Autophagy-modulated human bone marrow-derived mesenchymal stem cells accelerate liver restoration in mouse models of acute liver failure

Background: Mesenchymal stem cells [MSCs] have been recently received increasing attention for cell-based therapy, especially in regenerative medicine. However, the low survival rate of these cells restricts their therapeutic applications. It is hypothesized that autophagy might play an important role in cellular homeostasis and survival. This study aims to investigate the regenerative potentials of autophagy-modulated MSCs for the treatment of acute liver failure [ALF] in mice

Methods: ALF was induced in mice by intraperitoneal injection of 1.5 ml/kg carbon tetrachloride. Mice were intravenously infused with MSCs, which were suppressed in their autophagy pathway. Blood and liver samples were collected at different intervals [24, 48 and 72 h] after the transplantation of MSCs. Both the liver enzymes and tissue necrosis levels were evaluated using biochemical and histopathological assessments. The survival rate of the transplanted mice was also recorded during one week

Results: Biochemical and pathological results indicated that 1.5 ml/kg carbon tetrachloride induces ALF in mice. A significant reduction of liver enzymes and necrosis score were observed in autophagy-modulated MSC-transplanted mice compared to sham [with no cell therapy] after 24 h. After 72 h, liver enzymes reached their normal levels in mice transplanted with autophagy-suppressed MSCs. Interestingly, normal histology without necrosis was also observed

Conclusion: Autophagy suppression in MSCs ameliorates their liver regeneration potentials due to paracrine effects and might be suggested as a new strategy for the improvement of cell therapy in ALF

interaction between Sertoli cells and leukemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro

Background: Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells [SSCs] self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility

Objective: This study investigated the role of luekemia inhibitory factor [LIF] on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells

Materials and Methods: SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction

Results: Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression [p< 0.05]

Conclusion: Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment

Isolation and enrichment of mouse female germ line stem cells

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. In this experimental study, after digesting neonate ovary from C57Bl/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 immunocytochemistry. 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. We established a simple and an efficient pre-plating technique to culture and to enrich FGSCs from neonatal mouse ovaries

Down-regulation of the autophagy gene, ATG7, protects bone marrow-derived mesenchymal stem cells from stressful conditions

BACKGROUND: Mesenchymal stem cells (MSCs) are valuable for cell-based therapy. However, their application is limited owing to their low survival rate when exposed to stressful conditions. Autophagy, the process by which cells recycle the cytoplasm and dispose of defective organelles, is activated by stress stimuli to adapt, tolerate adverse conditions, or trigger the apoptotic machinery. This study aimed to determine whether regulation of autophagy would affect the survival of MSCs under stress conditions. METHODS: Autophagy was induced in bone marrow-derived MSCs (BM-MSCs) by rapamycin, and was inhibited via shRNA-mediated knockdown of the autophagy specific gene, ATG7. ATG7 expression in BM-MSCs was evaluated by reverse transcription polymerase chain reaction (RT-PCR), western blot, and quantitative PCR (qPCR). Cells were then exposed to harsh microenvironments, and a water-soluble tetrazolium salt (WST)-1 assay was performed to determine the cytotoxic effects of the stressful conditions on cells. RESULTS: Of 4 specific ATG7-inhibitor clones analyzed, only shRNA clone 3 decreased ATG7 expression. Under normal conditions, the induction of autophagy slightly increased the viability of MSCs while autophagy inhibition decreased their viability. However, under stressful conditions such as hypoxia, serum deprivation, and oxidative stress, the induction of autophagy resulted in cell death, while its inhibition potentiated MSCs to withstand the stress conditions. The viability of autophagy-suppressed MSCs was significantly higher than that of relevant controls (P<0.05, P<0.01 and P<0.001). CONCLUSION: Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.

MS 14 down-regulates lipocalin2 expression in spinal cord tissue in an animal model of multiple sclerosis in female C57BL/6

Experimental autoimmune encephalomyelitis [EAE] is an animal model of multiple sclerosis, which is a demyelinating and an inflammatory disease of central nervous system. Recent studies have established that some molecules such as Lipocaline2 [LCN2], which expresses during inflammatory conditions, play an important role in EAE pathogenesis and might involve in its treatment process. Recently, it has been proved that MS 14, an herbal-marine drug, has anti-inflammatory properties through reduction of TNF-a and IL-lp. Thus, the present study investigated the effects of MS 14 on the course of EAE and its relation to LCN2 expression in both protein and gene levels. EAE was induced in female C57BL/6 mice using Hooke kits. Animals were scored for clinical signs of the disease according to a 10-point EAE scoring system. On 21[st] and 35[th] days after immunization, mice [n = 4/group] were deeply anesthetized, and the spinal cords were removed. Inflammatory cell infiltration and LCN2 expression in spinal cord were assessed by hematoxylin and eosin staining, immuno-histochemistry, and real-time PCR methods. MS 14 significantly ameliorated EAE symptoms and decreased lymphocyte infiltration into the spinal cord [P<0.05]. Our data also revealed that LCN2 expression was significantly down-regulated in acute and chronic phases of EAE both at protein and gene levels after MS 14 treatment [P<0.05]. The results demonstrated that MS 14 regulatory effect on EAE is accompanied by LCN2 down-regulation after treatment with the herb; however, more studies are required for clarifying the other involved mechanisms

