central nervous system has been known as an important predisposing factor for Parkinson’s disease (PD). Increased expression of OX40 protein (CD134) is a known factor for increased inflammation and initiation of NF-kappa-B signaling pathway in different diseases. We aimed to investigate the expression of OX40 at the transcript and serum protein levels. Materials and Methods: Twenty individuals with PD and 20 healthy individuals, as controls, were enrolled in this casecontrol study. Expression of OX40 at the transcript level and serum protein levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assays respectively. Results: The mean expression level of OX40 was increased in patients but not at a significant level (P>0.05). Consistently, the mean serum concentration of OX40 showed a mild, but non-significant, increase in the patients (P>0.05). Conclusion: We conclude that OX40 expression at either the transcript or protein level has no diagnostic utility in asymptomatic PD. This shows the need for clinical, cellular and interventional research to detect new robust biomarkers.

188-194

Authors: Milad Ahani-Nahayati, Saeed Solali, Karim Shams Asanjan, Ali Akbar Movassaghpour Akbari, Mehdi Talebi, Milad Zadi Heydarabad, Sina Baharaghdam, Majid Farshdousti Hagh

Objective: DNA methylation is a well-studied epigenetic mechanism that is a potent arm of the gene expression controlling machinery. Since the hypoxic situation and the various cells of bone marrow microenvironment, e.g. mesenchymal stem cells, play a role in the in vivo and in vitro biology of leukemic cells, we decided to study the effects of hypoxia and mesenchymal stem cells (MSCs) on the promoter methylation pattern of BAX and BCL2 genes. Materials and Methods: In this experimental study, the co-culture of MOLT-4 cells with MSCs and treatment with CoCl2 was done during 6, 12, and 24 hour periods. Total DNA was extracted using commercial DNA extraction kits, and sodium bisulfite (SBS) treatment was performed on the extracted DNA. Methylation specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the selected genes’ promoter regions. Results: The BAX and BCL2 promoters of untreated MOLT-4 cells were in partial methylated and fully unmethylated states, respectively. After incubating the cancer cells with CoCl2 and MSCs, the MSP results after 6, 12, and 24 hours were the same as untreated MOLT-4 cells. In other words, the exposure of MOLT-4 cells to the hypoxia-mimicry agent and MSCs in various modes and different time frames showed that these factors have exerted no change on the methylation signature of the studied fragments from the promoter region of the mentioned genes. Conclusion: Hypoxia and MSCs actually have no notable effect on the methylation status of the promoters of BAX and BCL2 in the specifically studied regions. DNA methylation is probably not the main process by which MSCs and CoCl2 induced hypoxia regulate the expression of these genes. Finally, we are still far from discovering the exact functional mechanisms of gene expression directors, but these investigations can provide new insights into this field for upcoming studies.

204-210

Authors: Hossein Pashaiefar, Marjan Yaghmaie, Javad Tavakkoly-Bazzaz, Seyed Hamidollah Ghaffari, Kamran Alimoghaddam, Pantea Izadi, Ardeshir Ghavamzadeh

Objective: Chromosomal translocations are among the most common mutational events in cancer development, especially in hematologic malignancies. However, the precise molecular mechanism of these events is still not clear. It has been recently shown that alternative non-homologous end-joining (alt-NHEJ), a newly described pathway for double-stranded DNA break repair, mediates the formation of chromosomal translocations. Here, we examined the expression levels of the main components of alt-NHEJ (PARP1 and LIG3) in acute myeloid leukemia (AML) patients and assessed their potential correlation with the formation of chromosomal translocations. Materials and Methods: This experimental study used reverse transcription-quantitative polymerase chain reaction (RTqPCR) to quantify the expression levels of PARP1 and LIG3 at the transcript level in AML patients (n=78) and healthy individuals (n=19). Results: PARP1 was the only gene overexpressed in the AML group when compared with healthy individuals (P=0.0004), especially in the poor prognosis sub-group. Both genes were, however, found to be up-regulated in AML patients with chromosomal translocations (P=0.04 and 0.0004 respectively). Moreover, patients with one isolated translocation showed an over-expression of only LIG3 (P=0.005), whereas those with two or more translocations over-expressed both LIG3 (P=0.002) and PARP1 (P=0.02). Conclusion: The significant correlations observed between PARP1 and LIG3 expression and the rate of chromosomal translocations in AML patients provides a molecular context for further studies to investigate the causality of this association.

