Cell Journal (Yakhteh)

133-139

Authors: Salahadin Bahrami, Amir Amiri-Yekta, Abbas Daneshipour, Seyedeh Hoda Jazayeri, Paul Mozdziak, Mohammad Hossein Sanati, Hamid Gourabi

Specific developmental characteristics of the chicken make it an attractive model for the generation of transgenic organisms. Chicken possess a strong potential for recombinant protein production and can be used as a powerful bioreactor to produce pharmaceutical and nutritional proteins. Several transgenic chickens have been generated during the last two decades via viral and non-viral transfection. Culturing chicken primordial germ cells (PGCs) and their ability for germline transmission ushered in a new stage in this regard. With the advent of CRISPR/Cas9 system, a new phase of studies for manipulating genomes has begun. It is feasible to integrate a desired gene in a predetermined position of the genome using CRISPR/Cas9 system. In this review, we discuss the new approaches and technologies that can be applied to generate a transgenic chicken with regards to recombinant protein productions.

149-157

Authors: Mohammad Salehi, Beheshteh Abouhamzeh, Ahmad Hosseini, Zohreh Zare, Azizollah Bakhtari

Objective Regarding that undifferentiated mesenchymal stem cells, as donor cells, require less epigenetic reprogramming, possibility of using bovine adipose tissue-derived stem cells (BASCs) with low level of DNMTs and HDACs expression was evaluated. Materials And Methods In this experimental study, we examined gene expression of epigenetic modifiers including DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) and histone deacetylases (HDAC1-3), as well as protein levels of histone H3 acetylation at lysine 9 (H3K9ac) and POU5F1 (also known as OCT4) at two stages of preimplantation development among in vitro fertilization (IVF), parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) groups. Results The results revealed that developmental competence of IVF embryos was higher than SCNT embryos (P<0.05). In the PA and SCNT groups, DNMT1, HDAC2 and HDAC3 mRNA were overexpressed (P<0.05), and proteins levels of H3K9ac and POU5F1 were reduced at 6-8 cells and blastocyst stages compared to IVF (P<0.05). The mRNA expression of DNMT1 an<0.05) in both developmental stages (except HDAC1 in blastocyst stage). Conclusion The SCNT embryos derived from BASCs have endured considerable nuclear reprogramming during early embryo development. Comparison of PA and SCNT blastocysts demonstrated that HDAC1 and DNMT1 may attribute to developmental competence variability of bovine embryos.

165-170

Authors: Afshin Moradi, Mahdis Rahimi Naiini, Nasrin Yazdanpanahi, Hossein Tabatabaeian, Fariba Nabatchian, Masoud Baghi, Mansoureh Azadeh, Kamran Ghaedi

Objective Multiple sclerosis (MS) is a chronic disorder involving both inflammatory and neurodegenerative responses. Long non-coding RNAs (lncRNAs) have been had an emerging role as the biomarkers of different disorders, including autoimmune diseases. Previous studies have shown that NR_003531.3 (MEG3a), AC000061.1_201, and AC007182.6 play a role in the pathogenesis of human autoimmune diseases. However, the potential significance of these lncRNAs, as the diagnostic biomarkers of MS, has not been studied yet. We aimed to quantitatively evaluate the expression levels of NR_003531.3, AC000061.1_201, and AC007182.6 in peripheral blood samples of MS patients in comparison with healthy controls. Materials And Methods In this case-control study, the blood samples from 20 MS patients and 10 healthy controls were collected. Total RNA was extracted, and the expression levels of three selected lncRNAs were quantitatively measured using the quantitative real time-polymerase chain reaction (qRT-PCR) method. Results We detected a significant down-regulation in the expression of NR_003531.3 in MS patients, while no marked changes were observed in the expression of AC000061.1_201 and AC007182.6 in patients compared with controls. Based on the receiver operating characteristic (ROC) curve analysis, NR_003531.3 could discriminate MS patients from healthy subjects effectively. Regarding the prognosis of MS patients, NR_003531.3 is significantly and inversely correlated with the expanded disability status scale (EDSS). Conclusion The potential role of NR_003531.3 lncRNA as a diagnostic biomarker to distinguish MS patients is proposed. Prognostically, NR_003531.3 correlates with lower disability rates in MS patients.

