Cell Journal (Yakhteh)

392-405

Authors: Nahid Nasiri, Poopak Eftekhari-Yazdi

Assessment of embryo quality in order to choose the embryos that most likely result in pregnancy is the critical goal in assisted reproductive technologies (ART). The current trend in human in vitro fertilization/embryo transfer (IVF/ET) protocols is to decrease the rate of multiple pregnancies after multiple embryo transfer with maintaining the pregnancy rate at admissible levels (according to laboratory standards). Assessment of morphological feathers as a reliable non-invasive method that provides valuable information in prediction of IVF/intra cytoplasmic sperm injection (ICSI) outcome has been frequently proposed in recent years. This article describes the current status of morphological embryo evaluation at different pre-implantation stages.

416-425

Authors: Sareh Asadi, Samaneh Dehghan, Maryam Hajikaram, Seyed Javad Mowla, Abolhassan Ahmadiani, Mohammad Javan

Objective: Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid (VPA) are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. Materials and Methods: In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction (PCR) was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell (NSC) markers. Results: Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog (p<0.05). Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc (p<0.001). VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc (p<0.01), Pax6 and Sox1 (p<0.001). Conclusion: These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4.

440-447

Authors: Qin Yu, Lizhen Liu, Jie Lin, Yan Wang, Xiaobo Xuan, Ying Guo, Shaojun Hu

Objective: Transplantation of mesenchymal stem cells (MSCs) can promote functional recovery of the brain after hypoxic-ischemic brain damage (HIBD). However, the mechanism regulating MSC migration to a hypoxic-ischemic lesion is poorly understood. Interaction between stromal cell-derived factor-1α (SDF-1α) and its cognate receptor CXC chemokine receptor 4 (CXCR4) is crucial for homing and migration of multiple stem cell types. In this study, we investigate the potential role of SDF-1α/CXCR4 axis in mediating MSC migration in an HIBD model. Materials and Methods: In this experimental study, we first established the animal model of HIBD using the neonatal rat. Bone marrow MSCs were cultured and labeled with 5-bromo-21-deoxyuridine (BrdU) after which 6×106 cells were intravenously injected into the rat. BrdU positive MSCs in the hippocampus were detected by immunohistochemical analyses. The expression of hypoxia-inducible factor-1α (HIF-1α) and SDF-1α in the hippocampus of hypoxic-ischemic rats was detected by Western blotting. To investigate the role of hypoxia and SDF-1α on migration of MSCs in vitro, MSCs isolated from normal rats were cultured in a hypoxic environment (PO2=1%). Migration of MSCs was detected by the transwell assay. The expression of CXCR4 was tested using Western blotting and flow cytometry. Results: BrdU-labeled MSCs were found in the rat brain, which suggested that transplanted MSCs migrated to the site of the hypoxic-ischemic brain tissue. HIF-1α and SDF- 1α significantly increased in the hippocampal formations of HIBD rats in a time-dependent manner. They peaked on day 7 and were stably expressed until day 21. Migration of MSCs in vitro was promoted by SDF-1α under hypoxia and inhibited by the CXCR4 inhibitor AMD3100. The expression of CXCR4 on MSCs was elevated by hypoxia stimulation as well as microdosage treatment of SDF-1α. Conclusion: This observation illustrates that SDF-1α/CXCR4 axis mediate the migration of MSCs to a hypoxic-ischemic brain lesion in a rat model.

456-465

Authors: Faezeh Alipour, Abbas Parham, Hossein Kazemi Mehrjerdi, Hesam Dehghani

Objective: Because of the therapeutic application of stem cells (SCs), isolation and characterization of different types of SCs, especially mesenchymal stem cells (MSCs), have gained considerable attention in recent studies. Adipose tissue is an abundant and accessible source of MSCs which can be used for tissue engineering and in particular for treatment of musculoskeletal disorders. This study was aimed to isolate and culture equine adipose-derived MSCs (AT-MSCs) from little amounts of fat tissue samples and determine some of their biological characteristics. Materials and Methods: In this descriptive study, only 3-5 grams of fat tissue were collected from three crossbred mares. Immediately, cells were isolated by mechanical means and enzymatic digestion and were cultured in optimized conditions until passage 3 (P3). The cells at P3 were evaluated for proliferative capacities, expression of specific markers, and osteogenic, chondrogenic and adipogenic differentiation potentials. Results: Results showed that the isolated cells were plastic adherent with a fibroblast-like phenotype. AT-MSCs exhibited expression of mesenchymal cluster of differentiation (CD) markers (CD29, CD44 and CD90) and not major histocompatibility complex II (MHC-II) and CD34 (hematopoietic marker). Cellular differentiation assays demonstrated the chondrogenic, adipogenic and osteogenic potential of the isolated cells. Conclusion: Taken together, our findings reveal that equine MSCs can be obtained easily from little amounts of fat tissue which can be used in the future for regenerative purposes in veterinary medicine.

