Notch signalling inhibits the adipogenic differentiation of single‐cell‐derived mesenchymal stem cell clones isolated from human adipose tissue

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.     

Wnt/β-Catenin Pathway Regulates Cementogenic Differentiation of Adipose Tissue-Deprived Stem Cells in Dental Follicle Cell-Conditioned Medium

The formation and attachment of new cementum is crucial for periodontium regeneration. Tissue engineering is currently explored to achieve complete, reliable and reproducible regeneration of the periodontium. The capacity of multipotency and self-renewal makes adipose tissue-deprived stem cells (ADSCs) an excellent cell source for tissue regeneration and repair. After rat ADSCs were cultured in dental follicle cell-conditioned medium (DFC-CM) supplemented with DKK-1, an inhibitor of the Wnt pathway, followed by 7 days of induction, they exhibited several phenotypic characteristics of cementoblast lineages, as indicated by upregulated expression levels of CAP, ALP, BSP and OPN mRNA, and accelerated expression of BSP and CAP proteins. The Wnt/β-catenin signaling pathway controls differentiation of stem cells by regulating the expression of target genes. Cementoblasts share phenotypical features with osteoblasts. In this study, we demonstrated that culturing ADSCs in DFC-CM supplemented with DKK-1 results in inhibition of β-catenin nuclear translocation and down-regulates TCF-4 and LEF-1 mRNA expression levels. We also found that DKK-1 could promote cementogenic differentiation of ADSCs, which was evident by the up-regulation of CAP, ALP, BSP and OPN gene expressions. On the other hand, culturing ADSCs in DFC-CM supplemented with 100 ng/mL Wnt3a, which activates the Wnt/β-catenin pathway, abrogated this effect. Taken together, our study indicates that the Wnt/β-catenin signaling pathway plays an important role in regulating cementogenic differentiation of ADSCs cultured in DFC-CM. These results raise the possibility of using ADSCs for periodontal regeneration by modifying the Wnt/β-catenin pathway.     

组蛋白去乙酰化酶（HDAC）在间充质干细胞C3H10T1/2成脂分化过程中的表达及HDAC11的作用

组蛋白去乙酰化酶（histone deacetylase, HDAC）通过参与调节组蛋白乙酰化修饰调控基因表达. 研究发现多种HDAC参与成脂分化,但其机制尚不清楚. 本研究旨在探讨间充质干细胞C3H10T1/2成脂分化过程中组蛋白去乙酰化酶（HDAC）的表达变化及其对成脂分化的影响. 本研究首先建立了C3H10T1/2体外成脂分化的模型并以油红O染色鉴定成功诱导成脂分化. PCR检测C3H10T1/2细胞成脂分化过程中11种HDAC的变化趋势,发现成脂分化过程中,HDAC1、2、5、9和10的mRNA表达量下降而HDAC3、6、8和11的mRNA表达量明显上升,其中HDAC11上升最为显著. 进一步通过RNA干扰沉默HDAC11表达, PCR检测成脂分化的关键转录因子PPARγ2和成脂标志物Perilipin、Adipoq 的mRNA表达量下降,但Fabp4表达变化不明显. 油红O染色结果表明,诱导C3H10T1/2成脂分化过程中,干扰HDAC11表达,胞浆内脂滴形成数量减少,成脂分化受到抑制. 总之,我们实验的结果提示C3H10T1/2细胞成脂分化伴随着多种HDAC表达的变化,其中HDAC11的增加最显著,干扰HDAC11的表达可以抑制C3H10T1/2细胞的成脂分化.     

Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-β1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-β1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-β1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-β1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-β1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.     

The possible role of TGF-beta-induced suppressors of cytokine signaling expression in osteoclast/macrophage lineage commitment in vitro

Osteoclast formation is dependent on the ability of TGF-beta to enable receptor activator of NF-kappaB ligand (RANKL)-induced commitment of hemopoietic precursors to the osteoclastic lineage. The mechanism by which TGF-beta enables formation is unknown. One possibility is that TGF-beta opposes Janus kinase (JAK)/STAT signals generated by inhibitory cytokines such as IFN-beta. The JAK/STAT pathway is activated by cytokines that induce resistance to osteoclast formation, such as IFN-gamma and M-CSF, and the effect of these is opposed by TGF-beta. Recently, a group of STAT-induced factors, termed suppressors of cytokine signaling (SOCS), has been identified that inhibit JAK/STAT signals. Therefore, we tested the ability of TGF-beta to induce SOCS expression in osteoclast precursors and examined the effect of SOCS expression on osteoclast/macrophage lineage commitment. We found that while SOCS mRNA is undetectable in macrophages, osteoclasts express SOCS-3, and TGF-beta up-regulates this expression. Furthermore, TGF-beta rapidly induces sustained SOCS-3 expression in macrophage/osteoclast precursors. To determine whether SOCS-3 plays a role in osteoclast differentiation we expressed SOCS-3 in precursors using a retroviral system. We found that osteoclast differentiation was significantly enhanced in SOCS-3-infected precursors, and SOCS-3 expression enables formation in the presence of anti-TGF-beta Ab. On the other hand, antisense knockdown of SOCS-3 strongly suppressed osteoclast formation and significantly blunted the response to TGF-beta. Moreover, like TGF-beta, SOCS-3 expression opposed the inhibitory effect of IFN-beta. These data suggest that TGF-beta-induced expression of SOCS-3 may represent a mechanism by which TGF-beta suppresses inhibitory cytokine signaling, priming precursors for a role in bone resorption.     

