Cell Journal (Yakhteh)

187-192

Authors: Atefeh Namipashaki, Zahra Razaghi-Moghadam, Naser Ansari-Pour

Population-based genetic association studies have proven to be a powerful tool in identifying genes implicated in many complex human diseases that have a huge impact on public health. An essential quality control step in such studies is to undertake Hardy-Weinberg equilibrium (HWE) calculations. Deviations from HWE in the control group may reflect important problems including selection bias, population stratification and genotyping errors. If HWE is violated, the inferences of these studies may thus be biased. We therefore aimed to examine the extent to which HWE calculations are reported in genetic association studies published in Cell Journal(Yakhteh) (Cell J). Using keywords pertaining to genetic association studies, eleven relevant articles were identified of which ten provided full genotypic data. The genotype distribution of 16 single nucleotide polymorphisms (SNPs) was re-analyzed for HWE by using three different methods where appropriate. HWE was not reported in 60% of all articles investigated. Among those reporting, only one article provided calculations correctly and in detail. Therefore, 90% of articles analyzed failed to provide sufficient HWE data. Interestingly, three articles had significant HWE deviation in their control groups of which one highly deviated from HWE expectations (P= 9.8×10-12). We thus show that HWE calculations are under-reported in genetic association studies published in this journal. Furthermore, the conclusions of the three studies showing significant HWE in their control groups should be treated cautiously as they may be potentially misleading. We therefore recommend that reporting of detailed HWE calculations should become mandatory for such studies in the future.

201-210

Authors: Farhad Oubari,Naser Amirizade,Hemn Mohammadpour,Mozhdeh Nakhlestani,Mahin Nikougoftar Zarif

Objective: Hematopoietic stem cells (HSCs) transplantation using umbilical cord blood (UCB) has improved during the last decade. Because of cell limitations, several studies focused on the ex vivo expansion of HSCs. Numerous investigations were performed to introduce the best cytokine cocktails for HSC expansion The majority used the Fms-related tyrosine kinase 3 ligand (FLT3-L) as a critical component. According to FLT3-L biology, in this study we have investigated the hypothesis that FLT3-L only effectively induces HSCs expansion in the presence of a mesenchymal stem cell (MSC) feeder. Materials and Methods: In this experimental study, HSCs and MSCs were isolated from UCB and placenta, respectively. HSCs were cultured in different culture conditions in the presence and absence of MSC feeder and cytokines. After ten days of culture, total nucleated cell count (TNC), cluster of differentiation 34+ (CD34+) cell count, colony forming unit assay (CFU), long-term culture initiating cell (LTC-IC), homeobox protein B4 (HoxB4) mRNA and surface CD49d expression were evaluated. The fold increase for some culture conditions was compared by the t test. Results: HSCs expanded in the presence of cytokines and MSCs feeder. The rate of expansion in the co-culture condition was two-fold more than culture with cytokines (P<0.05). FLT3-L could expand HSCs in the co-culture condition at a level of 20-fold equal to the presence of stem cell factor (SCF), thrombopoietin (TPO) and FLT3-L without feeder cells. The number of extracted colonies from LTC-IC and CD49d expression compared with a cytokine cocktail condition meaningfully increased (P<0.05). Conclusion: FLT3-L co-culture with MSCs can induce high yield expansion of HSCs

231-242

Authors: Amir Allahverdi, Saied Abroun, Arefeh Jafarian, Masoud Soleimani,, Mohammad Taghikhani, Fatemeh Eskandari

Objective: Type I diabetes is an immunologically-mediated devastation of insulin producing cells (IPCs) in the pancreatic islet. Stem cells that produce β-cells are a new promising tool. Adult stem cells such as mesenchymal stem cells (MSCs) are self renewing multi potent cells showing capabilities to differentiate into ectodermal, mesodermal and endodermal tissues. Pancreatic and duodenal homeobox factor 1 (PDX1) is a master regulator gene required for embryonic development of the pancreas and is crucial for normal pancreatic islets activities in adults. Materials and Methods: We induced the over-expression of the PDX1 gene in human bone marrow MSCs (BM-MSCs) by Lenti-PDX1 in order to generate IPCs. Next, we examine the ability of the cells by measuring insulin/c-peptide production and INSULIN and PDX1 gene expressions. Results: After transduction, MSCs changed their morphology at day 5 and gradually differentiated into IPCs. INSULIN and PDX1 expressions were confirmed by real time polymerase chain reaction (RT-PCR) and immunostaining. IPC secreted insulin and C-peptide in the media that contained different glucose concentrations. Conclusion: MSCs differentiated into IPCs by genetic manipulation. Our result showed that lentiviral vectors could deliver PDX1 gene to MSCs and induce pancreatic differentiation.

