7 research outputs found

    Isolation and Enrichment of Mouse Female Germ Line Stem Cells

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    Objective: The existence of female germ-line stem cells (FGSCs) has been the subject of a wide range of recent studies. Successful isolation and culture of FGSCs could facilitate studies on regenerative medicine and infertility treatments in the near future. Our aim in the present study was evaluation of the most commonly used techniques in enrichment of FGSCs and in establishment of the best procedure. Materials and Methods: In this experimental study, after digesting neonate ovary from C57BI/6 mice, we performed 2 different isolation experiments: magnetic activated cell sorting (MACS) and pre-plating. MACS was applied using two different antibodies against mouse vasa homolog (MVH) and stage-specific embryonic antigen-1 (SSEA1) markers. After the cells were passaged and proliferated in vitro, colony-forming cells were characterized using reverse transcription-polymerase chain reaction (RT-PCR) (for analysis of expression of Oct4, Nanog, C-kit, Fragilis, Mvh, Dazl, Scp3 and Zp3), alkaline phosphatase (AP) activity test and imnnunocytochemistry. Results: Data showed that colonies can be seen more frequently in pre-plating technique than that in MACS. Using the SSEA1 antibody with MACS, 1.98 +/- 0.49% (Mean +/- SDV) positive cells were yield as compared to the total cells sorted. The colonies formed after pre-plating expressed pluripotency and germ stem cell markers (Oct4, Nanog, C-kit, Fragilis, Mvh and Dazl) whereas did not express Zp3 and Scp3 at the mRNA level. Immunocytochemistry in these colonies further confirmed the presence of OCT4 and MVH proteins, and AP activity measured by AP-kit showed positive reaction. Conclusion: We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries

    Anticancer activity of ethanolic extract of propolis on AGS cell line

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    Introduction: Propolis is a natural product derived from various plant resins collected by honeybees, and has been used as a folk medicine for centuries. Propolis has been reported to exhibit a broad spectrum of activities including antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, hepatoprotective, and anticancer properties. The aim of this study was to investigate the effect of ethanolic extract of propolis (EEP) obtained from Dinaran area (Iran) on AGS human gastric cancer cell line. Methods: The ethanolic extract of samples was obtained by ethanol 96 and pure extract was dissolved in dimethyl sulfoxide (DMSO) and used for experiments. The cytotoxic effects of various concentrations of EEP on AGS cells were investigated by MTT assay test after 24, 48, and 72 hours and compared with control cells. Results: The EEP inhibited the growth and proliferation of AGS human gastric cancer cell line. The antiproliferative effects were revealed in a dose and time-dependent manner. The IC50 values were recorded as 60, 30, and 15 (&mu;g/ml) in treatment times of 24, 48 and 72 hours, respectively. Conclusion: These findings indicated that the native EEP has strong antiproliferative effects against cancerous AGS cells. Thus, propolis and related products may provide a novel approach to the chemoprevention and treatment of human gastric cancer.</p

    Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques

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    Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3-4) and late (10-15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other

    The assessment of lentiviral vectors application for gene transformation in human dermal fibroblasts (HDFs)

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    چکیده: زمینه و هدف: سلول های بنیادی پرتوان القایی Induced pluripotent stem cells= iPSc))، سلول های اولیه و تمایز نیافته ای هستند که قادر به ایجاد تقریباً هر نوع سلولی در بدن می باشند. هدف از پژوهش حاضر تولید و استفاده مؤثر از وکتور لنتی ویروسی TetO-FUW-OSKM جهت انتقال ژن ها به سلول های فیبروبلاست انسانی ((HDFs= Human fibroblast cells و در نهایت ارزیابی عملکرد این وکتور بود. روش بررسی: در این مطالعه تجربی پس از جداسازی و کشت سلول های HDF، وکتور لنتی ویروسی TetO-FUW-OSKM (به عنوان پلاسمید انتقال دهنده) حاوی ژن های برنامه ریزی مجدد همراه با پلاسمیدهای PsPAX2 و PMDG2 (به عنوان پلاسمیدهای کمکی لازم برای بسته بندی ویروس) به لاین سلولی HEK-293T جهت تولید ویروس ها ترانسفکت شدند. محیط رویی سلول های HEK-293T حاوی ویروس های تولید شده پس از 48 و 72 ساعت برداشت شد و این ویروس ها جهت برنامه ریزی مجدد سلول های HDF به این سلول ها ترانس داکت گردیدند. یافته ها: نتایج این مطالعه نشان دهنده تولید موفقیت آمیز وکتور لنتی ویروسی TetO-FUW-OSKM، کارایی مؤثر روش انتقال ژن با استفاده از وکتورهای لنتی ویروسی و بیان مناسب فاکتورهای رونویسی در سلول های HDF پس از ترانس داکشن بود. نتیجه گیری: با توجه به این یافته ها می توان از وکتورهای لنتی ویروسی جهت انتقال ژن و برنامه ریزی مجدد سلول های بالغ از قبیل HDF در مطالعات بعدی و تولید سلول های iPS استفاده کرد

