Cell Journal (Yakhteh)

235-244

Authors: Abdolreza Ardeshirylajimi , Masoud Soleimani , Saman Hosseinkhani , Kazem Parivar , Parichehr Yaghmaei

Objective: Human induced pluripotent stem cells (iPSCs) have been shown to have promising capacity for stem cell therapy and tissue engineering applications. Therefore, it is essential to compare the ability of these cells with the commonly used mesenchymal stem cells (MSC) for bone tissue engineering in vitro. Materials and Methods: In this experimental study, the biological behavior and osteogenic capacity of the iPSCs were compared with MSCs isolated from human adipose tissue (AT-MSCs) using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Alizarin red staining, alkaline phosphatase (ALP) activity measurements, calcium content assay and common osteogenic-related genes. Data were reported as the mean ± SD. One-way analysis of variance (ANOVA) was used to compare the results. A p value of less than 0.05 was considered statistically significant. Results: There was a significant difference between the rate of proliferation of the two types of stem cells; iPSCs showed increased proliferation compared to AT-MSCs. During osteogenic differentiation, ALP activity and mineralization were demonstrated to be significantly higher in iPSCs. Although AT-MSCs expressed higher levels of Runx2, iPSCs expressed higher levels of osteonection and osteocalcin during differentiation. Conclusion: iPSCs showed a higher capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.

255-262

Authors: Shohreh Hajizadeh-Sikaroodi, Ahmad Hosseini, Ali Fallah, Hajar Estiri, Zahra Noormohammadi, Mohammad Salehi, Sayyed Mohammad Hossein Ghaderian, Haleh Akhavan Niaki, Masoud Soleimani, Bahram Kazemi

Objective: Autoimmune diseases precede a complex dysregulation of the immune system. T helper17 (Th17) and interleukin (IL)-17 have central roles in initiation of inflammation and subsequent autoimmune diseases. IL-27 significantly controls autoimmune diseases by Th17 and IL-17 suppression. In the present study we have created genetic engineered mesenchymal stem cells (MSCs) that mediate with lentiviral vectors to release IL-27 as an adequate vehicle for ex vivo gene therapy in the reduction of inflammation and autoimmune diseases. Materials and Methods: In this experimental study, we isolated adipose-derived MSCs (AD-MSCs) from lipoaspirate and subsequently characterized them by differentiation. Two subunits of IL-27 (p28 and EBI3) were cloned in a pCDH-513B-1 lentiviral vector. Expressions of p28 and EBI3 (Epstein-Barr virus induced gene 3) were determined by real time polymerase chain reaction (PCR). MSCs were transduced by a pCDH-CMV-p28-IRESEBI3- EF-copGFP-Pur lentiviral vector and the bioassay of IL-27 was evaluated by IL-10 expression. Results: Cell differentiation confirmed true isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified successful cloning of both p28 and EBI3 in the pCDH-513B-1 lentiviral vector. Real time PCR showed high expressions level of IL-27 and IL-10 as well as accurate activity of IL-27. Conclusion: The results showed transduction of functional IL-27 to AD-MSCs by means of a lentiviral vector. The lentiviral vector did not impact MSC characteristics.

271-278

Authors: Zahra Oryan Abkenar, Amir Eghbal Khajehrahimi, Mohammad Hadi Bahadori

Objective: Cryopreservation of ovarian tissue or follicles has been proposed as an alternative method for fertility preservation. Although successful vitrification of follicles has been reported in several mammalian species, the survival rate is generally low. The aim of this study was to investigate the effects of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on in vitro preantral follicle development after vitrification. Materials and Methods:In this experimental study, preantral follicles with diameter of 150-180 μm were mechanically isolated from ovaries of 18-21 days old NMRI mice. Follicles were vitrified and warmed, then cultured in α-minimal essential medium (α-MEM) without growth factor supplementation as control group (group I), while supplemented with 20 ng/ml FGF (group II), 20 ng/ml EGF (group III), and 20 ng/ml FGF +20 ng/ml EGF (group IV). After 12 days, human chorionic gonadotrophin (hCG)/EGF was added to culture medium, and after 18-20 hours, the presence of cumulus oocyte complexes (COCs) and oocyte maturation were assessed. The chi-square (χ2) test was used to analyze survival and ovulation rates of the follicles. Results: Our results showed that the rate of metaphase II (MII) oocytes in FGF group increased in comparison with control and other treatment groups (p<0.027), but there was no difference between control with EGF and EGF+FGF groups in oocyte maturation rate (p>0.05). There was a significant decrease in survival rate of follicles in EGF+FGE group in comparison with other groups (p<0.008). After in vitro ovulation induction, the follicles in EGF group showed a higher ovulation rate (p<0.008) than those cultured in other groups. Conclusion: FGF has beneficial effect on oocyte maturation, and EGF increases COCs number in vitro. Combination of EGF and FGE decreases the number of survived follicles.