253-263

Authors: Marahaini Musa, Khadijah Mohd Ali, Thirumulu Ponnuraj Kannan, Ahmad Azlina, Nor Shamsuria Omar,Anil Chatterji,Khairani Idah Mokhtar

Objective: Perivitelline fluid (PVF) of the horseshoe crab embryo has been reported to possess an important role during embryogenesis by promoting cell proliferation. This study aims to evaluate the effect of PVF on the proliferation, chromosome aberration (CA) and mutagenicity of the dental pulp stem cells (DPSCs). Materials and Methods: This is an in vitro experimental study. PVF samples were collected from horseshoe crabs from beaches in Malaysia and the crude extract was prepared. DPSCs were treated with different concentrations of PVF crude extract in an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (cytotoxicity test). We choose two inhibitory concentrations (IC50 and IC25) and two PVF concentrations which produced more cell viability compared to a negative control (100%) for further tests. Quantitative analysis of the proliferation activity of PVF was studied using the AlamarBlue® assay for 10 days. Population doubling times (PDTs) of the treatment groups were calculated from this assay. Genotoxicity was evaluated based on the CA and Ames tests. Statistical analysis was carried out using independent t test to calculate significant differences in the PDT and mitotic indices in the CA test between the treatment and negative control groups. Significant differences in the data were P<0.05. Results: A total of four PVF concentrations retrieved from the MTT assay were 26.887 mg/ml (IC50), 14.093 mg/ml (IC25), 0.278 mg/ml (102% cell viability) and 0.019 mg/ml (102.5% cell viability). According to the AlamarBlue® assay, these PVF groups produced comparable proliferation activities compared to the negative (untreated) control. PDTs between PVF groups and the negative control were insignificantly different (P>0.05). No significant aberrations in chromosomes were observed in the PVF groups and the Ames test on the PVF showed the absence of significant positive results. Conclusion: PVF from horseshoe crabs produced insignificant proliferative activity on treated DPSCs. The PVF was non-genotoxic based on the CA and Ames tests.

273-287

Authors: Doan Chinh Chung, Le Thanh Long, Hoang Nghia Son, Le Tri Bao, Do Minh Si, Le Van Dong

Objective: Hepatocellular carcinoma (HCC), one of the most common cancers worldwide, is resistant to anticancer drugs. Angiogenesis is a major cause of tumor resistance to chemotherapy, and vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The purpose of this study is to investigate the impact of small-interfering RNA targeting VEGF gene (VEGF-siRNA) on chemosensitivity of HCC cells in vitro. Materials and Methods: In this experimental study, transfection was performed on Hep3B cells. After transfection with siRNAs, VEGF mRNA and protein levels were examined. Cell proliferation, apoptosis and anti-apoptotic gene expression were also analyzed after treatment with VEGF-siRNA in combination with doxorubicin in Hep3B cells. Results: Transfection of VEGF-siRNA into Hep3B cells significantly reduced the expression of VEGF at both mRNA and protein levels. Combination therapy with VEGF-siRNA and doxorubicin more effectively suppressed cell proliferation and induced apoptosis than the respective monotherapies. This could be explained by the significant downregulation of B-cell lymphoma 2 (BCL-2) and SURVIVIN. Conclusion: VEGF-siRNA enhanced the chemosensitivity of doxorubicin in Hep3B cells at least in part by suppressing the expression of anti-apoptotic genes. Therefore, the downregulation of VEGF by siRNA combined with doxorubicin treatment has been shown to yield promising results for eradicating HCC cells.