220-230

Authors: Sara Eslamizadeh, Mansour Heidari, Shahram Agah, Ebrahim Faghihloo, Hossein Ghazi, Alireza Mirzaei, Abolfazl Akbari

Objective: Colorectal cancer (CRC) is one of the most common cancers and a major cause of cancer-related death worldwide. The early diagnosis of colorectal tumors is one of the most important challenges in cancer management. MicroRNAs (miRNAs) have provided new insight into CRC development and have been suggested as reliable and stable biomarkers for diagnosis and prognosis. The aim of this study was to analyze the differential expression of miRNAs at different stages of CRC searching for possible correlation with clinicopathological features to examine their potential value as diagnostic biomarkers. Materials and Methods: In this case-control study, plasma and matched tissue samples were collected from 74 CRC patients at stage II-IV as well as blood samples from 32 healthy controls. After exhaustive study of the current literature, eight miRNAs including miR-200c, 20a, 21, 31,135b, 133b,145 and let-7g were selected. The expression level of the miRNAs was assayed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Statistical analysis, including t test , Mann-Whitney U, Kruskall-Wallis tests and receiver operating characteristic (ROC) curve was applied, where needed. Results: Significantly elevated levels of miR-21, miR-31, miR-20a, miR-135b, and decreased levels of miR- 200c, miR-145 and let-7 g were detected in both plasma and matched tissue samples compared to the healthy group (P<0.05). However, no significant differences were observed in the expression level of plasma and tissue miR-133b (P>0.05). ROC for tissue miRNAs showed an area under the ROC curve (AUC) of 0.98 and P<0.001 for miR-21, 0.91 and P<0.001 for miR-135b, 0.91 and P<0.001 for miR-31, and 0.92 and P<0.001 for miR-20a. Conclusion: Our results indicate that the expression levels of microRNAs are systematically altered in CRC tissue and plasma. In conclusion, detection of miR-21, miR-135b, miR-31 and miR-20a levels in the tissue might be helpful to illuminate the molecular mechanisms underlying CRC carcinogenesis and serve as tumor-associated biomarkers for diagnosis.

244-249

Authors: Somayeh Keshavarzi, Mohammad Salehi, Fttaneh Farifteh-Nobijari, Taher Hosseini, Sara Hosseini, Alaleh Ghazifard, Marefat Ghaffari Novin, Vahid Fallah-Omrani, Mohsen Nourozian, Ahmad Hosseini

Objective: We evaluated the effect of melatonin, as a potent antioxidant agent, on glutathione (GSH) and reactive oxygen species (ROS) levels, as well as histone H3 lysine 9 (H3K9), and H4 lysine 12 (H4K12) acetylation when added to oocytes culture medium. Materials and Methods: In this experimental study, two in vitro and in vivo groups were used. In the in vitro group, cumulus oocyte complexes (COCs) from the ovaries of B6D2F1 mice were cultured in maturation medium containing two doses of melatonin (10-9 and 10-6 M) and without melatonin [control group treated with dimethyl sulfoxide (DMSO)] for 22-24 hour. The cumulus expansion and nuclear status were monitored by an inverted microscope. Next, COCs were isolated from the oviducts of superovulated mice and studied as the in vivo group. In in vitro and in vivo matured oocytes, GSH and ROS levels were assessed by monochlorobimane (MCB) and 2-7-dichlorodihydrofluorescein diacetate (H2DCFDA) staining, respectively. Changes in histone acetylation were examined by immunofluorescent staining with specific antibodies against acetylated H3K9 and H4K12. Results: The H4K12 acetylation and ROS levels were significantly higher in the oocytes matured in the in vitro group compared to the in vivo group (P<0.05). Furthermore, glutathione levels in the in vitro group were considerably lower than that of the in vivo group (P<0.05). Melatonin at the concentration of 10-6 M had the most substantial effect on nuclear maturation and histone acetylation as well as glutathione and ROS levels in the in vitro group (P<0.05). Conclusion: Exogenous melatonin improves the competence of mouse oocytes during in vitro maturation (IVM).

