Qiping Shi

Morphology and mechanics of chondroid cells from human adipose-derived Stem cells detected by atomic force microscopy

Chondroid cell from human adipose-derived stem cells (ADSCs) has emerged as an alternative treatment option for articular cartilage defects. Herein, we successfully compared ADSCs, normal chondrocytes, and chondroid cells. The comparative study of ADSCs and chondroid cells revealed type II collagen (COL II) and glycosaminoglycans expression of chondroid cells were similar to those in normal chondrocytes, and much higher than ADSCs. Using atomic force microscope (AFM) and laser confocal scanning microscopy (LCSM), we compared the differences in morphology, mechanical properties, and F-actin distribution between chondroid cells and normal chondrocytes. Our results showed no differences observed between these two types of cells regarding morphology, stiffness, and F-actin distribution. However, found that the adhesion force in chondroid cells was lower than that in normal chondrocytes. Taken together, our AFM and LCSM analyses suggest that the lower adhesion force in chondroid cells may contribute to the dedifferentiation of ADSC-derived chondroid cells. Future examination of surface adhesion force-related protein expression will likely provide new insight into the molecular mechanisms underlying the dedifferentiation of ADSC-derived chondroid cells.

The roles of integrin β1 in phenotypic maintenance and dedifferentiation in chondroid cells differentiated from human adipose-derived stem cells

ObjectiveThe aim of this study is to probe the intrinsic mechanism of chondroid cell dedifferentiation in order to provide a feasible solution for this in cell culture.MethodsMorphological and biomechanical properties of cells undergoing chondrogenic differentiation from human adipose-derived stem cells (ADSCs) were measured at the nanometer scale using atomic force microscopy and laser confocal scanning microscopy. Gene expression was determined by real-time quantitative polymerase chain reaction.ResultsThe expression of COL II, SOX9, and Aggrecan mRNA began to increase gradually at the beginning of differentiation and reach a peak similar to that of normal chondrocytes on the 12th day, then dropped to the level of the 6th day at 18th day. Cell topography and mechanics trended resembled those of the genes’ expression. Integrin β1 was expressed in ADSCs and rapidly upregulated during differentiation but downregulated after reaching maturity.ConclusionsThe amount and distribution of integrin β1 may play a critical role in mediating both chondroid cell maturity and dedifferentiation. Integrin β1 is a possible new marker and target for phenotypic maintenance in chondroid cells.

Wnt/β-catenin signaling may be involved with the maturation, but not the differentiation, of insulin-producing cells.

Wnt/β-catenin signaling (WNT) has widespread roles during stem cell differentiation. Whether WNT suppresses or promotes insulin-producing cell (IPC) differentiation and function is still not known. In this study, we investigated the role of WNT signaling during human adipose-derived stem cell (hADSC) differentiation into IPCs. Western blot analysis revealed that several key components of WNT were dynamically regulated in a 12-day IPC differentiation assay. Specifically, protein levels of Wnt1, β-catenin, and GSK3β steadily increased from day 0 to day 9 and rapidly decreased by day 12 of differentiation. Similarly, endonuclear β-catenin levels peaked at day 9 and then, fell to pre-differentiation levels. The expression of two WNT pathway targets, TCF-1 and cyclin D1, closely followed the same pattern of regulation, confirming that WNT signaling was transiently activated during IPC differentiation. Interestingly, the inhibition of WNT signaling did not block IPC differentiation; instead, it resulted in the upregulation of IPC-specific markers, including PDX-1, insulin, IRS-1, and IRS-2. Notably, another IPC marker, glucokinase, remained downregulated since it is a direct target of WNT signaling. Next, we examined the effect of maintaining active WNT signaling from day 9 to day 12 of IPC differentiation. Differentiating cells were treated with Wnt1 on day 9, when WNT signaling is typically turned off, and subjected to gene expression analysis on day 12. Remarkably, Wnt1 treatment resulted in reduced expression of IPC-specific markers. Taken together, these data indicate that WNT may not be necessary for IPC differentiation but may be involved in IPC maturation.

Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

ObjectiveThe aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level.MethodsThe levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy.ResultsIPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs.ConclusionsOur results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells.

