A Novel Age-Related Circular RNA Circ-ATXN2 Inhibits Proliferation, Promotes Cell Death and Adipogenesis in Rat Adipose Tissue-Derived Stromal Cells

Adipose tissue-derived stromal cells are promising candidates investigating the stem cell-related treatment. However, their proportion and utility in the human body decline with time, rendering stem cells incompetent to complete repair processes in vivo. The involvement of circRNAs in the aging process is poorly understood. Rat subcutaneous adipose tissue from 10-week-old and 27-month-old rats were used for hematoxylin and eosin (H and E) staining, TUNEL staining, and circRNA sequencing. Rat adipose tissue-derived stromal cells were cultured and overexpressed with circ-ATXN2. Proliferation was examined using xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Apoptosis was induced by CoCl2 and examined using flow cytometry. RT-PCR assay and Oil Red O staining were used to measure adipogenesis at 48 h and 14 days, respectively. H and E staining showed that the diameter of adipocytes increased; however, the number of cells decreased in old rats. TUNEL staining showed that the proportion of apoptotic cells was increased in old rats. A total of 4,860 and 4,952 circRNAs was detected in young and old rats, respectively. Among them, 67 circRNAs exhibited divergent expression between the two groups (fold change ≥2, p ≤ 0.05), of which 33 were upregulated (49.3%) and 34 were downregulated (50.7%). The proliferation of circ-ATXN2-overexpressing cells decreased significantly in vitro, which was further validated by xCELLigence real-time cell analysis, EdU staining, and cell cycle assay. Overexpression of circ-ATXN2 significantly increased the total apoptotic rate from 5.78 ± 0.46% to 11.97 ± 1.61%, early apoptotic rate from 1.76 ± 0.22% to 5.50 ± 0.66%, and late apoptosis rate from 4.02 ± 0.25% to 6.47 ± 1.06% in adipose tissue-derived stromal cells. Furthermore, in circ-ATXN2-overexpressing cells, RT-PCR assay revealed that the expression levels of adipose differentiation-related genes PPARγ and CEBP/α were increased and the Oil Red O staining assay showed more lipid droplets. Our study revealed the expression profile of circRNAs in the adipose tissue of old rats. We found a novel age-related circular RNA—circ-ATXN2—that inhibits proliferation and promotes cell death and adipogenesis in rat adipose tissue-derived stromal cells.

The Initial Cardiac Tissue Response to Cryopreserved Allogeneic Adipose Tissue-Derived Mesenchymal Stromal Cells in Rats with Chronic Ischemic Cardiomyopathy

Mesenchymal stromal cells have proven capable of improving cardiac pump function in patients with chronic heart failure, yet little is known about their mode of action. The aim of the study was to investigate the short-term effect of cryopreserved allogeneic rat adipose tissue-derived stromal cells (ASC) on cardiac composition, cellular subpopulations, and gene transcription in a rat model of chronic ischemic cardiomyopathy (ICM). Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. After 6 weeks, the rats were treated with ASCs, saline, or no injection, using echo-guided trans-thoracic intramyocardial injections. The cardiac tissue was subsequently collected for analysis of cellular subpopulations and gene transcription 3 and 7 days after treatment. At day 3, an upregulation of genes associated with angiogenesis were present in the ASC group. On day 7, increases in CCR2+ and CD38+ macrophages (p = 0.047 and p = 0.021), as well as in the CD4/CD8 lymphocyte ratio (p = 0.021), were found in the ASC group compared to the saline group. This was supported by an upregulation of genes associated with monocytes/macrophages. In conclusion, ASC treatment initiated an immune response involving monocytes/macrophages and T-cells and induced a gene expression pattern associated with angiogenesis and monocyte/macrophage differentiation.