289-298

Authors: AliReza Alizadeh, Ph.D.1*, Vahid Esmaeili,Abdolhossein Shahverdi, Ladan Rashidi

Objective: The effects of dietary fish oil on semen quality and sperm fatty acid profiles during consumption of n-3 fatty acids as well as the persistency of fatty acids in ram’s sperm after removing dietary oil from the diet were investigated. Materials and Methods: In this experimental study, we randomly assigned 9 Zandi rams to two groups (isoenergetic and isonitrogenous diets): control (CTR; n=5) and fish oil (FO; n=4) for 70 days with a constant level of vitamin E in both groups. Semen was collected at the first week and at the last week of the feeding period (phase 1). After the feeding period, all rams were fed a conventional diet and semen samples were collected one and two months after removal of FO (phase 2). The sperm parameters and fatty acid profiles were measured by computer assisted semen analyzer (CASA) and gas chromatography (GC), respectively. The completely randomized design was used and data were analyzed with SPSS version 16. Results: Dietary FO had significant positive effects on all sperm quality and quantity parameters compared with the CTR during the feeding period (p<0.05). The positive effects of FO on sperm concentration and total sperm output were observed at one and two months after removal of FO (p<0.05), whereas other sperm parameters were unaffected. Before feeding, C14 (myristic acid), C16 (palmitic acid), C18 (stearic acid), C18:1 (oleic acid) and C22:6 (docosahexaenoic acid: DHA) were the primary sperm FA. FO in the diet increased sperm DHA, C14:0 and C18:0 during the feeding period (p<0.05). Conclusion: The present study showed not only manipulation of ram sperm fatty acid profiles by dietary FO and sperm parameters during the feeding period, but also the persistency of unique effects of dietary omega-3 fatty acids up to two months following its removal from the diet. Also, we recommend that sperm fatty acid profiles should be comprehensively analyzed and monitored.

309-314

Authors: Majid Motovali-Bashi, Hojatollah Rezaei, Fariba Dehghanian, Halimeh Rezaei

Objective: People are usually susceptible to carcinogenic aromatic amines, present in cigarrette smoke and polluted environment, which can cause DNA damage. Therefore, maintenance of genomic DNA integrity is a direct result of proper function of DNA repair enzymes. Polymorphic diversity could affect the function of repair enzymes and thus augment the risk of different cancers. Xeroderma pigmentosum group D (XPD) gene encodes one of the most prominent repair enzymes and the polymorphisms of this gene are thought to be of importance in lung cancer risk. This gene encodes the helicase, which is a component of transcription factor IIH and an important part of the nucleotide excision repair system. Studies reveal that individuals with Lys751Gln polymorphism of XPD gene have a low repairing capacity to delete the damages of ultraviolet light among other XPD polymorphisms. Materials and Methods: In this case-control study, first Lys751Gln polymorphism was genotyped, then its association with lung cancer risk was analyzed. Genomic DNA was extracted from the whole blood sample of 640 individuals from Iran (352 healthy individuals and 288 patients). Allele frequencies and heterozygosity of Lys751Gln polymorphism were determined using polymerase chain reaction-restriction fragment length polymorphism method. Results: According to statistical analyses, lung cancer risk in individuals with Lys751Gln polymorphism (Odd Ratio=1.8, 95% Confidence Interval 0.848-3.819) is approximately twice as high as that of Lys/Lys genotype, however 751Gln/Gln genotype did not relate to lung cancer risk (Odd Ratio=0.7, 95% Confidence Interval 0/307-1/595). Conclusion: This study suggests that heterozygous polymorphism (Lys/Gln) increases the sensitivity of lung cancer risk, while homozygous polymorphism (Lys/Lys) probably decreases its risk and C allele frequency shows no remarkable increase in the patients.

325-334

Authors: Mohammad Amin Edalatmanesh,Haniyeh Nikfarjam,Marzieh Moghadas,Aliakbar Haddad-Mashadrizeh,Reza Robati,Mohammad Reza Hashemzadeh

Objective: The cerebellum is a key structure involved in coordinated motor planning, cognition, learning and memory functions. This study presents a permanent model of a toxin produced cerebellar lesion characterized according to contemporary motor and cognitive abnormalities. Materials and Methods: In this experimental study, slow administration of quinolinic acid (QA, 5 μl of 200 μmol, 1 μl/minute) in the right cerebellar hemisphere (lobule VI) caused noticeable motor and cognitive disturbances along with cellular degeneration in all treated animals. We assessed behavioral and histopathological studies over ten weeks after QA treatment. The data were analyzed with ANOVA and the student’s t test. Results: The QA treated group showed marked motor learning deficits on the rotating rod test (p≤0.0001), locomotor asymmetry on the cylinder test (p≤0.0001), dysmetria on the beam balance test (p≤0.0001), abnormalities in neuromuscular strength on the hang wire test (p≤0.0001), spatial memory deficits in the Morris water maze (MWM, p≤0.001) and fear conditioned memory on the passive avoidance test (p≤0.01) over a ten-week period compared with the control animals. Histopathological analysis showed loss of Purkinje cells (p≤0.001) and granular cell density (p≤0.0001) in the lesioned hemisphere of the cerebellum. Conclusion: Results of the present study show that QA can remove numerous cells which respond to this toxin in hemispheric lobule VI and thus provide a potential model for functional and cell-based studies.

