Cell Journal (Yakhteh)

485-492

Authors: Masoumeh Mohammadi, Mehdi Hedayati

Approximately 5-10% of all thyroid cancers are medullary thyroid carcinomas (MTC). MTC is mainly sporadic in nature, but 20-30% of cases are hereditary. Genetic testing for hereditary MTC is very important for the patient and his family, but the patients must be receiving appropriate genetic counseling. About 98% of patients with hereditary MTC have germline mutations in exons 10, 11, 13, 14, 15, 16 and intron 16 of the REarrangement during transfection (RET) proto-oncogene, but the etiology of the more frequent sporadic form of MTC (sMTC) is not well understood. Recently, it has been reported that apparently sporadic MTC may involve point mutations in BRAF and RAS genes, with an overall prevalence of almost 10%. Also alteration and abnormal expression of miRNA has been described in MTC. In this review, we attempted to mention some mutations and molecular changes in sporadic and hereditary MTC pathogenesis.

503-513

Authors: Forouzan Absalan, Sadegh Saremy, Esrafil Mansouri, Mahin Taheri Moghadam, Ali Reza Eftekhari Moghadam, Razie Ghanavati

Objective Phthalates, which are commonly used to render plastics into soft and flexible materials, have also been determined as developmental and reproductive toxicants in human and animals. The purpose of this study was to evaluate the effect of mono-(2- ethylhexyl) phthalate (MEHP) and di-(2-ethylhexyl) phthalate (DEHP) oral administrations on maturation of mouse oocytes, apoptosis and gene transcription levels. Materials and Methods In this experimental study, immature oocytes recovered from Naval Medical Research Institute (NMRI) mouse strain (6-8 weeks), were divided into seven different experimental and control groups. Control group oocytes were retrieved from mice that received only normal saline. The experimental groups I, II or III oocytes were retrieved from mice treated with 50, 100 or 200 µl DEHP (2.56 µM) solution, respectively. The experimental groups IV, V or VI oocytes were retrieved from mouse exposed to 50, 100 or 200 µl MEHP (2.56 µM) solution, respectively. Fertilization and embryonic development were carried out in OMM and T6 medium. Apoptosis was assessed by annexin V-FITC/Dead Cell Apoptosis Kit, with PI staining. In addition, the mRNA levels of Pou5f1, Ccna1 and Asah1 were examined in oocytes. Finally, mouse embryo at early blastocyst stage was stained with acridine-orange (AO) and ethidium-bromide (EB), in order to access their viability. Results The proportion of oocytes that progressed up to metaphase II (MII) and 2-cells embryo formation stage was significantly decreased by exposure to MEHP or DEHP, in a dose-dependent manner. Annexin V and PI positive oocytes showed greater quantity in the treated mice than control. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) revealed that expression levels of Pou5f1, Asah1 and Ccna1 were significantly lower in the treated mouse oocytes than control. The total cell count for blastocyst developed from the treated mouse oocytes was lower than the controls. Conclusion These results indicate that oral administration of MEHP and DEHP could negatively affect mouse oocyte meiotic maturation and development in vivo, suggesting that phthalates could be risk factors for mammalians’ reproductive health. Additionally, phthalate-induced changes in Pou5f1, Asah1 and Ccna1 transcription level could explain in part, the reduced developmental ability of mouse-treated oocytes.

532-539

Authors: Mohsen Basiri, Mehrdad Behmanesh, Yaser Tahamtani, Keynoosh Khalooghi, Azadeh Moradmand, Hossein Baharvand

Objective CRISPR/Cas9 technology provides a powerful tool for targeted modification of genomes. In this system, a donor DNA harboring two flanking homology arms is mostly used for targeted insertion of long exogenous DNA. Here, we introduced an alternative design for the donor DNA by incorporation of a single short homology arm into a circular plasmid. Materials and Methods In this experimental study, single homology arm donor was applied along with a single guide RNA (sgRNA) specific to the homology region, and either Cas9 or its mutant nickase variant (Cas9n). Using Pdx1 gene as the target locus the functionality of this system was evaluated in MIN6 cell line and murine embryonic stem cells (ESCs). Results Both wild type Cas9 and Cas9n could conduct the knock-in process with this system. We successfully applied this strategy with Cas9n for generation of Pdx1GFP knock-in mouse ESC lines. Altogether, our results demonstrated that a combination of a single homology arm donor, a single guide RNA and Cas9n is capable of precisely incorporating DNA fragments of multiple kilo base pairs into the targeted genomic locus. Conclusion While taking advantage of low off-target mutagenesis of the Cas9n, our new design strategy may facilitate the targeting process. Consequently, this strategy can be applied in knock-in or insertional inactivation studies.