Mitochondrial ROS activate interleukin‑1β expression in allergic rhinitis

Allergic rhinitis (AR) is the most common cause of inflammation of the nasal mucosa. It is also the most common form of non-infectious rhinitis associated with an immunoglobulin E (IgE)-mediated immune response against allergens. Previous studies have indicated that interleukin-1β (IL-1β) has a pathological role in the development of allergic asthma. The present study was designed to assess whether IL-1β participates in the pathogenesis of AR. A total of 45 patients with AR were enrolled in the present study and were identified to have increased IL-1β expression expressed by peripheral blood mononuclear cells (PBMCs), and the mitochondrial reactive oxygen species (ROS) and NLRP3 are required for IL-1β synthesis in monocytes/macrophages and PBMCs from patients with AR. The levels of IL-1β and interleukin-17 (IL-17) were increased in patients with AR and were positively correlated with each other. The results of the present study suggested that patients with AR have raised mitochondrial ROS levels, which may upregulate the expression of IL-1β, affecting IL-17-production and serving a role in the pathogenesis of AR.

Expression and Significance of MMPs in Synovial Fluid, Serum and PBMC Culture Supernatant Stimulated by LPS in Osteoarthritis Patients With or Without Diabetes

OBJECTIVE Patients with Type 2 Diabetes mellitus (T2DM) are prone to osteoarthritis (OA). Matrix metalloproteinases (MMPs), an essential modulator in cartilage matrix homeostasis, increase in T2DM and OA. We aimed to ascertain the expression difference of MMPs and function in mononuclear cells after stimulating by lipopolysaccharide (LPS) in OA patients with or without diabetes. METHODS 30 knee OA patients without T2DM (OA group), 20 knee OA patients with T2DM (DM-OA group) and 5 healthy volunteers recruited as control were enrolled from January 2016 to January 2017. The expression levels of MMPs in both serum and synovial fluid were initially detected in three groups by enzyme-linked immunosorbent assay (ELISA). After stimulation of peripheral blood mononuclear cell (PBMC) with LPS, the release of MMPs were determined and evaluated. RESULTS The expression of MMP-1, -7, -8, -9, -10 and -12 in synovial fluid in DM-OA group were significantly higher than in OA group and healthy control. The expression of MMP-1 and -7 in serum were highest in DM-OA group. LPS significantly promotes the production of MMP-1, -8, -9 and -10 in PBMC of each group after 4 h stimulation. It is worth to note that the LPS-stimulated MMP-8 and -9 elevations were more prominent in DM-OA group compared with their counterparts. CONCLUSION High levels of MMP-1, -7, -8, -9, -10, and -12 in the synovial fluid might be one of important reasons that diabetes patients are more frequently suffered from OA. Inflammation-induced malfunction of mononuclear cells would stimulate MMP-8 and -9 secretion to various extents.

GLP-1 could improve the similarity of IPCs and pancreatic beta cells in cellular ultrastructure and function.

Transplantation of functional insulin‐producing cells (IPCs) provides a novel mode for insulin replacement, but is often accompanied by many undesirable side effects. Our previous studies suggested that IPCs could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. To obtain a better method through which to acquire more similar IPCs, we compared the difference between IPCs of the GLP‐1 group and IPCs of the non‐GLP‐1 group in the morphological features in cellular level and physiological function. The levels of insulin secretion were measured by ELISA. The insulin and glucagon‐like peptide‐1 (GLP‐1) mRNA gene expression was determined by real‐time quantitative PCR. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy (LCSM). Intracellular Ca2+ levels and Glucagon‐like peptide‐1 receptor (GLP‐1R) levels were determined by flow cytometer (FCM). We found that IPCs of the GLP‐1 group had bigger membrane particle size and average roughness (Ra) than IPCs of the non‐GLP‐1 group but still smaller than normal human pancreatic beta cells. The physiology function of IPCs of the GLP‐1 group were much closer to normal human pancreatic beta cells than IPCs of the non‐GLP‐1 group. GLP‐1 could improve the similarity of IPCs from human adipose tissue‐derived mesenchymal stem cells and pancreatic beta cells in cellular ultrastructure and function. J. Cell. Biochem. 114: 2221–2230, 2013.