Lipocalin-2-mediated upregulation of various antioxidants and growth factors protects bone marrow-derived mesenchymal stem cells against unfavorable microenvironments

Despite many advantages of mesenchymal stem cells (MSCs) that make them suitable for cell therapy purposes, their therapeutic application has been limited due to their susceptibility to several stresses (e.g., nutrient-poor environment, oxidative stress, and hypoxic and masses of cytotoxic factors) to which they are exposed during their preparation and following transplantation. Hence, reinforcing MSCs against these stresses is a challenge for both basic and clinician scientists. Recently, much attention has been directed toward equipping MSCs with cytoprotective factors to strengthen them against unfavorable microenvironments. Here, we engineered MSCs with lipocalin 2 (Lcn2), a cytoprotective factor that is naturally induced following exposure of cells to stresses imposed by the microenvironment. Lcn2 overexpression not only did not interfere with the multidifferentiation capacity of the MSCs but also granted many protective properties to them. Lcn2 potentiated MSCs to withstand oxidative, hypoxia, and serum deprivation (SD) conditions via antagonizing their induced cytotoxicity and apoptosis. Adhesion rate of MSCs to coated culture plates was also enhanced by Lcn2 overexpression. In addition, Lcn2 induced antioxidants and upregulated some growth factors in MSCs. Our findings suggested a new strategy for prevention of graft cell death in MSC-based cell therapy.     

Neutrophil Gelatinase-Associated Lipocalin induces the expression of heme oxygenase-1 and superoxide dismutase <Subscript>1, 2</Subscript>

Lipocalin-2 (Lcn2, NGAL) is a member of the lipocalin super family with diverse function such as the induction of apoptosis, the suppression of bacterial growth, and modulation of inflammatory response. Much interest has recently been focused on the physiological/pathological role of the lipocalin-2 that is considered to be a novel protective factor against oxidative stress. However, its precise biological roles in this protection are not fully understood. In this report we intended to test the effect of lipocalin-2 on the expression of heme oxygenase _{(1, 2)} and superoxide dismutase _{(1, 2)} which are two strong antioxidants. NGAL was cloned to pcDNA3.1 plasmid by using genetic engineering method. The recombinant vector was transfected to CHO and HEK293T to establish stable cell expressing NGAL and the expression of HO-1, 2 and SOD_{1, 2} were compared with appropriate controls by RT-PCR and western blot. On the other hand, expression of NGAL was suppressed by siRNA transfection in order to study the effect of lipocalin-2 on mentioned genes/proteins. The results showed that the expression of HO-1 and SOD_{1, 2} enzymes were higher in cells expressing recombinant lipocalin-2 compared with the control cells. Although the expression of HO-1 was lower in NGAL silencing cells, the expression of SOD₁ and SOD₂ were higher. Our data suggest that NGAL is a potent inducer of HO-1 and somewhat SOD₁ and SOD₂ and it appears that part of antioxidant property of NGAL could be attributed to the induction of HO-1and SOD_{1, 2}.     

Lipocalin 2 decreases senescence of bone marrow-derived mesenchymal stem cells under sub-lethal doses of oxidative stress

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.     

Harnessing an Integrative In Silico Approach to Engage Highly Immunogenic Peptides in an Antigen Design Against Epsilon Toxin (ETX) of Clostridium perfringens

International Journal of Peptide Research and Therapeutics - Epsilon toxin (ETX) is one of four lethal toxins of Clostridium perfringens produced by types B and D of the pathogen. This pore-forming...     

The effect of female sex steroid hormones on osteogenic differentiation of endometrial stem cells

Molecular Biology Reports - Bone regeneration is a significant and crucial health issue worldwide. Tissue bioengineering has shown itself to be the best substitute for common clinical treatment of...     

