BMP3 Alone and Together with TGF-β Promote the Differentiation of Human Mesenchymal Stem Cells into a Nucleus Pulposus-Like Phenotype

Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)-β on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-β, and compared with cultures in basal and TGF-β media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF-β on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-β, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-β, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF-β induced positive effects. These actions are likely related to the activation of TGF-β signaling pathway.     

Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules

Human pluripotent stem cells, including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), hold promise as novel therapeutic tools for diabetes treatment because of their self-renewal capacity and ability to differentiate into beta (β)-cells. Small and large molecules play important roles in each stage of β-cell differentiation from both hESCs and hiPSCs. The small and large molecules that are described in this review have significantly advanced efforts to cure diabetic disease. Lately, effective protocols have been implemented to induce hESCs and human mesenchymal stem cells (hMSCs) to differentiate into functional β-cells. Several small molecules, proteins, and growth factors promote pancreatic differentiation from hESCs and hMSCs. These small molecules (e.g., cyclopamine, wortmannin, retinoic acid, and sodium butyrate) and large molecules (e.g. activin A, betacellulin, bone morphogentic protein (BMP4), epidermal growth factor (EGF), fibroblast growth factor (FGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), noggin, transforming growth factor (TGF-α), and WNT3A) are thought to contribute from the initial stages of definitive endoderm formation to the final stages of maturation of functional endocrine cells. We discuss the importance of such small and large molecules in uniquely optimized protocols of β-cell differentiation from stem cells. A global understanding of various small and large molecules and their functions will help to establish an efficient protocol for β-cell differentiation.     

A Novel Human TGF-β1 Fusion Protein in Combination with rhBMP-2 Increases Chondro-Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells

Transforming growth factor-beta (TGF-β) is involved in processes related to the differentiation and maturation of osteoprogenitor cells into osteoblasts. Rat bone marrow (BM) cells were cultured in a collagen-gel containing 0.5% fetal bovine serum (FBS) for 10 days in the presence of rhTGF (recombinant human TGF)-β1-F2, a fusion protein engineered to include a high-affinity collagen-binding decapeptide derived from von Willebrand factor. Subsequently, cells were moderately expanded in medium with 10% FBS for 4 days and treated with a short pulse of rhBMP (recombinant human bone morphogenetic protein)-2 for 4 h. During the last 2 days, dexamethasone and β-glycerophosphate were added to potentiate osteoinduction. Concomitant with an up-regulation of cell proliferation, DNA synthesis levels were determined. Polymerase chain reaction was performed to reveal the possible stemness of these cells. Osteogenic differentiation was evaluated in terms of alkaline phosphatase activity and mineralized matrix formation as well as by mRNA expression of osteogenic marker genes. Moreover, cells were placed inside diffusion chambers and implanted subcutaneously into the backs of adult rats for 4 weeks. Histological study provided evidence of cartilage and bone-like tissue formation. This experimental procedure is capable of selecting cell populations from BM that, in the presence of rhTGF-β1-F2 and rhBMP-2, achieve skeletogenic potential in vitro and in vivo.     

Lipofundin Mediates the Major Inhibition of Intravenous Propofol in IL-1β Secretion and Phagocytosis of Staphylococcus aureus-Infected Macrophages

Lipofundin is the solvent for propofol in the intravenous injection of Propofol-Lipuro® and is used in patients who need intravenous feeding to provide fatty acids and fat for energy. In addition to propofol, Lipofundin also affects the immune modulation of phagocytes. In a previous study, we reported that intravenous propofol effectively decreased Staphylococcus aureus-stimulated reactive oxygen species (ROS) levels, IL-1β secretion, and phagocytosis in RAW264.7 macrophages. It is important to separately assess the effects of pure propofol, Lipofundin, and Propofol-Lipuro. By using an S. aureus-infected RAW264.7 macrophage model, the levels of secreted IL-1β in cell supernatants were determined by ELISA. IL-1β mRNA in cell pellets was further analyzed by quantitative polymerase chain reaction (qPCR), and Western blotting was performed to detect pro-IL-1β synthesis. Total ROS levels were determined by a luminol chemiluminescence assay. Compared with pure propofol, treatment with clinically relevant concentrations of Propofol-Lipuro and Lipofundin obviously reduced IL-1β secretion (>85% inhibition), S. aureus-stimulated ROS production (50% inhibition), and phagocytosis (>60% inhibition) to similar levels. Treatment with pure propofol alone significantly decreased IL-1β mRNA levels and pro-IL-1β protein synthesis, and slightly inhibited phagocytosis. In contrast, treatment with Propofol-Lipuro did not influence IL-1β mRNA or pro-IL-1β protein expression, even though treatment with Lipofundin increased the levels of both IL-1β mRNA and its precursor protein. In conclusion, IL-1β secretion is regulated at the posttranslational level. Lipofundin mediated the major effect of Propofol-Lipuro on the inhibition of IL-1β secretion, ROS production, and phagocytosis in S. aureus-infected RAW264.7 cells.