In vitro cardiomyogenic differentiation of adipose‐derived stromal cells using transforming growth factor‐β1

Transplanting stem cells differentiated towards a cardiac lineage can regenerate cardiac muscle tissues to treat myocardial infarction. In this study, we tested the hypothesis that transforming growth factor‐β1 (TGF‐β1) induces cardiomyogenic differentiation of adipose‐ derived stromal cells (ADSCs) in vitro. Rat ADSCs were cultured with TGF‐β1 (10 ng ml^?1) for 2 weeks in vitro. ADSCs cultured without TGF‐β1 served as a control. The mRNA expression of cardiac‐specific gene was induced by TGF‐β1, while the control culture did not show cardiac‐specific gene expression. Immunocytochemical analyses showed that a small fraction of ADSCs cultured with TGF‐β1 for 2 weeks stained positively for cardiac myosin heavy chain (MHC) and α‐sarcomeric actin. Flow cytometric analyses showed that the proportion of cells expressing cardiac MHC increased with TGF‐β1. However, no mesenchymal differentiation (e.g., osteogenic and adipogenic differentiation) was detected other than cardiomyogenic differentiation. These results showed that TGF‐β1 induce ADSC cardiomyogenic differentiation in vitro, which could be useful for myocardial infarction stem cell therapy. Copyright © 2009 John Wiley & Sons, Ltd.     

MicroRNA 140 Promotes Expression of Long Noncoding RNA NEAT1 in Adipogenesis

More than 40% of the U.S. population are clinically obese and suffer from metabolic syndrome with an increased risk of postmenopausal estrogen receptor-positive breast cancer. Adipocytes are the primary component of adipose tissue and are formed through adipogenesis from precursor mesenchymal stem cells. While the major molecular pathways of adipogenesis are understood, little is known about the noncoding RNA signaling networks involved in adipogenesis. Using adipocyte-derived stem cells (ADSCs) isolated from wild-type and microRNA 140 (miR-140) knockout mice, we identify a novel miR-140/long noncoding RNA (lncRNA) NEAT1 signaling network necessary for adipogenesis. miR-140 knockout ADSCs have dramatically decreased adipogenic capabilities associated with downregulation of NEAT1 expression. We identified a miR-140 binding site in NEAT1 and found that mature miR-140 in the nucleus can physically interact with NEAT1, leading to increased NEAT1 expression. We demonstrated that reexpression of NEAT1 in miR-140 knockout ADSCs is sufficient to restore their ability to undergo differentiation. Our results reveal an exciting new noncoding RNA signaling network that regulates adipogenesis and that is a potential new target in the prevention or treatment of obesity.     

Effects of BMP-2 and vitamin D3 on the osteogenic differentiation of adipose stem cells

A theoretical inverse relationship has long been postulated for osteogenic and adipogenic differentiation (bone versus adipose tissue differentiation). This inverse relationship in theory at least partially underlies the clinical entity of osteoporosis, in which marrow mesenchymal stem cells (MSCs) have a predilection for adipose differentiation that increases with age. In the present study, we assayed the potential anti-adipogenic effects of Nell-1 protein (an osteoinductive molecule). Using 3T3-L1 (a human preadipocyte cell line) cells and human adipose-derived stromal cells (ASCs), we observed that adenoviral delivered (Ad)-Nell-1 or recombinant NELL-1 protein significantly reduced adipose differentiation across all markers examined (Oil red O staining, adipogenic gene expression [Pparg, Lpl, Ap2]). In a prospective fashion, Hedgehog signaling was assayed as potentially downstream of Nell-1 signaling in regulating osteogenic over adipogenic differentiation. In comparison to Ad-LacZ control, Ad-Nell-1 increased expression of hedgehog signaling markers (Ihh, Gli1, Ptc1). These studies suggest that Nell-1 is a potent anti-adipogenic agent. Moreover, Nell-1 signaling may inhibit adipogenic differentiation via a Hedgehog dependent mechanism.     

Regulation of suppressor of cytokine signaling 3 (SOCS3) mRNA stability by TNF-alpha involves activation of the MKK6/p38MAPK/MK2 cascade

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-alpha. In contrast to IL-6, TNF-alpha increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38(MAPK)-cascade is required for TNF-alpha-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38(MAPK) cascade, basal SOCS3-expression is strongly reduced and TNF-alpha-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38(MAPK)-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3' untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-alpha. In summary, these data indicate that TNF-alpha regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38(MAPK) cascade and that the activation of MK2, a downstream target of p38(MAPK), is important for the regulation of SOCS3 expression.     

SOCS3 inhibits insulin signaling in porcine primary adipocytes

Insulin resistance is a major player in the pathogenesis of type II diabetes, the metabolic syndrome, and obesity. SOCS3 plays an important role in the development of insulin resistance. To investigate the role of SOCS3 in porcine adipocyte insulin signaling, we first detected the effect of insulin on SOCS3 mRNA and protein expression in porcine primary adipocytes by real-time RT-PCR and Western blotting. Then, we constructed a recombinant adenovirus encoding SOCS3 gene (Ad-SOCS3) which was used to infect differentiated porcine primary adipocytes for 3 days. The expression and phosphorylation of main insulin signaling components were detected by Western blotting. The results showed that 100 nM insulin could induce SOCS3 mRNA expression but not protein expression, and overexpression of SOCS3 decreased IRS1 protein level, insulin-stimulated IRS1 tyrosine phosphorylation, PI3K activation, and Akt phosphorylation, but increased IRS1 serine phosphorylation in porcine primary adipocytes. These results indicate that SOCS3 is an important negative regulator of insulin signaling in porcine adipocytes. Thus, SOCS3 may be a novel therapeutic target for the prevention or treatment of insulin resistance and type II diabetes.