259-266

Authors: Abdolkhaleg Deezagi, Samira Shomali

Objective: Tissue engineering today uses factors that can induce differentiation of mesenchymal stem cells (MSCs) into other cell types. However, the problem of angiogenesis in this differentiated tissue remains an unresolved area of research interest. The aim of this study was to investigate the effects of prostaglandin F-2α (PGF-2α) on the expression of vascular endothelial growth factor (VEGF) in human adipose tissue derived MSCs. Materials and Methods: In this experimental research, human adipose tissue was digested using collagenase. The isolated MSCs cells were treated with PGF-2α (up to 5 μg/ml) and incubated for 96 hours. Cell proliferation, secretion of VEGF and cell migration were spontaneously assayed by MTT, BrdU, ELISA, RT-PCR and scratching methods. Results: Cell growth at 1.0, 2.5, 5 μg/ml of PGF-2α was not significantly reduced compared to control cells, suggesting that these concentrations of PGF-2α are not toxic to cell growth. The results of the BrdU incorporation assay indicated that, in comparison to untreated cells, BrdU incorporation was respectively 1.08, 1.96, 2.0 and 1.8 fold among cells treated with 0.1, 1.0, 2.5 and 5.0 μg/ml of PGF-2α. The scratching test also demonstrated a positive influence on cell proliferation and migration. Cells treated with 1.0 μg/ml of PGF-2α for 12 hours showed the highest relative migration and coverage in comparison to untreated cells. Quantitative VEGF ELISA and RTPCR results indicated an increase in VEGF expression and secretion in the presence of PGF-2α. The amount of VEGF produced in response to 0.1, 1.0, 2.5 and 5.0 μg/ml of PGF-2α was 62.4 ± 3.2 , 66.3 ± 3.7, 53.1 ± 2.6 and 49.0 ± 2.3 pg/ml, respectively, compared to the 35.2 ± 2.1 pg/ml produced by untreated cells. Conclusion: Stimulation of VEGF secretion by PGF-2α treated MSCs could be useful for the induction of angiogenesis in tissue engineering in vitro.

278-283

Authors: Mohammad Amin Behmanesh, Hosein Najafzadehvarzi, Seyyedeh Mahsa Poormoosavi

Objective: Bisphenol A (BPA), an endocrine-disrupting chemical, has been considered as a possible risk factor for fertility because it induces testicular toxicity. Thus, we sought to analyze the effect of Aloe vera as plant with antioxidant properties on tissues and oxidative stress parameters in male rats. Materials and Methods: In this experimental study, 50 adult male Wistar rats (200 ± 20 g) have been used in this 56 day study. Animals were completely randomized and divided into five groups: A1 (control), A2 (vehicle control), A3 (Aloe vera gel 300 mg/kg), B1 (BPA 20 μg/kg bw) and B2 (Aloe vera gel+ BPA). At the end of the study, the rats were anesthetized and 2 ml blood samples were obtained for evaluation of oxidative stress markers. Also, both testes were collected for histological examinations. Results: BPA significantly decreased (P<0.05) body and testis weights. Seminiferous tubule diameter (STD) and height of seminiferous epithelium (HSE), were significantly decreased (P<0.05) in the groups receiving BPA as compared to the control. There was also a reduction in the quantity of spermatocyte and spermatids. Moreover, malondialdehyde (MDA) increased and thiol protein (G-SH) decreased. But, co-administration of Aloe vera with BPA accelerated the total antioxidant capacity and testicular tissue structure healing. Conclusion: According to our findings, Aloe vera gel extract can overcome the damaging effects of BPA on the reproductive system of rats and protects rats’ testes against BPA-induced toxicity.

290-292

Authors: Emad Nikkhah, Reza safaralizadeh, Javad mohammadiasl, Maryam Tahmasebi birgani, Mohammad Ali Hosseinpour Feizi, Neda Golchin

Norrie disease (ND) is a rare X-linked recessive disorder, which is characterized by congenital blindness and, in several cases, accompanied with mental retardation and deafness. ND is caused by mutations in NDP, located on the proximal short arm of the X chromosome (Xp11.3). The disease has been observed in many ethnic groups worldwide, however, no such case has been reported from Iran. In this study, we present the molecular analysis of two patients with ND and the subsequent prenatal diagnosis (PND). Screening of NDP identified a hemizygous missense mutation (p.Ser133Cys) in the affected male siblings of the family. The mother was the carrier for the mutation (p.Ser133Cys). In a subsequent chorionic amniotic pregnancy, we carried out PND by sequencing NDP in the chorionic villi sample at 11 weeks of gestation. The fetus was carrying the mutation and thus unaffected. This is the first mutation report and PND of an Iranian family with ND, and highlights the importance of prenatal diagnostic screening of this congenital disorder and relevant genetic counseling.