296-303

Authors: Kazem Parivar,Nasim Hayati Rudbari,Ramazan Khanbabaee,Mahya Khaleghi

Objective: There is a wide application of titanium dioxide (TiO2) nanoparticles (NPs) in industry. These particles are used in various products, and they also has biological effects on cells and organs through direct contact. Materials and Methods: In this experimental research, the effect of TiO2 on chondrogenesis of forelimb buds of mice embryos was assessed in in vivo condition. Concentrations of 30, 150 and 500 mg/kg body weight (BW) TiO2 NPs (20 nm size) dissolved in distilled water were injected intraperitoneally to Naval Medical Research Institute (NMRI) mice on day 11.5 of gestation. On day 15, limb buds were amputated from the embryos and skeletogeneis of limb buds were studied. Results: TiO2 NPs caused the significant changes in chondrocytes in the following developmental stages: resting, proliferating, hypertrophy, degenerating, perichondrium and mesenchymal cells. Decreased number of mesenchymal cells and increased level of chondrocytes were observed after the injection of different concentrations of TiO2, which proves the unpredictable effects of TiO2 on limb buds. Conclusion: Results of the present study showed TiO2 NPs accelerated the chondrogenesis of limb buds, but further studies are recommended to predict TiO2 toxicity effects on organogenesis.

312-321

Authors: Nazila Eyvazzadeh,Ali Neshasteh-Riz,Seyed Rabee Mahdavi,Afshin Mohsenifar

Objective: To explore the cumulative genotoxic damage to glioblastoma (GBM) cells, grown as multicellular spheroids, following exposure to 6 MV X-rays (2 Gy, 22 Gy) with or without, 2- methoxy estradiol (2ME2), iododeoxyuridine (IUDR) or topotecan (TPT), using the Picogreen assay. Materials and Methods: The U87MG cells cultured as spheroids were treated with 6 MV X-ray using linear accelerator. Specimens were divided into five groups and irradiated using X-ray giving the dose of 2 Gy after sequentially incubated with one of the following three drug combinations: TPT, 2-ME2/TPT, IUDR/TPT or 2ME2/IUDR/ TPT. One specimen was used as the irradiated only sample (R). The last group was also irradiated with total dose of 22 Gy (each time 2 Gy) of 6 MV X-ray in 11 fractions and treated for three times. DNA damage was evaluated using the Picogreen method in the experimental study. Results: R/TPT treated group had more DNA damage [double strand break (DSB)/single strand break (SSB)] compared with the untreated group (P<0.05). Moreover the R/ TPT group treated with 2ME2 followed by IUDR had maximum DNA damage in spheroid GBM indicating an augmented genotoxicity in the cells. The DNA damage was induced after seven fractionated irradiation and two sequential treatments with 2ME2/IUDR/TPT. To ensure accuracy of the slope of dose response curve the fractionated radiation was calculated as 7.36 Gy with respect to α/β ratio based on biologically effective dose (BED) formulae. Conclusion: Cells treated with 2ME2/IUDR showed more sensitivity to radiation and accumulative DNA damage. DNA damage was significantly increased when GBM cells treated with TPT ceased at S phase due to the inhibition of topoisomerase enzyme and phosphorylation of Chk1 enzyme. These results suggest that R/TPTtreated cells increase sensitivity to 2ME2 and IUDR especially when they are used together. Therefore, due to an increase in the level of DNA damage (SSB vs. DSB) and impairment of DNA repair machinery, more cell death will occur. This in turn may improve the treatment of GBM.

332-338

Authors: Roya Fatehi, Bita Ebrahimi

Objective: This study was conducted to assess survival of follicles, their oocyte maturation and fertilization potential as well as expression of early embryo developmental genes in in vitro cultured pre-antral follicles derived from vitrified-warmed mouse ovary. Materials and Methods: In this experimental study, ovaries of 12-day old Naval Medical Research Institute (NMRI) female mice were placed into non-vitrified and vitrifiedwarmed groups. Isolated preantral follicles from experimental groups were cultured in vitro for 12 days. On the 12th day of culture, oocyte maturation was induced and then matured oocytes were in vitro fertilized. The rates of oocyte maturation and two-cell stage embryo formation were assessed. Relative expression of Mater and Zar1 was evaluated on days 1, 6, 10 and 12 of culture. Data analysis was performed by t test and two-way ANOVA (P<0.05). Results: Our data showed no significant difference between the control and vitrification groups in the rate of follicular survival, oocyte maturation and two-cell stage embryo formation. The level of gene expression was higher on the 6th and 10th days of culture for Mater and Zar1 in vitrified-warmed group compared with non-vitrified group, however, there was no significant difference between the two groups. Conclusion: It seems that the applied vitrification method did not reveal any negative effect on maturation and developmental competence of oocytes surrounded in preantral follicles and therefore could preserve follicular reserves efficiently.