178-184

Authors: Asghar Arshi, Farzane Raeisi, Esmaeil Mahmoudi, Fatemeh Mohajerani, Hamidreza Kabiri, Razieh Fazel, Maedeh Zabihian Langroudi, Amela Jusic

Objective Recent data suggest that increased levels of the HOTAIR long non-coding RNA (lncRNA) are involved in the development of various types of malignancy, including breast cancer. The aim of present study was to investigate HOTAIR lncRNA expression profile in breast cancer (BC) patients and cell lines. Materials And Methods In this experimental study, expression level of HOTAIR lncRNA was evaluated in BC and normal tissues of 15 patients as well as MDA-MB-231, MCF-7 and MCF-10A cell lines, using quantitative reverse- transcription polymerase chain reaction (qRT-PCR). HOTAIR lncRNA expression levels were estimated using 2-ΔΔCt method. Further, receiver operating characteristic (ROC) curve analysis was done to evaluate the selected lncRNA diagnostic potential. The Cox’s proportional hazards regression model was performed to evaluate the predictive value of this lncRNA level in BC patients. Results The results of present study demonstrated no significant difference in the expression of HOTAIR lncRNA in MCF7 and MDA-MB-231 cancer cell lines compared to MCF-10A as normal cell line (P>0.05). However, we observed a significantly increase in the expression of HOTAIR in BC patients compared to normal tissues (P<0.001). Significant associations were found between gene expression and tumour size and margin. We found 91.1% sensitivity and 95.7% specificity of circulating HOTAIR with an area under the ROC curve of 0.969. The Kaplan-Meier analysis indicated significant correlation between HOTAIR expression and overall survival. Conclusion This study demonstrated that expression of HOTAIR is increased in BC and might be associated with its progression. According to these findings, HOTAIR expression could be proposed as biomarkers for BC early diagnosis and prognosis.

193-202

Authors: Mahnaz Mohammadi Kian, Atousa Haghi, Mahdieh Salami, Bahram Chahardouli, Saharbano Rostami, Kianoosh Malekzadeh, Hosein Kamranzadeh Foumani, Saeed Mohammadi, Mohsen Nikbakht

Objective Autophagy and apoptosis play key roles in cancer survival and pathogenesis and are governed by specific genes which have a dual role in both cell death and survival. Arsenic trioxide (ATO) and thalidomide (THAL) are used for treatment of many types of hematologic malignancies. ATO prevents the proliferation of cells and induces apoptosis in some cancer cells. Moreover, THAL has immunomodulatory and antiangiogenic effects in malignant cells. The aim of present study was to examine the effects of ATO and THAL on U937 and KG-1 cells, and evaluation of mRNA expression level of VEGFs genes, PI3K genes and some of autophagy genes. Materials And Methods In this in vitro experimental study, U937 and KG-1 cells were treated by ATO (0.4-5 µM) and THAL (5-100 µM) for 24, 48 and 72 hours. Cell viability was measured by MTT assay. The apoptosis rate and cell cycle arrest were evaluated by flow cytometry (Annexin/PI) and cell cycle flow cytometry analysis, respectively. The effect of ATO/THAL on mRNAs expression was evaluated by real-time polymerase chain reaction (PCR). Results ATO/THAL combination enhanced cell apoptosis in a dose-dependent manner. Also, ATO/THAL induced SubG1/ G1 phase arrest. mRNA expression levels of VEGFC (contrary to other VEGFs isoform), PI3K, AKT, mTOR, MEK1, PTEN, IL6, LC3 and P62 genes were upregulated in acute myeloid leukemia (AML) cells following treatment with ATO/THAL. Conclusion Combined treatment with ATO and THAL can inhibit proliferation and invasion of AML cells by down-regulating ULK1 and BECLIN1 and up-regulating PTEN and IL6, and this effect was more marked than the effects of ATO and THAL alone.

212-217

Authors: Vahid Baghdadi, Fatemeh Yari, Mahin Nikougoftar Zarif, Mohammad Hessam Rafiee