[Comparison of three cord blood processing methods: hydroxyethyl starch, simple centrifugation and Sepax automated system]

Background: Different processing methods are being used to improve the quality of hematopoietic stem cell transplantation. Using hydroxyethyl starch, simple centrifugation and Sepax automation, this study was aimed to compare these three conventional methods.

Material and Methods: 90 cord blood samples were taken and processed by hydroxyethyl starch, simple centrifugation and Sepax automation methods. Then they were subjected to total nucleated cell [TNC] counting and CD34 positive counting as well as colony assay. Finally, all data were analyzed using one-way analysis of variance [ANOVA] and ps less than 0.05 were considered statistically significant.

Results: The TNC recoveries in hydroxyethyl starch, simple centrifugation and Sepax automation methods were 76%, 71% and 80%, respectively [p> 0.05]. The CD34+ cell recoveries in the Sepax automation and in the other two methods were 91% and 85%, respectively [p> 0.05]. Also, the colony assay recoveries were not significantly different among the three methods [p> 0.05].

Conclusion: No significant difference was seen in TNC number, CD34 positive counting and colony formation among the three different methods.

Role of somatic testicular cells during mouse spermatogenesis in three-dimensional collagen gel culture system

Spermatogonial stem cells [SSCs] are the only cell type that can restore fertility to an infertile recipient following transplantation. Much effort has been made to develop a protocol for differentiating isolated SSCs in vitro. Recently, three-dimensional [3D] culture system has been introduced as an appropriate microenvironment for clonal expansion and differentiation of SSCs. This system provides structural support and multiple options for several manipulation such as addition of different cells. Somatic cells have a critical role in stimulating spermatogenesis. They provide complex cell to cell interaction, transport proteins and produce enzymes and regulatory factors. This study aimed to optimize the culture condition by adding somatic testicular cells to the collagen gel culture system in order to induce spermatogenesis progression. In this experimental study, the disassociation of SSCs was performed by using a two-step enzymatic digestion of type I collagenase, hyaluronidase and DNase. Somatic testicular cells including Sertoli cells and peritubular cells were obtained after the second digestion. SSCs were isolated by Magnetic Activated Cell Sorting [MACS] using GDNF family receptor alpha-1 [Gfr Alpha -1] antibody. Two experimental designs were investigated. 1. Gfr Alpha -1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfr Alpha -1 positive SSCs in a collagen solution. Spermatogenesis progression was determined after three weeks by staining of synaptonemal complex protein 3 [SCP3]-positive cells. Semi-quantitative Reverse Transcription PCR was undertaken for SCP3 as a meiotic marker and, Crem and Thyroid transcription factor-1 [TTF1] as post meiotic markers. For statistical analysis student t test was performed Testicular supporter cells increased the expression of meiotic and post meiotic markers and had a positive effect on extensive colony formation. Collagen gel culture system supported by somatic testicular cells provides a microenvironment that mimics seminiferous epithelium and induces spermatogenesis in vitro

Human plasma derived drugs separation by fractionation of plasma with polyethylene glycol

There are variety of purification techniques for separation of human plasma proteins such as salting out, ion exchange chromatography and ethanol fractionation. There are limitations for each method, for example in salting out method, the salt has to be removed in an additional step. Ion exchange chromatography is difficult for scaling up, and plasma fractionation is a time consuming method and it needs machinery and plants. In the present study the fractionation of human plasma by polyethylene glycol was investigated. The purpose of this study was to investigate the possibility of the fractionation of human plasma by polyethylene glycol. Human plasma fractionation was carried out by using polyethylene glycol with different concentrations from five to twenty percent, followed by centrifugation. After centrifugation the supernatant was used for further fractionation by addition of a higher concentration of polyethylene glycol. Suitable intermediate sources for protein purification were obtained by fractionation of human plasma by polyethylene glycol. Fibrinogen in fraction 5%, IgG and IgM in fraction 10%, IgA in fraction 20%, and finally albumin and alpha1- Antitrypsin in supernatant 20% of polyethylene glycol were achieved. By our study we could obtain four different fractions as intermediate sources for protein purification which cannot be easily obtained from plasma fractionation by cold ethanol fractionation

Co-culture of spermatogonial stem cells with sertoli cells in the presence of testosterone and FSH improved differentiation via up-regulation of post meiotic genes