355-360

Authors: Sobia Mushtaq,Tahir Ali,Qamar Javed,Sobia Tabassum, Iram Murtaza

Objective: Oxidative stress down regulates antioxidant enzymes including superoxide dismutase (SOD) and contributes to the development of cardiac hypertrophy. N-Acetyl cysteine (NAC) can enhance the SOD activity, so the aim of this study is to highlight the inhibitory role of NAC against endothelin-1 (ET-1)-induced cardiac hypertrophy. Materials and Methods: In this experimental study at QAU from January, 2013 to March, 2013. ET-1 (50 μg/kg) and NAC (50 mg/kg) were given intraperitoneally to 6-day old neonatal rats in combination or alone. All rats were sacrificed 15 days after the final injection. Histological analysis was carried out to observe the effects caused by both drugs. Reactive oxygen species (ROS) analysis and SOD assay were also carried out. Expression level of hypertrophic marker, brain natriuretic peptide (BNP), was detected by western blotting. Results: Our findings showed that ET-1-induced cardiac hypertrophy leading towards heart failure was due to the imbalance of different parameters including free radical-induced oxidative stress and antioxidative enzymes such as SOD. Furthermore NAC acted as an antioxidant and played inhibitory role against ROS-dependent hypertrophy via regulatory role of SOD as a result of oxidative response associated with hypertrophy. Conclusion: ET-1-induced hypertrophic response is associated with increased ROS production and decreased SOD level, while NAC plays a role against free radicals-induced oxidative stress via SOD regulation.

372-378

Authors: Rahim Alijani

Objective: In recent years emphasis has been placed on evaluation studies and the publication of scientific papers in national and international journals. In this regard the publication of scientific papers in journals in the Institute for Scientific Information (ISI) database is highly recommended. The evaluation of scientific output via articles in journals indexed in the ISI database will enable the Iranian research authorities to allocate and organize research budgets and human resources in a way that maximises efficient science production. The purpose of the present paper is to publish a general and valid view of science production in the field of stem cells. Materials and Methods: In this research, outputs in the field of stem cell research are evaluated by survey research, the method of science assessment called Scientometrics in this branch of science. A total of 1528 documents was extracted from the ISI database and analysed using descriptive statistics software in Excel. Results: The results of this research showed that 1528 papers in the stem cell field in the Web of Knowledge database were produced by Iranian researchers. The top ten Iranian researchers in this field have produced 936 of these papers, equivalent to 61.3% of the total. Among the top ten, Soleimani M. has occupied the first place with 181 papers. Regarding international scientific participation, Iranian researchers have cooperated to publish papers with researchers from 50 countries. Nearly 32% (452 papers) of the total research output in this field has been published in the top 10 journals. Conclusion: These results show that a small number of researchers have published the majority of papers in the stem cell field. International participation in this field of research unacceptably low. Such participation provides the opportunity to import modern science and international experience into Iran. This not only causes scientific growth, but also improves the research and enhances opportunities for employment and professional development. Iranian scientific

389-394

Authors: Alok Sharma,Hemangi Sane,Pooja Kulkarni,Myola D’sa,Nandini Gokulchandran,Prerna Badhe

Cerebral palsy (CP) is a non progressive, demyelinating disorder that affects a child’s development and posture and may be associated with sensation, cognition, communication and perception abnormalities. In CP, cerebral white matter is injured resulting in the loss of oligodendrocytes. This causes damage to the myelin and disruption of nerve conduction. Cell therapy is being explored as an alternate therapeutic strategy as there is no treatment currently available for CP. To study the benefits of this treatment we have administered autologous bone marrow mononuclear cells (BMMNCs) to a 12-year-old CP case. He was clinically re-evaluated after six months and found to demonstrate positive clinical and functional outcomes. His trunk strength, upper limb control, hand functions, walking stability, balance, posture and coordination improved. His ability to perform activities of daily living improved. On repeating the Functional Independence Measure (FIM), the score increased from 90 to 113. A repeat positron emission tomography- computed tomography (PET-CT) scan of the brain six months after intervention showed progression of the mean standard deviation values towards normalization which correlated to the functional changes. At one year, all clinical improvements have remained. This indicated that cell transplantation may improve quality of life and have a potential for treatment of CP.