[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: An increasing body of evidence has emerged regarding the existence and function of spermatogonial stem cells (SSCs); however, their female counterparts are the subject of extensive debate. Theoretically, ovarian germ stem cells (GSCs) have to reside in the murine ovary to support and replenish the follicle pool during the reproductive life span. Recently, various methods have been recruited to isolate and describe aspects of ovarian GSCs, but newer and more convenient strategies in isolation are still growing. Herein, a morphology-based method was used to isolate GSCs.
A cell suspension of mouse neonatal ovaries was cultured. Colonies of GSCs were harvested mechanically and cultivated on mouse embryonic fibroblasts (MEF). Alkaline phosphatase activity was assessed to verify stemness features of cells in colonies. Expression of germ and stem cell specific genes (Oct-4, Nanog, Fragilis, C-kit, Dazl, and Mvh) was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Immunofluorescence of Oct4, Dazl, Mvh, and SSEA-1 was also performed.
Small colonies without a clear border appeared during the first 4 days of culture, and the size of colonies increased rapidly. Cells in colonies were positive for alkaline phosphatase activity. Reverse transcription-polymerase chain reaction showed that Oct-4, Fragilis, C-kit, Nanog, Mvh, and Dazl were expressed in colony-forming cells. Immunofluorescence revealed a positive signal for Oct4, Dazl, Mvh, and SSEA-1 in colonies as well.
The applicability of morphological selection for isolation of GSCs was verified. This method is easier and more economical than other techniques. The availability of ovarian stem cells can motivate further studies in development of oocyte and cell-based therapies.
Archives of Medical Science 06/2015; 11(3):670-8. DOI:10.5114/aoms.2015.52374 · 1.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Purpose: Recent developments in the field of cell therapy have led to a renewed interest in treatment of acute kidney injury (AKI). However, the early death of transplanted mesenchymal stem cells (MSCs) in stressful microenvironment of a recipient tissue is a major problem with this kind of treatment. The objective of this study was to determine whether overexpression of a cytoprotective factor, nuclear factor erythroid-2 related factor 2 (Nrf2), in MSCs could protect rats against AKI.
Methods: The Nrf2 was overexpressed in MSCs by recombinant adenoviruses, and the MSCs were implanted to rats suffering from cisplatin-induced AKI.
Results: The obtained results showed that transplantation with the engineered MSCs ameliorates cisplatin-induced AKI. Morphologic features of the investigated kidneys showed that transplantation with the MSCs in which Nrf2 had been overexpressed significantly improved the complications of AKI.
Conclusion: These findings suggested that the engineered MSCs might be a good candidate to be further evaluated in clinical trials. However, detailed studies must be performed to investigate the possible carcinogenic effect of Nrf2 overexpression.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
Autophagy modulation in MSCs can be proposed as a new strategy to improve their survival rate in stressful microenvironments.
[Show abstract][Hide abstract] ABSTRACT: Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely. Materials and.
HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR.
Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress. This decrease was further found to be attributed to senescence.
Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence.