Spermatogonial stem cells [SSCs] maintain spermatogenesis throughout life in the male. Maintenance of SSCs and induction of spermiogenesis in vitro may provide a therapeutic strategy to treat male infertility. This study investigated in vitro differentiation of mouse SSCs in presence or absence of Sertoli cells, hormones and vitamins. Spermatogonial populations were enriched from testes of 4-6 week old males by magnetic activated cell sorting and anti-Thy-1 antibody. Sertoli cells isolated from 6-8 week old testes were enriched using lectin-DSA-coated plates. Isolated SSCs were cultured in the presence of Leukemia inhibitory factor [LIF] for 7 days in gelatin-coated dishes, then dissociated and cultured for 7 days in media lacking LIF in the presence or absence of Sertoli cells, with or without FSH, testosterone and vitamins. After one week, the effects of Sertoli cells +/- supplementary media on SSC differentiation was evaluated by microscopy and expression of meiotic and postmeiotic transcripts using RT-PCR. SSC colonies had limited development after LIF removal alone, exhibiting low expression of meiotic [Scp3, Th2b] but not postmeiotic transcript, and loss of Stra8 and Dazl expression. SSCs co-cultured with Sertoli cells, hormones and vitamins developed spermatid-like cells expressing postmeiotic markers [TP1, TP2, Prm1] at levels over 2-fold higher than Sertoli cells or hormone/vitamins alone. Our present SSC-Sertoli co-culture provides conditions that may allow efficient in vitro differentiation of SSCs for the treatment of male infertility

HESA-A exerts its cytoprotective effects through scavenging of free radicals: an in vitro study

Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect. Chinese hamster ovary [CHO] and human embryonic kidney [HEK293T] cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit. The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties

Production of Pentameric Cholera Toxin B Subunit in Escherichia coli

Cholera toxin B subunit [CTB] has been extensively studied as an immunogen, adjuvant, and inducer of oral tolerance in many investigations. Production of CTB has been carried out in the bacterial, plant, insect and yeast expression systems. In this study the expression of the CTB containing a 6XHis-tagged was performed by Escherichia coli [E.coli] M15. The yield of purified pentameric recombinant CTB was about 1 mg/l. Western blot analysis demonstrated that the recombinant CTB was antigenically active. In addition, GM1-ganglioside ELISA showed that recombinant CTB binds to GM1-gangelioside receptor, confirming disulfide bond formation and proper folding of the recombinant protein in E.coli. Overall, in regard to the vast applications of CTB in medicine, this bacterial expression system will be a fast, cost-effective and simple system for production of pentameric CTB and CTB conjugated proteins

[Effects of knotweet or polygonum aviculare herbal extract on proliferation of HeLa cell line]

Cervical cancer is the most common cancer in women living in developing countries. Recently, for treatment of diseases such as cancer, herbal medicine is used as a supplementary. The aim of this study was assessment of anticancerous effects of polygonum aviculare herbal extract on Hela cervical cancer cell line. HeLa cells were cultured in RPMI - 1640 with 10% Fetal Bovine Serum in 5% Co2 and at 37 [degree sign] C in different concentrations [0, 0.005, 0.05, 0.01, 0.025, 0.075, 0.1, 0.125, 0.15, 0.175, 0.2, 0.25, 0.3, 0.35, 0.5, 5 mg/ml] of polygonum aviculare. For assessment of viability of cells, trypan blue staining was performed. MTT assay was used for proliferation detection. Our results showed that in 0.15, 0.20 and 0.35 mg/ml proliferation of HeLa cells decreased according to MTT assay. It was proved that polygonum aviculare had antioxidant component and could be a scavenger of free radical. Because of high production of free radicals in diseases such as cancer, the use of the herbal medicine with high amount of antioxidant could be a supplementary treatment in cancer and other diseases

Down-regulation of metallothionein 1 and 2 after exposure to electromagnetic field in mouse testis

It is proved that testis is sensitive to electromagnetic field [EMF] and its damage results in infertility. Exposure to EMF induces reactive oxygen species production and affects on anti-oxidants defense mechanisms. Metallothionein [MT] is a name for a group of low molecular weight [6-7 kDa], sulfhydryl rich proteins. Expression of MT1 and MT2 genes in testis tissue after EMF exposure was aimed in this study. Male BALB/c mice [8 weeks old] were exposed to 3 MT EMF for 8 weeks, 4 hours/day. After 8 weeks, the mice were sacrificed and the testis tissue was removed. The testis pieces were stained with hematoxylin and eosin and analyzed under an optical microscope. Assessment of MT1 and MT2 genes and also protein expression was performed by real-time PCR and Western-blot, respectively. In light microscopic observation, the number of primary spermatocytes was increased significantly in EMF group [P<0.01]. In addition, in interstitial space, the number of leydig cells was increased significantly in EMF group [P<0.01] and basement membrane thickness was increased as well. MT1 and MT2 genes were down-regulated significantly in testis tissue of mice exposed to EMF both in mRNA and protein level compared to control. It is clear that MT is mediated in testis development and spermatogenesis. Down-regulation of MT1 and MT2 after EMF in mouse testis might be followed by some consequences that result in infertility