343-352

Authors: Mazdak Ganjalikhani Hakemi,Kamran Ghaedi,Vida Homayouni,Alireza Andalib,Mohsen Hosseini,Abbas Rezaei

Objective: Th17 cells are known to be involved in some types of inflammations and autoimmune disorders. RORC2 is the key transcription factor coordinating Th17 cell differentiation. Thus, blocking RORC2 may be useful in suppressing Th17-dependent inflammatory processes. The aim was to silence RORC2 by specific siRNAs in naïve T cells differentiating to Th17. Time-dependent expression of RORC2 as well as IL-17 and IL-23R were considered before and after RORC2 silencing. Materials and Methods: In this experimental study, naïve CD4+ T cells were isolated from human cord blood samples. Cytokines TGFβ plus IL-6 and IL-23 were used to polarize the naïve T cells to Th17 cells in X-VIVO 15 serum free medium. A mixture of three siRNAs specific for RORC2 was applied for blocking its expression. RORC2, IL-17 and IL-23R mRNA and protein levels were measured using qRT-PCR, ELISA and flow cytometry techniques. Pearson correlation and one-way ANOVA were used for statistical analyses. Results: Significant correlations were obtained in time-dependent analysis of IL-17 and IL-23R expression in relation with RORC2 (R=0.87 and 0.89 respectively, p<0.05). Silencing of RORC2 was accompanied with almost complete suppression of IL-17 (99.3%; p<0.05) and significant decrease in IL-23R gene expression (77.2%, p<0.05). Conclusion: Our results showed that RORC2 is the main and the primary trigger for upregulation of IL-17 and IL-23R genes in human Th17 cell differentiation. Moreover, we show that day 3 could be considered as the key day in the Th17 differentiation process.

361-366

Authors: Leili Aghebati Maleki, Jafar Majidi,Behzad Baradaran,Aliakbar Movassaghpour,Jalal Abdolalizadeh,

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs).

375-376

Authors: Gulcin Guler Simsek, Erdem Vargol, Hulya Simsek

Cytomorphological changes of mitomycin C on urothelial cells may be misinterpreted as a neoplastic process. A 60-year old male patient who was given an eight-week course of intravesical mitomycin C due to non-invasive low grade transitional cell carcinoma. During his follow-up care, the findings of a urine cytology exam were as follows: nuclear enlargement of cells, wrinkled nuclear membranes, little hyperchromasia, pleomorphism, abnormal nuclear morphology and disordered orientation of the urothelium. Furthermore, there were eosinophils nearby the atypical cells. This report aimed at reminding the cytomorphologic changes of mitomycin C may be misinterpreted as carcinoma, so the presence of eosinophils is required to predict the drug-induced changes.

383-384

Authors: Hamid Nasri,Mahmoud Rafieian-Kopaei

World Cancer Day (WCD) is celebrated on February 4th each year around the world to remind the efforts done by united nations, World Health Organization (WHO), and other governmental and non-governmental health organizations with the aim of delivering the real message about cancer and its treatments to fight against this fatal disease through uniting all the people on global basis (1-4). In brief, cancer is a large group of different disorders involving unregulated cell growth. In malignancy, cells divide and grow uncontrollably to form malignant tumors and to invade neighboring parts of the body. The cancer tumor may spread to more distant parts of the body through blood or lymphatic systems (5-8). It has been observed that most of the cancer cases and related deaths happen in less developed countries that this situation is expected to get worse by 2030. Therefore, it is very crucial to get control over such condition throughout the world. Furthermore, WCD aims to save millions of preventable deaths every year by raising alertness and by educating people about malignancy, while forcing the governments throughout the world to take action against this disease (2-8). The day is also a key chance for cancer patients to ensure that world leaders stick to the promises they made at the United Nations Summit for reducing the cancer and its impacts. During this particular occasion, participants try to promote healthy lifestyles, balanced diet, regular physical activity, weight management, as well as using antioxidants in order to diminish the risk of malignancies (1-8). Furthermore, this day is celebrated to plan certain new strategies and to imply various new programs in order to make people aware of this disorder. WCD is also celebrated to make non-patient people aware about the preventive methods and the risk factors of cancer (2-8). The theme of WCD of 2014 is "Debunk the Myths" because some people believe that if they contact or live with a patient who has cancer, they would get cancer as well. The day is, therefore, distinguished to eradicate such types of the social myths (5-13) and to make certain concepts about different aspects of the malignancy, such as its symptoms, causing factors and treatment (1, 3, 9). Furthermore, on this day, some of activities are organized to indicate that cancer patients should not be treated distinctly, while they are entitled to the same rights as normal people in the society (14-19). Although they have less chances of existence, they should be fulfilled their wishes by their relatives. It should not make them sense that the treatment remedies are given them for existence as they are dying. In this regards, it is very important to make them feel better like a normal person. They should be also prepared a normal environment in their home and society. It is essential that normal people avoid being overly sympathetic to them (20-27).