Iranian Journal of Basic Medical Science 05/2015; 18(5):459-64. · 0.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the major obstacles in cancer therapy is the lack of anticancer agent specificity to tumor tissues. The strategy of cell-based therapy is a promising therapeutic option for cancer treatment. The specific tumor-oriented migration of mesenchymal stem cells (MSCs) makes them a useful vehicle to deliver anticancer agents. In this study, we genetically manipulated bone marrow-derived mesenchymal stem cells with their lipocalin 2 (Lcn2) in order to inhibit liver metastasis of colon cancer in nude mice. Lcn2 was successfully overexpressed in transfected MSCs. The PCR results of SRY gene confirmed the presence of MSCs in cancer liver tissue. This study showed that Lcn2-engineered MSCs (MSC-Lcn2) not only inhibited liver metastasis of colon cancer but also downregulated the expression of vascular endothelial growth factor (VEGF) in the liver. Overall, MSCs by innate tropism toward cancer cells can deliver the therapeutic agent, Lcn2, and inhibit cancer metastasis. Hence, it could be a new modality for efficient targeted delivery of anticancer agent to liver metastasis.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are under intensive investigation for use in cell-based therapies because their differentiation abilities, immunomodulatory effects, and homing properties offer potential for significantly augmenting regenerative capacity of many tissues. Nevertheless, major impediments to their therapeutic application, such as low proliferation and survival rates remain as obstacles to broad clinical use of MSCs. Another major challenge to evolution of MSC-based therapies is functional degradation of these cells as a result of their exposure to oxidative stressors during isolation. Indeed, oxidative stress-mediated MSC depletion occurs due to inflammatory processes associated with chemotherapy, radiotherapy, and expression of pro-apoptotic factors, and the microenvironment of damaged tissue in patients receiving MSC therapy is typically therapeutic not favorable to their survival. For this reason, any strategies that enhance the viability and proliferative capacity of MSCs associated with their therapeutic use are of great value. Here, recent strategies used by various researchers to improve MSC allograft function are reviewed, with particular focus on in vitro conditioning of MSCs in preparation for clinical application. Preconditioning, genetic manipulation, and optimization of MSC culture conditions are some examples of the methodologies described in the present article, along with novel strategies such as treatment of MSCs with secretome and MSC-derived microvesicles. This topic material is likely to find value as a guide for both research and clinical use of MSC allografts and for improvement of the value that use of these cells brings to health care.
[Show abstract][Hide abstract] ABSTRACT: 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 MS14, an herbal-marine drug, has anti-inflammatory properties through reduction of TNF-α and IL-1β. Thus, the present study investigated the effects of MS14 on the course of EAE and its relation to LCN2 expression in both protein and gene levels.
[Show abstract][Hide abstract] ABSTRACT: Objective Wharton's jelly (WJ), an appropriate source of mesenchymal stem cells (MSCs), has been shown to have a wide array of therapeutic applications. However, the WJ-derived MSCs are very heterogeneous and have limited expression of pluripotency markers. Hence, improvement of their culture condition would promote the efficiency of WJ-MSCs. This study aims to employ a simple method of cultivation to obtain WJ-MSCs which express more pluripotency markers. Methods CD105(+) cells were separated by magnetic-associated (activated) cell sorting from umbilical cord mucous tissue. CD105(+) cells were added to Methocult medium diluted in α-minimum essential medium (α-MEM) and seeded in poly(2-hydroxyethyl methacrylate) (poly-HEMA)-coated plates for suspension culture preparation. Differentiation capacity of isolated cells was evaluated in the presence of differentiation-inducing media. The expression of pluripotency markers such as Oct3/4, Nanog, and Sox2 was also analyzed by RT-PCR and western blot techniques. Moreover, immunocytochemistry was performed to detect α-SMA protein. Results WJ-MSCs grew homogeneously and formed colonies when cultured under suspension culture conditions (Non-adhesive WJ-MSCs). They maintained their growth ability in both adherent and suspension cultures for several passages. Non-adhesive WJ-MSCs expressed Oct3/4, Nanog, and Sox2 both at transcriptional and translational levels in comparison to those cultured in conventional adherent cultures. They also expressed alpha-smooth muscle actin (antigene) (α-SMA) protein. Discussion In this study, we isolated WJ-MSCs using a slightly modified culture condition. Our simple non-genetic method resulted in a homogeneous population of WJ-MSCs, which highly expressed pluripotency markers. Conclusion In the future, more multipotent WJ-MSCs can be harnessed as a non-embryonic source of MSCs in MSC-based cell therapy.
[Show abstract][Hide abstract] ABSTRACT: 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 مقدار 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.