Genetically engineered mesenchymal stem cells stably expressing green fluorescent protein

Mesenchymal stem cells [MSCs] are nonhematopoietic stromal cells that are capable of differentiating into and contribute to the regeneration of mesenchymal tissues. Human mesenchymal stem cells [liMSCs] are ideal targets in cell transplantation and tissue engineering. Enhanced green fluorescent protein [EGFP] has been an important reporter gene for gene therapy. The aim of this study was establishment of MSCs expressing GFP. MSCs were isolated and characterized by Immunophenotyping. The pEGFP-Nl plasmid was extracted from previously transformed Escherichia, coli cells and transfected into MSCs using FuGENE HD transfection reagent. Stable cells were established in the presence of geneticin. Expression of GFP was detected by RT-PCR, western blot analysis and immunoflorecent microscope. MSCs were successfully isolated and characterized. The MSCs transfected with the pEGFP-Nl plasmid expressed GFP both in mRNA and protein levels while cells transfected with empty vector did not. The results suggested that this engineered cell line will be used in the future studies and can easily be traced in vivo

High frequency electromagnetic field induces lipocalin 2 expression in mouse liver

Neutrophil gelatinase-associated lipocalin [NGAL/Lcn2], comprise a group of small extracellular proteins with a common beta-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field [EMF] produces reactive oxygen species [ROS] in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Balb/c mice [8 weeks old] were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin-Eosin [H and E] and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 micro g of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time-PCR. The light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels. To the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards

Effects of leukemia inhibitory factor on gp130 expression and rate of metaphase II development during in vitro maturation of mouse oocyte

Leukemia inhibitory factor [LIF] is a 45-56 kDa glycoprotein that has an important role in proliferation and embryo implantation. Its effect on oocyte maturation and how to exert the function remained to be elucidated. Immature mice superovulated with human menopausal gonadotropin and germinal vesicle [GV] oocytes were obtained from ovary 48 hours after. GV oocytes were cultured in tissue culture medium 199 with 0, 100, 500 and 1,000 U/ml LIF. Cumulus expansion and in vitro maturation [IVM] rate were assessed after culture. For reverse transcriptase PCR, total RNA from GV and metaphase II [Mil] oocytes were extracted by Trizol reagent. The quantity and quality of RNA were determined by spectrophotometry and electrophoresis, respectively. Reverse transcription was performed by Super Script III reverse transcriptase with 1 micro g of total RNA followed by DNase I treatment and heat inactivation. Expression of gp130 was determined by RT-PCR. Our results showed that cumulus expansion was improved with 1,000 U/ml in culture medium compared to others. GV breakdown and Mil rate in groups with LIF were higher than control group and were dose dependant. In 1,000 U/ml, LIF rate of Mil was significantly higher than control group [P<0.05]. Our results also showed that gp130 is expressed neither in GV nor in Mil oocytes during IVM of mouse oocytes. gp130 is expressed in human oocyte but not in mouse. Our results suggest that in mouse, LIF could affect the oocyte via another receptor or via cumulus cells; however, further studies are warranted

influence of meiotic spindle configuration by cysteamine during in vitro maturation of mouse oocytes

The aim of this study was to assess effects of cysteamine as an anti-oxidant on rate of in vitro maturation of oocyte and determination of its effects on spindle size and shape. Pre-mature mice were primed with pregnant mare stimulating gonadotrophin [PMSG] and germinal vesicle [GV] stage oocytes were obtained 48 h after. The oocytes were cultured in tissue culture medium [TCM 199] with 50, 100, 200 and 500 microM cysteamine. Experiments also included a group of ovulated oocytes [in vivo matured] after priming with PMSG and human chorionic gonadotrophin. For spindle immuno-cytochemistry of metaphase II [MII], oocytes alpha and beta tubulin antibody were performed. Chromosome staining was performed with Hoechst. Our results showed that the rate of GV breakdown [GVBD] and MII increased in all cysteamine groups except in group cultured with 500 microM cysteamine. Immuno-cytochemistry analysis showed that spindle area in all in vitro Maturation oocytes increased when compared to in vivo oocytes. Spindle shape and size [spindle area] in 100 microM cysteamine in comparison to in vivo group was insignificant [P>0.05]. Our results revealed that cysteamine improved IVM rate in a dose dependant manner. The size and shape of spindle in the presence of given concentration of cysteamine was similar to in vivo. Therefore, cysteamine improved microtubule organization, rate of GVBD and MIl development