476-487

Authors: Ehsan Taghiabad, Sima Nasri, Saeed Shafieyan, Sasan Jalili Firoozinezhad, Nasser Aghdami

Objective: As a biological tissue material, amniotic membrane (AM) has low immunogenicity and to date has been widely adopted in clinical practice. However, some features such as low biomechanical consistency and rapid biodegradation is limited the application of AM. Therefore, in this study, we fabricated a novel three-dimensional (3D) spongy scaffold made of the extracellular matrix (ECM) of denuded AM. Due to their unique characteristics which are similar to the skin, these scaffolds can be considered as an alternative option in skin tissue engineering. Materials and Methods: In this experimental study, cellular components of human amniotic membrane (HAM) were removed with 0.03% (w/v) sodium dodecyl sulphate (SDS). Quantitative analysis was performed to determine levels of Glycosaminoglycans (GAGs), collagen, and deoxyribonucleic acid (DNA). To increase the low efficiency and purity of the ECM component, especially collagen and GAG, we applied an acid solubilization procedure hydrochloridric acid (HCl 0.1 M) with pepsin (1 mg/ml). In the present experiment 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross linker agent was used to improve the mechanical properties of 3D lyophilized AM scaffold. The spongy 3D AM scaffolds were specified, by scanning electron microscopy, hematoxylin and eosin (H&E) staining, a swelling test, and mechanical strength and in vitro biodegradation tests. Human fetal fibroblast culture systems were used to establish that the scafolds were cytocompatible. Results: Histological analysis of treated human AM showed impressive removal of cellular components. DNA content was diminished after treatment (39 ± 4.06 μg/ml vs. 341 ± 29.60 μg/ml). Differences were observed between cellular and denude AM in matrix collagen (478 ± 18.06 μg/mg vs. 361 ± 27.47 μg/mg).With the optimum concentration of 1 mM NHS/EDC ratio1:4, chemical cross-linker agent could significantly increase the mechanical property, and resistance to collagenase digestion. The results of 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) test showed that cross-linking efficiency of AM derived ECM scaffolds was about 65% ± 10.53. Scaffolds treated with NHS/EDC cross-linker agent by 100 μg/ml collagenase, lost 75% of their dry weight after 14 days. The average pore size of 3D spongy scaffold was 160 μm measured from scanning electron microscope (SEM) images that it is suitable for cell penetration, nutrients and gas change. In addition, the NHS/ EDC cross-linked AM scaffolds were able to support human fetal fibroblast cell proliferation in vitro. Extracts and contact prepared from the 3D spongy scaffold of AM showed a significant increase in the attachment and proliferation of the human fetal fibroblasts cells. Conclusion: The extra-cellular matrix of denuded AM-based scaffold displays the main properties required for substitute skin including natural in vitro biodegradation, similar physical and mechanical characterization, nontoxic biomaterial and no toxic effect on cell attachment and cell proliferation.

494-505

Authors: Mahmoud-Reza Rafiee,Afsaneh Malekzadeh Shafaroudi,Sara Rohban, Hamid Khayatzadeh,Hamid Reza Kalhor,Seyed Javad Mowla

Objective: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. Materials and Methods: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. Results: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. Conclusion: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues.

514-527

Authors: Massoud Seifi,Ali Arayesh,Nafise Shamloo,Roya Hamedi

Objective: Orthodontically induced inflammatory root resorption (OIIRR) is considered to be an important sequel associated with orthodontic tooth movement (OTM). OTM after Socket preservation enhances the periodontal condition before orthodontic space closure. The purpose of this study is to investigate the histologic effects of NanoBone®, a new highly nonsintered porous nano-crystalline hydroxyapatite bone on root resorption following OTM. Materials and Methods: This experimental study was conducted on four male dogs. In each dog, four defects were created at the mesial aspects of the maxillary and mandibular first premolars. The defects were filled with NanoBone®. We used the NiTi closed coil for mesial movement of the first premolar tooth. When the experimental teeth moved approximately halfway into the defects, after two months, the animals were sacrificed and we harvested the area of interest. The first premolar root and adjacent tissues were histologically evaluated. The three-way ANOVA statistical test was used for comparison. Results: The mean root resorption in the synthetic bone substitute group was 22.87 ± 11.25×10-4 mm2 in the maxilla and 21.41 ± 11.25×10-4 mm2 in the mandible. Statistically, there was no significant difference compared to the control group (p>0.05). Conclusion: The use of a substitution graft in the nano particle has some positive effects in accessing healthy periodontal tissue following orthodontic procedures without significant influence on root resorption (RR). Histological evaluation in the present study showed osteoblastic activity and remodeling environment of nanoparticles in NanoBone®.