[Show abstract][Hide abstract] ABSTRACT: Conditioned medium of mesenchymal stem cells (MSCs) is now being used for its cytoprotective effects, especially when the cells are equipped with cytoprotective factors to strengthen them against unfavorable microenvironments. Overexpression of Lcn2 in MSCs mimics in vivo kidney injury. Hence, unraveling how Lcn2-engineered MSCs affect kidney cells has been investigated. Cisplatin treated HK-2 or HEK293 kidney cells were co-cultivated with Lcn2 overexpressing MSCs in upper and lower chambers of transwell plates. Proliferation, apoptosis, and expression of growth factors and cytokines were assessed in the kidney cells. Co-cultivation with the MSCs-Lcn2 not only inhibited cisplatin-induced cytotoxicity in the HK-2 and HEK293 cells, but increased proliferation rate, prevented cisplatin-induced apoptosis, and increased expression of growth factors and the amount of antioxidants in the kidney cells. Thus Lcn2-engineered MSCs can ameliorate and repair injured kidney cells in vitro, which strongly suggests there are beneficial effects of the MSCs-Lcn2 in cell therapy of kidney injury.
Cell Biology International 07/2014; 39(2). DOI:10.1002/cbin.10344 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 capacity. Therefore, we hypothesized that over-expression of Nrf2 in MSCs can
improve cellular cardiomyoplasty
[Show abstract][Hide abstract] ABSTRACT: Background Bacterial contamination of platelet products is the major infectious risk in blood transfusion medicine, which can result in life-threatening sepsis in recipient. Lipocalin 2 (Lcn2) is an iron-sequestering protein in the antibacterial innate immune response, which inhibit bacterial growth. This study was aimed to evaluate the antibacterial property of Lcn2 in preventing bacterial contamination of platelets. Methods Recombinant Lcn2 was expressed in a eukaryotic expression system and following purification and characterization of the recombinant Lcn2, its minimum inhibitory concentration was determined. Then, platelet concentrates were inoculated with various concentrations of Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis, and the antibacterial effects of Lcn2 was evaluated at 20-24°C. Results Results revealed that Lcn2 effectively inhibited the growth of 1.5 × 10(4) CFU/ml S. epidermidis, P. aeruginosa, K. pneumoniae, E. coli, and E. faecalis at 40 ng/ml. At this concentration, Lcn2 also inhibited the growth of 1.5 × 10(3) CFU/ml Staphylococcus aureus and Proteus mirabilis. Conclusion Recombinant Lcn2 inhibited growth of a variety of platelet-contaminating bacteria. Therefore, supplementation of platelet concentrates with Lcn2 may reduce bacterial contamination.
[Show abstract][Hide abstract] ABSTRACT: 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 (Gfrα-1) antibody. Two experimental designs were investigated. 1. Gfrα-1 positive SSCs were cultured in a collagen solution. 2. Somatic testicular cells were added to the Gfrα-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.
[Show abstract][Hide abstract] ABSTRACT: Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy.
[Show abstract][Hide abstract] ABSTRACT: The regenerative potential of mesenchymal stem cells (MSCs) is impaired by cellular senescence, a multi factorial process that has various functions. However, pathways and molecules involved in senescence have not been fully identified. Lipocalin 2 (Lcn2) has been the subject of intensive research, due to its contribution to many physiological and pathophysiological conditions. The implication of Lcn2 has been reported in many conditions where senescence also occurs. In the present study, we evaluated the role of Lcn2 in the occurrence of senescence in human bone marrow-derived mesenchymal stem cells (hB-MSCs) under oxidative conditions. When hB-MSCs were genetically engineered to over-express Lcn2 (MSC-Lcn2) and exposed to H2O2, the proliferation rate of the cells increased. However, the number of colonies and the number of cells that made up each colony in both MSC-V and MSC-Lcn2 cells decreased compared to those cultivated under normal conditions. Our results revealed that over-expression of recombinant Lcn2 in hB-MSCs decreases senescence induced by H2O2 treatment. Senescent cells were observed in aged hB-MSCs; however, no alteration in the expression level of Lcn2 was detected compared to earlier passages. Finally, a higher amount of Lcn2 protein was detected in the plasma of the elderly than in young people. Our findings suggest that Lcn2 might restore the health and regeneration potential of MSCs by decreasing senescence.