Palmitic Acid Methyl Ester Enhances Adipogenic Differentiation in Rat Adipose Tissue-Derived Mesenchymal Stem Cells through a G Protein-Coupled Receptor-Mediated Pathway

Adipogenic differentiation from stem cells has become a research target due to the increasing interest in obesity. It has been indicated that adipocytes can secrete palmitic acid methyl ester (PAME), which is able to regulate stem cell proliferation. However, the effects of PAME on adipogenic differentiation in stem cell remain unclear. Here, we present that the adipogenic differentiation medium supplemented with PAME induced the differentiation of rat adipose tissue-derived mesenchymal stem cells (rAD-MSCs) into adipocyte. rAD-MSCs were treated with PAME for 12 days and then subjected to various analyses. The results from the present study show that PAME significantly increased the levels of adipogenic differentiation markers, PPARγ and Gpd1, and enhanced adipogenic differentiation in rAD-MSCs. Furthermore, the level of GPR40/120 protein increased during induction of adipocyte differentiation in rAD-MSCs. Cotreatment with PAME and a GPR40/120 antagonist together inhibited the PAME-enhanced adipogenic differentiation. Moreover, PAME significantly increased phosphorylation of extracellular signal-regulated kinases (ERK), but not AKT and mTOR. Cotreatment with PAME and a GPR40/120 antagonist together inhibited the PAME-enhanced ERK phosphorylation and adipogenic differentiation. PAME also increased the intracellular Ca2+ levels. Cotreatment with PAME and a Ca2+ chelator or a phospholipase C (PLC) inhibitor prevented the PAME-enhanced ERK phosphorylation and adipogenic differentiation. Our data suggest that PAME activated the GPR40/120/PLC-mediated pathway, which in turn increased the intracellular Ca2+ levels, thereby activating the ERK, and eventually enhanced adipogenic differentiation in rAD-MSCs. The findings from the present study might help get insight into the physiological roles and molecular mechanism of PAME in regulating stem cell differentiation.

Effects of different doses of melatonin on rat adipose derived mesenchymal stem cells

Objectives Stem cell treatment is based on Melatonin which is crucial for lots of pathological and physiological pathways. Our aim is determining the most appropriate dose of melatonin affecting the rat adipose tissue mesenchymal stem cells. Methods Stem cells were isolated from male rat adipose tissue. Differentiation and characterization experiments were performed. Cell viability analyses in stem cells were used the XTT [2,3-Bis-(2-methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide] assay. After 24 h incubation, different concentrations (0.5, 1, 5, 10, 50 µM) of extract were treated to the stem cells for 24 h, 48 and 72 h considering time and dose dependent manner. Total antioxidant status (TAS) and the total oxidant status (TOS) in control cells and melatonin treated cells (5, 10 µM) were determined Rel Assay commercial kits. Results In 24 h, melatonin increased cell viability in all groups. When we evaluate the effect of melatonin in 48 h, the most proliferation increase was seen at 5, 10 µM doses. When the total oxidant activity melatonin was found to be significantly lower in 5 and 10 µM dose groups of melatonin. Conclusions Melatonin increases the survivor of stem cells and the most effective dose is 5 and 10 µM. The reduction of the oxidative stress index as a result of treating melatonin to mesenchymal stem cells showed that melatonin is a powerful antioxidant for stem cells.

Transduction of rat and human adipose-tissue derived mesenchymal stromal cells by adeno-associated viral vector serotype DJ

Ex vivo, gene therapy is a powerful approach holding great promises for the treatment of both genetic and acquired diseases. Adeno-associated virus (AAV) vectors are safe and efficient delivery system for modification of mesenchymal stem cells (MSC) that could maximize their therapeutic benefits. Assessment to MSC viability and functional activity after infection with new AAV serotypes is necessary, due to AAV tropism to specific cell types. We infected human and rat adipose-tissue MSC with hybrid AAV-DJ serotype vectors carrying GFP and SCF genes. GFP expression from AAV-DJ was about 1.5-fold superior to that observed with AAV-2 and lasted for at least 21 days as was evaluated by flow cytometry and fluorescence microscopy. AAV-DJ proves to be suitable for the infection of rat and human MSC with a similar efficiency. Infected MSC were still viable however showing 25-30%. growth rate slowdown. Moreover, we found increase of SERPINB2 mRNA expression in human MSC whereas expression of other oxidative stress markers and extracellular matrix proteins was not affected. These results suggest that there is a differential cellular response in MSC infected with AAV viral vectors, which should be taken into account as it can affect the expected outcome for the therapeutic application.