538-545

Authors: Elham Amirchaghmaghi,Abbas Rezaei,Ashraf Moini,Mohammad Ali Roghaei,Maryam Hafezi,Reza Aflatoonian

Objective: Unexplained recurrent spontaneous abortion (URSA) is one of the main complications of pregnancy which is usually defined as three or more consecutive pregnancy losses before the 20th week of gestation without a known cause. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and shown, along with its receptors (VEGFR1, 2), to play important roles in several physiologic processes including reproduction. The aim of the present study was to analyze gene expression of VEGF and VEGF receptors in endometrium of patients with a history of URSA compared with normal fertile women. In addition, serum VEGF concentration was assessed and compared between the two groups at the same time. Materials and Methods: In this case control study, endometrial and blood samples were obtained between day 19th and 24th of menstrual cycle (window of implantation) from 10 women with a history of URSA (case group) and 6 fertile women who had at least one successful pregnancy (control group). Expression of VEGF and VEGFRs was studied by reverse transcription- polymerase chain reaction (RT-PCR) and then quantified by real time PCR. Normalization of expression levels was done by comparison with beta-actin expression level as an internal control. Relative VEGF, VEGFR1 and VEGFR2 expression quantities were compared between the two groups. Enzyme linked immunosorbent assay (ELISA) was used for serum VEGF assay. Results: VEGF, VEGFR1 and VEGFR2 gene expression was detected in endometrial samples of both groups. The mean relative expression of VEGF gene was lower in the case group compared with control women, however, both VEGF receptors were expressed higher in endometrium of the case group. In addition, the serum level of VEGF was significantly higher in the case group compared with the controls. Conclusion: Alteration in gene expression of VEGF and its receptors in endometrium and changes of serum VEGF might play important roles in pathogenesis of unexplained RSA.

554-559

Authors: Issa Layali,Eisa Tahmasbpour,Manijeh Joulaei,Seyed Gholam Ali Jorsaraei,Parvin Farzanegi

Semen hyperviscosity (SHV) is one of the factors involved in deficiency in sperm function. This research aimed to evaluate seminal plasma total antioxidant capacity (TAC) and malondialdehyde (MDA) levels in infertile patients with hyperviscous and non-hyperviscous semen samples to understand whether hyperviscous semen is associated with oxidative damage in infertile subjects. In this cross sectional study, 59 semen samples were provided by fertile (n=12) individuals as control, infertile patients with normal viscosity (n=25) and infertile patients with hyperviscosity (n=22). After semen parameters examination, semen viscosity was studied by glass pipettes. Seminal plasma TAC and MDA levels were measured by ferric reducing of antioxidant power (FRAP) and thiobarbituric acid reaction (TBAR) methods, respectively. A probability less than 0.05 was considered statistically significant throughout the article. The mean of sperm parameters including: counts, motility and normal morphology in patients with hyperviscosity were significantly lower than those in non-hyperviscosity patients (p<0.05, p<0.01 and p<0.001, respectively). The mean of seminal plasma TAC value in seminal plasma of non-hyperviscosity patients (1710.31 ± 458.67 μmol/l) was significantly (p<0.01) higher than that of hyperviscosity group (1230.25 ± 352 μmol/l). A trend toward a higher mean of seminal plasma MDA value was estimated for hyperviscous group compared with non-hyperviscous (1.01 ± 0.41 nmol/ml vs. 0.94 ± 0.28 nmol/l); however, it was nonsignificant. Hyperviscous semen impairs seminal plasma TAC which is eventually associated with sperm membrane lipid peroxidation.

564-567

Authors: Muhammad Irfan-Maqsood

Chromosome-centric human proteome project (C-HPP) is a recent initiative to rationalize and analyze gene-protein and protein-protein interactions in normal and disease conditions. This initiative is aimed to generate the proteomic atlas explaining the molecular architecture of the human body and was initiated in response to the hurdles identified during analyzing the human genome project (HGP). A need for the experimental observation of translated proteins was felt to analyze precisely what is going on in the cell. 25 countries around the world are participating in the C-HPP. This symposium report will introduce the Y-chromosome HPP which is undergoing in Iran by eminent molecular biologists of Royan Institute, Tehran and its collaborates.