Serum regulates adipogenesis of mesenchymal stem cells via MEK/ERK‐dependent PPARγ expression and phosphorylation

Mesenchymal stem cells (MSCs) provide us an excellent cellular model to uncover the molecular mechanisms underlying adipogenic differentiation of adult stem cells. PPARγ had been considered as an important molecular marker of cells undergoing adipogenic differentiation. Here, we demonstrated that expression and phosphorylation of PPARγ could be found in bone marrow–derived MSCs cultured in expansion medium without any adipogenic additives (dexamethasone, IBMX, insulin or indomethacin). Then, PPARγ was dephosphorylated in MSCs during the process of adipogenic differentiation. We then found that inhibition of MEK activation by specific inhibitor (PD98059) counteracted the PPARγ expression and phosphorylation. However, expression and phosphorylation of PPARγ did not present in MSCs cultured in medium with lower serum concentration. When these MSCs differentiated into adipocytes, no phosphorylation could be detected to accompany the expression of PPARγ. Moreover, exposure of MSCs to higher concentration of serum induced stronger PPARγ expression, and subsequently enhanced their adipogenesis. These data suggested that activation of the MEK/ERK signalling pathway by high serum concentration promoted PPARγ expression and phosphorylation, and subsequently enhanced adipogenic differentiation of MSCs.     

Induction of γδT cells from HSC‐enriched BMCs co‐cultured with iPSC‐derived thymic epithelial cells

T cells bearing γδ antigen receptors have been investigated as potential treatments for several diseases, including malignant tumours. However, the clinical application of γδT cells has been hampered by their relatively low abundance in vivo and the technical difficulty of inducing their differentiation from hematopoietic stem cells (HSCs) in vitro. Here, we describe a novel method for generating mouse γδT cells by co‐culturing HSC‐enriched bone marrow cells (HSC‐eBMCs) with induced thymic epithelial cells (iTECs) derived from induced pluripotent stem cells (iPSCs). We used BMCs from CD45.1 congenic C57BL/6 mice to distinguish them from iPSCs, which expressed CD45.2. We showed that HSC‐eBMCs and iTECs cultured with IL‐2 + IL‐7 for up to 21 days induced CD45.1⁺ γδT cells that expressed a broad repertoire of Vγ and Vδ T‐cell receptors. Notably, the induced lymphocytes contained few or no αβT cells, NK1.1⁺ natural killer cells, or B220⁺ B cells. Adoptive transfer of the induced γδT cells to leukemia‐bearing mice significantly reduced tumour growth and prolonged mouse survival with no obvious side effects, such as tumorigenesis and autoimmune diseases. This new method suggests that it could also be used to produce human γδT cells for clinical applications.     

Characterization of age‐associated exhausted CD8+ T cells defined by increased expression of Tim‐3 and PD‐1

Aging is accompanied by altered T‐cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD‐1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T‐cell exhaustion and aging‐associated immune dysfunction. In this study, we demonstrate that T‐cell immunoglobulin mucin domain‐3 (Tim‐3), another exhaustion marker, is up‐regulated on aged T cells, especially CD8⁺ T cells. Tim‐3‐expressing cells also produced PD‐1, but Tim‐3⁺PD‐1⁺ CD8⁺ T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim‐3^?PD‐1⁺ cells. Tim‐3⁺PD‐1⁺ CD8⁺ T cells showed more evident properties associated with exhaustion than Tim‐3^?PD‐1⁺ CD8⁺ T cells: an exhaustion‐related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL‐10 and induced normal CD8⁺ T cells to produce IL‐10, which might contribute to immune dysregulation in aged mice. The generation of Tim‐3‐expressing CD8⁺ T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8⁺ T‐cell population with age‐associated exhaustion was distinguishable by its expression of Tim‐3. These results provide clues for understanding the alterations that occur in T‐cell populations with age and for improving dysfunctions related to the aging of the immune system.     