[Show abstract][Hide abstract] ABSTRACT: Background: Increased mesenchymal stem cells (MSCs) survival after transplantation can improve their therapeutic potential. Therefore, manipulation of the MSCs with cytoprotective genes such as NF-E2 Related Factor-2 (NRF2) is important to improve cell therapy efficiency. The aim of this study was cloning and transient over-expression of the NRF2 gene in MSCs by adenoviral expression system.
Methods: NRF2 was isolated from MSCs and cloned into pENTR/TOPO/D vector by TOPO cloning reaction. NRF2 was translocated from pENTR/TOPO/D vector to adenoviral vector using the Gateway technology. The recombinant adenovirus vector was transformed into 293A cells to package recombinant adenovirus particles. Recombinant adenoviruses harboring NRF2 infected MSCs.
Results: NRF2 was successfully isolated, cloned and its accuracy was confirmed by Deoxyribonucleic acid (DNA) sequencing. NRF2 over-expression was evaluated. This over-expression by adenoviral expression system was transient.
Conclusion: NRF2 over-expression in MSCs with adenoviral expression system could be a valuable device because of its transient expression. Stable over-expression of genes in MSCs could not be permitted.
[Show abstract][Hide abstract] ABSTRACT: Abstract Spermatogonial stem cells (SSCs) represent a unique testicular cell type that has the capacity for proliferating, differentiating, and transmitting genetic information. This particular cell type is a strong focus of stem cell research, with isolation and maintenance of SSCs as an important issue. Therefore, we attempted to optimize SSCs handling and to analyze different media and feeder layers, such as adult and embryonic Sertoli cells. The expression patterns of SSC-specific proteins (α6 and β1 integrins, Stra8, and DAZL) and restoration of spermatogenesis were chosen as parameters to demonstrate the efficacy of the protocol. SSCs were isolated from testes of 3- to 6-day-old mice using a magnetic activated cell-sorting system and Thy-1 antibody. After enrichment, SSCs were cultured for 7 days with different media and feeder layers. Then, SSCs were transplanted to recipient mice. Culturing on adult and embryonic Sertoli cells and in the presence of different growth factors [glial cell line-derived neurotrophic factor (GDNF), GDNF family receptor α1 (GFR-α1), and basic fibroblast growth factor (bFGF) resulted in an undifferentiated SSC phenotype with typical stem cell characteristics observed in vivo. The established co-culture model could help to improve the recovery and quality of stem cell preparation of mammalian testis.
[Show abstract][Hide abstract] ABSTRACT: Sulfur mustard (SM) has been identified as an important chemical weapon. During the Iran-Iraq war of 1980-88, the extensive usage of SM against Iranian civilians and military forces was proven. This agent has been shown to cause severe damage mainly in the skin, eyes, lungs, and respiratory tract in Iranian veterans. The most common disease is bronchiolitis obliterans (BO)). SM increases the endogenous production of reactive oxygen species (ROS). Superoxide dismutases (SODs) are known as protective antioxidants against the harmful effects of ROS. Twenty exposed SM individuals (43.2±6.4 years), and 10 normal controls (41.3±2.5 years) were enrolled in this study. Evaluation of SODs was performed by semiquantitative RT-PCR and immunohistochemistry. Our results demonstrated that CuZnSOD and MnSOD mRNA were up-regulated 2.79±1.09 and 2.49±1.11 folds, respectively in SM-injured patients in comparison with control levels. In contrast, Immunohistochemistry results showed downregulation of CuZnSOD protein expression in SM injured patients. Our results revealed that SODs may play an important role in cellular protection against oxidative stress due to mustard gas toxicity in airway wall of SM exposed patients.
Iranian journal of allergy, asthma, and immunology 06/2013; 12(2):153-60. · 1.01 Impact Factor