Nrf-2 overexpression in mesenchymal stem cells reduces oxidative stress-induced apoptosis and cytotoxicity

The most prominent capabilities of mesenchymal stem cells (MCSs) which make them promising for therapeutic applications are their capacity to endure and implant in the target tissue. However, the therapeutic applications of these cells are limited due to their early death within the first few days following transplantation. Therefore, to improve cell therapy efficacy, it is necessary to manipulate MSCs to resist severe stresses imposed by microenvironment. In this study, we manipulated MSCs to express a cytoprotective factor, nuclear factor erythroid-2 related factor 2 (Nrf2) to address this issue. Full-length human Nrf2 cDNA was isolated and TOPO cloned into TOPO cloning vector and then transferred to gateway adapted adenovirus expression vector by LR recombination reaction. Afterwards, the Nrf2 bearing recombinant virus was prepared in appropriate mammalian cell line and used to infect MSCs. The viability and apoptosis of the Nrf2 expressing MSCs were evaluated following hypoxic and oxidative stress conditions. Transient expression of Nrf2 by MSCs protected them against cell death and the apoptosis triggered by hypoxic and oxidative stress conditions. Nrf2 also enhanced the activity of SOD and HO-1. These findings could be used as a strategy for prevention of graft cell death in MSC-based cell therapy. It also indicates that management of cellular stress responses can be used for practical applications.     

Isolation of a new ssDNA aptamer against staphylococcal enterotoxin B based on CNBr‐activated sepharose‐4B affinity chromatography

Staphylococcus aureus are potent human pathogens possessing arsenal of virulence factors. Staphylococcal food poisoning (SFP) and respiratory infections mediated by staphylococcal enterotoxin B (SEB) are common clinical manifestations. Many diagnostic techniques are based on serological detection and quantification of SEB in different food and clinical samples. Aptamers are known as new therapeutic and detection tools which are available in different ssDNA, dsDNA and protein structures. In this study, we used a new set of ssDNA aptamers against SEB. The methods used included preparation of a dsDNA library using standard SEB protein as the target analyte, affinity chromatography matrix in microfuge tubes, SELEX procedures to isolate specific ssDNA‐aptamer as an affinity ligand, aptamer purification using ethanol precipitation method, affinity binding assay using ELISA, aptamer cloning and specificity test. Among 12 readable sequences, three of them were selected as the most appropriate aptamer because of their affinity and specificity to SEB. This study presents a new set of ssDNA aptamer with favorable selectivity to SEB through 12 rounds of SELEX. Selected aptamers were used to detect SEB in infected serum samples. Results showed that SEB c1 aptamer (2 µg SEB/100 nM aptamer) had favorable specificity to SEB (k_d = 2.3 × 10^?11). In conclusion, aptamers can be considered as useful tools for detecting and evaluating SEB. The results showed that affinity chromatography was an affordable assay with acceptable accuracy to isolate sensitive and selective novel aptamers. Copyright © 2016 John Wiley & Sons, Ltd.     

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions

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.     

Lipocalin 2 regulation by thermal stresses: Protective role of Lcn2/NGAL against cold and heat stresses

Environmental temperature variations are the most common stresses experienced by a wide range of organisms. Lipocalin 2 (Lcn2/NGAL) is expressed in various normal and pathologic conditions. However, its precise functions have not been fully determined. Here we report the induction of Lcn2 by thermal stresses in vivo, and its role following exposure to cold and heat stresses in vitro. Induction of Lcn2 in liver, heart and kidney was detected by RT-PCR, Western blot and immunohistochemistry following exposure of mice to heat and cold stresses. When CHO and HEK293T cells overexpressing NGAL were exposed to cold stress, cell proliferation was higher compared to controls. Down-regulatrion of NGAL by siRNA in A549 cells resulted in less proliferation when exposed to cold stress compared to control cells. The number of apoptotic cells and expression of pro-apoptotic proteins were lower in the NGAL overexpressing CHO and HEK293T cells, but were higher in the siRNA-transfected A549 cells compared to controls, indicating that NGAL protects cells against cold stress. Following exposure of the cells to heat stress, ectopic expression of NGAL protected cells while addition of exogenous recombinant NGAL to the cell culture medium exacerbated the toxicity of heat stress specially when there was low or no endogenous expression of NGAL. It had a dual effect on apoptosis following heat stress. NGAL also increased the expression of HO-1. Lcn2/NGAL may have the potential to improve cell proliferation and preservation particularly to prevent cold ischemia injury of transplanted organs or for treatment of some cancers by hyperthermia.