Objective Platelet (PLT) storage at 4˚C has several benefits, however, it is accompanied by increased clearance of PLTs after transfusion. In this study, we evaluated the potential of sodium octanoate (SO) for reducing apoptosis and clearance rate of PLTs after long-term storage in cold. Materials And Methods In this experimental study, PLT concentrates (PCs) were stored for 5 days under the following three conditions: 20-24˚C, 4˚C, and 4˚C in the presence of SO. To measure the viability of PLTs, the water-soluble tetrazolium salt (WST-1) assay was performed. Phosphatidylserine (PS) exposure was determined on PLTs using flow cytometry technique. The amount of human active caspase-3 was determined in PLTs using an enzyme-linked immunosorbent assay. Additionally, the amount of PLT ingestion or clearance was determined by using HepG2 cell line. Results The viability was higher in the SO-treated PLTs compared to the other groups. The level of PS exposure on PLTs was lower in the SO-treated PLTs compared to the other groups. The amount of active caspase-3 increased in all groups during 5-day storage. The highest increase in the amount of caspase-3 levels was observed at cold temperature. However, PLTs kept at 4˚C in the presence of SO had a lower amount of active caspase-3 compared to PLTs kept at 4˚C. The amount of PLTs removal by HepG2 cells was increased for 4˚C-kept PLTs but it was lower for PLTs kept at 4˚C in the presence of SO but, the differences were not significant (P>0.05). Conclusion SO could partially moderate the effects of cold temperature on apoptosis and viability of platelets. It also decreases the ingestion rate of long-time refrigerated PLTs in vitro. Further studies using higher numbers of samples are required to demonstrate the effect of SO on reducing the clearance rate of PLTs.

227-235

Authors: Leila Mirzaeian, Farideh Eivazkhani, Maryam Hezavehei, Ashraf Moini, Fereshteh Esfandiari, Mojtaba Rezazadeh Valojerdi, Rouhollah Fathi

ABSTRACT Objective Decellularized tissue scaffolds provide an extracellular matrix to control stem cells differentiation toward specific lineages. The application of mesenchymal stem cells for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells. Thus, the development of ovarian engineered constructs (OVECs) requires the use of efficient methods for seeding of the cells into the ovarian and other types of scaffolds. The main goal of the present study was to develop an optimized culture system for efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold. Materials And Methods In this experimental study, three methods were used for cellular seeding including rotational (spinner flask) and static (conventional and injection) seeding cultures. OVECs were evaluated with Hematoxylin and Eosin staining and viability analyses for the seeded PMSCs. Then, immunohistochemistry analysis was performed using the best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Stereology analysis was also performed for the number of penetrated cells into the OVECs. Results Our results showed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs, comparing to the other methods. On the other hand, rotationally seeded PMSCs had a more favorable capability of proliferation with Ki67 expression and differentiation to ovarian specific cells with expression of primordial germ cell line markers without mesenchymal stem cells markers production. Furthermore, stereology showed a more favorable distribution of PMSCs along the outer surfaces of the OVEC with further distribution at the central part of the scaffold. The average total cell values were determined 2142187 cells/mm3 on each OVEC. Conclusion The rotational seeding method is a more favorable approach to cell seeding into ovarian decellularized tissue than static seeding.

245-252

Authors: Mohammad Hossein Mohammadi Mahdi Abadi Hassani, Mohammad Nabiuni, Kazem Parivar, Siamak Yari, Alireza Sahebi, Jaleel A. Miyan

Objective The embryonic cerebrospinal fluid (e-CSF) contains various growth factors and morphogens. Recent studies showed that e-CSF plays significant roles in embryonic brain development. Adipose tissue-derived stem cells (ADSCs) have a mesodermal origin that can be differentiated into mesodermal and ectodermal lineages. This study aimed to evaluate the effects of e-CSF on the proliferation, viability, and neural differentiation of ADSCs in rats. Materials And Methods In this experimental study, adipose tissue was dissected out from the inguinal region of adult male rats. Then, ADSCs were isolated by enzymatic digestion from adipose tissues and mesenchymal cells were confirmed using the flow cytometry analysis that measured the cell surface markers including CD90, CD44, CD73, CD105, CD34, CD45, and CD11b. The multi-potential characteristics of ADSCs were assessed by osteogenic and adipogenic potentials of these cells. Under suitable in vitro conditions, ADSCs were cultured in DMEM supplemented with and without additional 10% e-CSF. These fluids were collected from Wistar rats at the E17, E18, and E19 gestational ages. Cellular proliferation and viability were determined using the MTT assay. Immunocytochemistry was used to study the expression of β-III tubulin in ADSCs. The neurite outgrowth of cultured cells was assessed using the ImageJ software. Results The results of the present study demonstrated that the viability of ADSCs in cell culture conditioned with E17 and E18 e-CSF were significantly increased in comparison with controls. Cultured cells treated with e-CSF from E18 and E19 established neuronal-like cells bearing long process, whereas no process was observed in the control groups or cultured cells treated with E17 e-CSF. Conclusion This study showed that e-CSF has the ability to induce neuronal differentiation and viability in ADSCs. Our data support a significant role of e-CSF as a therapeutic strategy for the treatment of neurodegenerative diseases.