Isolation and Enrichment of Mouse Female Germ Line Stem Cells

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

Discrepancy between mRNA and Protein Expression of Neutrophil Gelatinase-Associated Lipocalin in Bronchial Epithelium Induced by Sulfur Mustard

Sulfur mustard (SM) is a potent vesicant that has been employed as a chemical weapon in various conflicts during the 20th century. More recently, mustard was used in the Iraq conflict against Iranian troops and civilians. At the present time there are more than 40.000 people suffering from pulmonary lesions special bronchiolitis obliterans (BOs) due to mustard gas. SM increases the endogenous production of reactive oxygen species (ROS). Neutrophil Gelatinase-associated Lipocalin 2 (Lcn2, NGAL) is a member of the lipocalin superfamily for which a variety of functions such as cellular protection against oxidative stress have been reported. Ten normal and Twenty SM-induced COPD patient individuals were studied. Assessment of NGAL expressions in healthy and the patients endobrinchial biopsies were performed by semiquantitative RT-PCR, real-time RT-PCR, and Immunohistochemistry analysis. While Normal control samples expressed same level of mRNA NGAL, expression level of mRNA-NGAL was upregulated about 1.4- to 9.8-folds compared to normal samples. No significant immunoreactivity was revealed in both samples. As we are aware this is the first report of induction of NGAL in patients exposed to SM. NGAL may play an important role in cellular protection against oxidative stress toxicity induced by mustard gas in airway wall of patients

HO1 mRNA and Protein do not Change in Parallel in Bronchial Biopsies of Patients After Long Term Exposure to Sulfur Mustard

Sulfur mustard (SM), is an alkylating agent and has been emerged as a chemical weapon in various battlefields. More recently, SM was employed in the Iraq conflict against Iranian military forces and civilians. Nowadays there are more than 40,000 people suffering from pulmonary lesions special chronic obstructive pulmonary disease (COPD) due to mustard gas in Iran. SM causes the endogenous production of reactive oxygen species (ROS)

High-levelexpression of functional recombinant human coagulation factor VII in insect cells

Abstract: Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future. Keywords: Baculovirus; γ-carboxylase; Coagulation FVII; Factor VII; Insect cel

NF-E2-related factor 2 over-expression in mesenchymal stem cells to improve cellular cardiomyoplasty

Myocardial infarction (MI) is the leading cause of death worldwide. Various therapeutic strategies have been introduced for MI treatment. In recent years, interest in utilizing mesenchymal stem cells (MSCs) for MI therapy has increased. In fact, the use of MSCs for MI treatment, known as cellular cardiomyoplasty, is in the clinical trial stage. However, despite promising results, most MSCs die after transplantation as a result of exposure to various stresses. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a well-known cytoprotective transcription factor, protects MSCs against some stresses. Over-expression of Nrf2 in MSCs decreases their apoptosis in vitro without any adverse effects on their differentiation ca

Artificial Blood Substitutes: First Steps on the Long Route to Clinical Utility

The 21st century is challenging for human beings. Increased population growth, population aging, generation of new infectious agents, and natural disasters are some threatening factors for the current state of blood transfusion. However, it seems that science and technology not only could overcome these challenges but also would turn many human dreams to reality in this regard. Scientists believe that one of the future evolutionary innovations could be artificial blood substitutes that might pave the way to a new era in transfusion medicine. In this review, recent status and progresses in artificial blood substitutes, focusing on red blood cells substitutes, are summarized. In addition, steps taken toward the development of artificial blood technology and some of their promises and hurdles will be highlighted. However, it must be noted that artificial blood is still at the preliminary stages of development, and to fulfill this dream, ie, to routinely transfuse artificial blood into human vessels, we still have to strengthen our knowledge and be patient