    Mesenchymal stem cells versus their conditioned medium in the treatment of ischemia/reperfusion injury: Evaluation of efficacy and hepatic specific gene expression in mice

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    Objective(s): The mechanisms underlying the beneficial effects of MSCs on hepatic I/R injury are still poorly described, especially the changes in hepatocyte gene expression. In this study, the effect of bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (AMSCs) and their conditioned medium on hepatocyte gene expression resulted by I/R shock were investigated. Materials and Methods: Liver ischemia models were induced by clamping in experimental groups. Experimental groups received MSCs or conditioned medium treatments and the control group received Dulbecco's Modified Eagle Medium (DMEM). During 1, 24 hr, and 1 week after treatment, the serum levels of alanine aminotransferase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) enzymes and tissue catalase activity (CAT) were measured. Gene expression of a number of hepatocyte-specific genes (Alb, Afp, and Ck8) and Icam-1 which is upregulated under inflammatory conditions were also evaluated in 5, 24 hr, and 1-week intervals after I/R insult. Results: In this study, liver enzymes showed a much more shift in the control group than treated groups and it was more noticeable 5 hr post-treatment. Moreover, gene expression pattern of the control group underwent changes after I/R injury. However, treated groups gene expression analysis met a steady trend after I/R insult. Conclusion: Our finding shows that stem cell treatment has better curative effects than conditioned medium. BMSCs, AMSCs or BMSC and AMSC-derived bioactive molecules injection have potential to be considered as a therapeutic approach for treating acute liver injury

    In vitro effects of melatonin on colonization of neonate mouse spermatogonial stem cells

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    We have recently reported that antioxidant supplements enhance the efficacy of cryopreserved spermatogonial stem cells. Melatonin is considered a free radical scavenger which has direct and indirect antioxidant effects in in vitro and in vivo microenvironments. Due to the anti-apoptotic properties of melatonin, researchers have proposed that melatonin may improve the efficiency of spermatogonial stem cell (SSC) transplantation. However, the appropriate methodology which facilitates SSC proliferation remains to be determined. Identification of a proper propagation system is essential for the future application of SSCs in the field of infertility. The aim of the present study was to investigate the effects of melatonin on the colonization of SSCs. SSCs were isolated from the testes of three to six day old mice, and their purities were assessed by cytometry using Plzf antibody. Isolated testicular cells were cultured in the absence or presence of melatonin extract for two weeks. Suppression of differentiation and maintenance of spermatogonial stem cells was confirmed by alkaline phosphatase staining and immunocytochemistry using Plzf antibody. The number and diameter of the colonies of SSCs were assessed during the 7th and 14th days of culture, and the expression of Id4, Plzf, and C-kit were evaluated using real-time PCR at the end of the culture period. The survival rate of the cultured cells in the presence of melatonin was significantly higher than the control group. The number and diameter of colonies also increased in the cells treated with melatonin. The results of our study suggest that culture of SSCs with 100 μM melatonin supplementation can increase SSCs proliferation significantly

    Mouse oocyte vitrification: the effects of two methods on maturing germinal vesicle breakdown oocytes

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    Purpose Evaluation of viability and subsequent developmental ability of mouse germinal vesicle breakdown oocytes vitrified in conventional straws. Methods Oocytes with compact cumulus cells were cultured for 3 h in TCM199 medium GVBD and vitrified by two methods: the step-wise and single-step. After vitrification, the oocytes were thawed, and subjected to in vitro maturation and in vitro fertilization. Oocyte survival (postthaw) was assessed by morphological appearance and staining, using propidium iodide (PI)/Hoechst 33342. The oocyte maturation and fertilization rates were examined in vitro. Results In the single-step method the rates of post thaw survival, maturation to metaphase II and cleavage (2-cell embryos) were 58.68%, 56.41% and 38.63%, respectively. In the step-wise method, the corresponding rates were 81.75%, 68.59% and 51.80%, respectively. Conclusion Vitrification of mouse germinal vesicle breakdown oocytes by the step-wise method had the advantag
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