547-555

Authors: Samireh Ghafouri, Yaghoub Fathollahi, Saeed Semnanian, Amir Shojaei, Javad Mirnajafi-Zadeh

Objective Low-frequency stimulation (LFS) exerts suppressive effects in kindled animals. It is believed that overstimulated glutamatergic and decreased GABAergic transmission have long been associated with seizure activity. In this study, we investigated the effect of electrical LFS on different parameters of spontaneous excitatory and inhibitory post-synaptic currents (sEPSCs and sIPSCs) in hippocampal CA1 pyramidal cells in kindled animals. Materials and Methods In this experimental study, rats were kindled by electrical stimulation of the hippocampal CA1 area in a semi-rapid manner (12 stimulations/day). The animals were considered fully kindled when they showed stage 5 seizures on three consecutive days. One group of animals received LFS 4 times at 30 seconds, 6 hours, 18 and 24 hours following the last kindling stimulation. Each LFS consisted of 4 packages at 5 minutes intervals. Each package of LFS consisted of 200 pulses at 1 Hz and each monophasic square wave pulse duration was 0.1 millisecond. At 2-3 hours post-LFS, acute hippocampal slices were prepared and a whole cell patch clamp recording was performed in all animals to measure the different parameters of sEPSCs and sIPSCs. Results In kindled animals, the inter-event interval (as an index of occurrence) of sEPSCs decreased, whereas sIPSC increased. In addition, the decay time constant of sIPSCs as an index of the duration of its activity decreased compared to the control group. There was no significant difference in other parameters between the kindled and control groups. Application of LFS in kindled animals prevented the observed changes. There was no significant difference between the measured parameters in kindled+LFS and control groups. Conclusion LFS application may prevent seizure-induced increase in the occurrence of sEPSCs and seizure-induced decrease in occurrence and activity duration of sIPSCs.

565-581

Authors: Kianoush Dormiani, Hamid Mir Mohammad Sadeghi, Hojjat Sadeghi-Aliabadi, Mahboobeh Forouzanfar, Hossein Baharvand, Kamran Ghaedi, Mohammad Hossein Nasr-Esfahani

Objective Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. Materials and Methods In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. Results In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. Conclusion According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

588-596

Authors: Marziyeh Tavalaee, Abbas Kiani-Esfahani, Mohammad Hossein Nasr-Esfahani

Objective The present study aimed to simultaneously evaluate the association between expression of three potential factors [post-acrosomal sheath WW domain-binding protein (PAWP), phospholipase Cζ (PLCζ), and truncated form of the kit receptor (TR-KIT)] as candidates of oocyte activation with fertilization rate and early embryonic development. Materials and Methods In this experimental study, semen samples were collected from 35 intra-cytoplasmic sperm injection (ICSI) candidates and analyzed according to World Health Organization criteria (2010). Each sample was divided into two parts. The first part was processed for insemination by density-gradient centrifugation (DGC) and the second part was prepared for assessment of sperm morphology (Papanicolaou staining), DNA fragmentation [transferase dUTP nick end labeling (TUNEL)], and three Sperm-borne oocyte-activating factor (s) (SOAFs)-PLCζ, PAWP, and TR-KIT. Results Significant positive correlations existed between the percentages of PLCζ, PAWP, and TR-KIT with fertilization rate. In addition, significant negative correlations existed between the percentage of DNA fragmentation with the percentages of PLCζ and PAWP. We did not find a relationship between percentages of PLCζ, PAWP, and TR-KIT with embryo quality and pregnancy rate (P>0.05). There was a significant negative correlation between percentage of DNA fragmentation with fertilization and embryo quality. Conclusion Oocyte activation was associated with the studied sperm factors (PAWP, PLCζ, and TR-KIT). These factors might hold the potential to be considered as diagnostic factors in the assessment of semen samples to evaluate their potential to induce oocyte activation. In addition, we observed a significant association between DNA fragmentation with fertilization, as well as embryo quality and expression of PAWP and PLCζ, which indicated that men with high degrees of DNA fragmentation might require artificial oocyte activation. Whether such action should take place, and its cost and benefits should be evaluated in the future.

609-618

Authors: Razieh Karamzadeh, Mohamadreza Baghaban Eslaminejad, Ali Sharifi-Zarchi

Objective Pulp and periodontal tissues are well-known sources of mesenchymal stem cells (MSCs) that provide a promising place in tissue engineering and regenerative medicine. The molecular mechanisms underlying commitment and differentiation of dental stem cells that originate from different dental tissues are not fully understood. In this study, we have compared the expression levels of pluripotency factors along with immunological and developmentally-related markers in the culture of human dental pulp stem cells (hDPSCs), human dental follicle stem cells (hDFSCs), and human embryonic stem cells (hESCs). Materials and Methods In this experimental study, isolated human dental stem cells were investigated using quantitative polymerase chain reaction (qPCR), immunostaining, and fluorescence-activated cell sorting (FACS). Additionally, we conducted gene ontology (GO) analysis of differentially expressed genes and compared them between dental stem cells and pluripotent stem cells. Results The results demonstrated that pluripotency (OCT4 and SOX2) and immunological (IL-6 and TLR4) factors had higher expressions in hDFSCs, with the exception of the JAGGED-1/NOTCH1 ratio, c-MYC and NESTIN which expressed more in hDPSCs. Immunostaining of OCT4, SOX2 and c-MYC showed cytoplasmic and nucleus localization in both groups at similar passages. GO analysis showed that the majority of hDFSCs and hDPSCs populations were in the synthesis (S) and mitosis (M) phases of the cell cycle, respectively. Conclusion This study showed different status of heterogeneous hDPSCs and hDFSCs in terms of stemness, differentiation fate, and cell cycle phases. Therefore, the different behaviors of dental stem cells should be considered based on clinical treatment variations.