Role of NF-κB signaling pathway in H2O2-induced oxidative stress of hiPSCs.

The balance between oxidation and antioxidation is crucial for the development of embryo. It is harmful to the early embryonic development if embryonic stem cells (ESCs) encounter the serious oxidative stress in vivo. Induced pluripotent stem cells (iPSCs) are very similar to ESCs and are the important cell source to replace ESCs for research and therapy. Studies show that iPSCs have better resistant ability to oxidative stress, but the involved mechanism remains unclear. In this study, we predicted that the NF-κB pathway might be involved in H2O2-induced developmental damage by network toxicology analysis. Then, the oxidative stress model was established with different concentrations of H2O2 to investigate the mechanism of NF-κB pathway in oxidative stress of human induced pluripotent stem cells (hiPSCs). The results showed as follows: With the increase of H2O2 concentration, the ROS level gradually went up leading to an increasing damage degree of hiPSCs; however, the MDA content was obviously high only in the 400 µM H2O2 group; the activities of some antioxidant indexes such as SOD2 and T-AOC were significantly upregulated in the 100 µM group, while most of antioxidant indexes showed downregulated tendency to different degrees with the increase of H2O2 concentration. The expression levels of P65, P50, IκB, SOD2, and FHC mRNA were upregulated in most H2O2-treated groups, showing a dose-dependent relationship. In subsequent experiments, the inhibitor of IκB-α phosphorylation, Bay11-7082, reversed the upregulation of P65, IκB, and FHC mRNA expression induced by 400 µM H2O2. The protein levels of P65, p-P65, P50, p-P50, IκB, p-IκB, SOD2, and FHC were upregulated in most H2O2-treated groups. However, the upregulation induced by 400 µM H2O2 could be reversed by BAY 11-7082, except for IκB and SOD2. In conclusion, H2O2 could promote the expressions and phosphorylations of NF-κB that could upregulate the expressions of its downstream antioxidant genes to minimize the damage of hiPSCs caused by oxidative stress. These results contribute to a fundamental understanding of the antioxidant mechanism of iPSCs and will further facilitate the application of iPSCs, as well as provide a reference for controlling the oxidative stress encountered in the early development stage of embryo.

Feline umbilical cord-derived mesenchymal stem cells: isolation, identification, and antioxidative stress role through NF-κB signaling pathway.

At present, the differentiation potential and antioxidant activity of feline umbilical cord-derived mesenchymal stem cells (UC-MSCs) have not been clearly studied. In this study, feline UC-MSCs were isolated by tissue adhesion method, identified by flow cytometry detection of cell surface markers (CD44, CD90, CD34, and CD45), and induced differentiation toward osteogenesis and adipogenesis in vitro. Furthermore, the oxidative stress model was established with hydrogen peroxide (H2O2) (100 µM, 300 µM, 500 µM, 700 µM, and 900 µM). The antioxidant properties of feline UC-MSCs and feline fibroblasts were compared by morphological observation, ROS detection, cell viability via CCK-8 assay, as well as oxidative and antioxidative parameters via ELISA. The mRNA expression of genes related to NF-κB pathway was detected via quantitative real-time polymerase chain reaction, while the levels of NF-κB signaling cascade-related proteins were determined via Western Blot. The results showed that feline UC-MSCs highly expressed CD44 and CD90, while negative for CD34 and CD45 expression. Feline UC-MSCs cultured under osteogenic and adipogenic conditions showed good differentiation capacity. After being exposed to different concentrations of H2O2 for eight hours, feline UC-MSCs exhibited the significantly higher survival rate than feline fibroblasts. A certain concentration of H2O2 could up-regulate the activities of SOD2 and GSH-Px in feline UC-MSCs. The expression levels of p50, MnSOD, and FHC mRNA in feline UC-MSCs stimulated by 300 µM and 500 µM H2O2 significantly increased compared with the control group. Furthermore, it was observed that 500 µM H2O2 significantly enhanced the protein levels of p-IκB, IκB, p-p50, p50, MnSOD, and FHC, which could be reversed by BAY 11-7,082, a NF-κB signaling pathway inhibitor. In conclusion, it was confirmed that feline UC-MSCs, with good osteogenesis and adipogenesis abilities, had better antioxidant property which might be related to NF-κB signaling pathway. This study lays a foundation for the further application of feline UC-MSCs in treating the various inflammatory and oxidative injury diseases of pets.

Optimization of piggyBac Transposon System Electrotransfection in Sheep Fibroblasts.

Electroporation is a non-viral mediated transfection technique, which has the advantages of being harmless, easy to operate, and less expensive. This transfection method can be used for almost all cell types and has gradually become the preferred transfection method for mammalian gene editing. However, further improvements are needed in electroporation efficiency. There is no universal standard electrotransfection step for different types of cells, and the inappropriate electroporation parameters will result in a low transfection efficiency and high cell mortality. Here, we systematically optimized the electrotransfection parameters of piggyBac transposon system into sheep fetal fibroblasts for the first time. We found that the cell transfection efficiency and cell viability could be improved by using traditional cell culture medium DMEM/F12 as an electroporation buffer, and simultaneously using the square-wave pulsing program of 200 V, 2 pulses, 20 ms length, and 20 µg DNA (3 µg/µL) in 4 mm cuvette, and the transfection efficiency and cell viability could eventually reach 78.0% and 40.9%, respectively. The purpose of this study is to provide a method reference and theoretical basis for the plasmid electrotransfection in mammal cells.

Expression patterns and correlation analyses of muscle-specific genes in the process of sheep myoblast differentiation.

The purpose of this study was to establish a system for the isolation, culture, and differentiation of sheep myoblasts, and to explore the expression patterns as well as mutual relationships of muscle-specific genes. Sheep fetal myoblasts (SFMs) were isolated by two-step enzymatic digestion, purified by differential adhesion and identified using immunofluorescence techniques. Two percent horse serum was used to induce differentiation in SFMs. Real-time quantitative and Western blot analyses were respectively used to detect the mRNA and protein expressions of muscle-specific genes including MyoD, MyoG, Myf5, Myf6, PAX3, PAX7, myomaker, desmin, MYH1, MYH2, MYH4, MYH7, and MSTN during the differentiation of SFMs. Finally, the correlation between muscle-specific genes was analyzed by the Pearson correlation coefficient method. The results showed that the isolated and purified SFMs could form myotubes after the induction for differentiation. The marker factors including MyoD, MyoG, myomaker, desmin, and MyHC were positively stained in SFMs. The mRNA expressions of MyoD, MyoG, and myomaker increased and then decreased, while Myf5, PAX3, and PAX7 decreased; Myf6, desmin, MYH1, MYH2, MYH4, and MYH7 increased; and MSTN fluctuated up and down during the differentiation of SFMs. The expression patterns of protein were basically consistent with those of mRNA except MSTN. There existed significant or highly significant correlations at mRNA or protein level among some genes. Some transcription factor proteins (MyoD, Myf5, Myf6, PAX3, PAX7) showed significant or highly significant correlations with the mRNA level of some other genes and/or themselves. In conclusion, SFMs with good myogenic differentiation ability were successfully isolated, and the expression patterns and correlations of muscle-specific genes during SFM differentiation were revealed, which laid an important foundation for elucidating the gene regulation mechanism of sheep myogenesis.