Long non‐coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR‐204‐5p and miR‐125a‐3p

A growing number of long non‐coding RNAs (lncRNAs) have been found to be involved in diverse biological processes such as cell cycle regulation, embryonic development, and cell differentiation. However, limited knowledge is available concerning the underlying mechanisms of lncRNA functions. In this study, we found down‐regulation of TCONS_00041960 during adipogenic and osteogenic differentiation of glucocorticoid‐treated bone marrow mesenchymal stem cells (BMSCs). Furthermore, up‐regulation of TCONS_00041960 promoted expression of osteogenic genes Runx2, osterix, and osteocalcin, and anti‐adipogenic gene glucocorticoid‐induced leucine zipper (GILZ). Conversely, expression of adipocyte‐specific markers was decreased in the presence of over‐expressed TCONS_00041960. Mechanistically, we determined that TCONS_00041960 as a competing endogenous RNA interacted with miR‐204‐5p and miR‐125a‐3p to regulate Runx2 and GILZ, respectively. Overall, we identified a new TCONS_00041960‐miR‐204‐5p/miR‐125a‐3p‐Runx2/GILZ axis involved in regulation of adipogenic and osteogenic differentiation of glucocorticoid‐treated BMSCs.     

Bone marrow‐derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion‐induced lung injury

Autophagy, a type II programmed cell death, is essential for cell survival under stress, e.g. lung injury, and bone marrow‐derived mesenchymal stem cells (BM‐MSCs) have great potential for cell therapy. However, the mechanisms underlying the BM‐MSC activation of autophagy to provide a therapeutic effect in ischaemia/reperfusion‐induced lung injury (IRI) remain unclear. Thus, we investigate the activation of autophagy in IRI following transplantation with BM‐MSCs. Seventy mice were pre‐treated with BM‐MSCs before they underwent lung IRI surgery in vivo. Human pulmonary micro‐vascular endothelial cells (HPMVECs) were pre‐conditioned with BM‐MSCs by oxygen‐glucose deprivation/reoxygenation (OGD) in vitro. Expression markers for autophagy and the phosphoinositide 3‐kinase/protein kinase B (PI3K/Akt) signalling pathway were analysed. In IRI‐treated mice, administration of BM‐MSCs significantly attenuated lung injury and inflammation, and increased the level of autophagy. In OGD‐treated HPMVECs, co‐culture with BM‐MSCs attenuated endothelial permeability by decreasing the level of cell death and enhanced autophagic activation. Moreover, administration of BM‐MSCs decreased the level of PI3K class I and p‐Akt while the expression of PI3K class III was increased. Finally, BM‐MSCs‐induced autophagic activity was prevented using the inhibitor LY294002. Administration of BM‐MSCs attenuated lung injury by improving the autophagy level via the PI3K/Akt signalling pathway. These findings provide further understanding of the mechanisms related to BM‐MSCs and will help to develop new cell‐based therapeutic strategies in lung injury.     

Significant curative functions of the mesenchymal stem cells on methotrexate‐induced kidney and liver injuries in rats

Mesenchymal stem cells (MSCs) curative effects on methotrexate (MTX)‐induced kidney and liver injuries remain elusive. Therefore, rats were divided into five groups, rats received MTX orally (14 mg/kg) as a single dose/week for 2 weeks, groups 3 and 4 were injected once with 2 × 10⁶ cells bone marrow MSCs and adipose‐derived MSCs, respectively. The last group administered dexamethasone (DEX) (0.5 mg/kg, p.o) for 7 days. MTX caused marked increase in malondialdehyde and nitrite/nitrate concentrations. However, MTX administration decreased reduced glutathione content plus catalase activity. In addition, MTX caused a significant increment in kidney and liver biomarkers levels. Moreover, MTX showed renal tubules vacuolation and necrosis of hepatocytes, as well expression of caspase‐3 and nuclear factor kappa beta in kidney and liver tissues were observed. MSCs treatment alleviated previous side effects induced by MTX. MSCs improved nephrotoxicity and hepatotoxicity induced by MTX to a better extent as compared with DEX.