Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Abstract. Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G₁ phase of the cell cycle. These include the predifferentiation arrest state (G_D) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (G_S), and the nutrient deficiency arrest state (G_N). Cells at states of reversible nonterminal differentiation (G_D?) and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at G_D, G_S and G_N states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [³⁵S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the G_S, G_D, G_D?. and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the G_D? and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.     

Antidiabetic AD4743 enhances adipocyte differentiation of 3T3 T mesenchymal stem cells

AD4743 is an antidiabetic agent that, when added to fetal bovine serum (FBS), has been shown to have adipogenic activity for some proadipocyte cell lines once they reach confluence. In the current study, the effects of AD4743 on the growth and adipocytic differentiation of 3T3 T multipotential mesenchymal stem cells have been tested. 3T3 T cells, unlike other cells capable of undergoing adipocyte differentiation, are routinely induced to differentiate at low cell density. This is done using platelet-poor human plasma (HP), a potent inducer of growth arrest and differentiation. AD4743 (0-200 micrograms/ml) was tested in varied concentrations of HP or FBS, at varied cell densities, and at various times during growth and differentiation. AD4743 slowed the growth rate of 3T3 T cells and it induced their differentiation in a dose-dependent manner in medium containing 10% FBS once they reached confluence. The data suggest that the ability of AD4743 to inhibit growth may also be coupled with its ability to enhance differentiation. In addition, AD4743 (1-10 micrograms/ml) in the presence of 25% HP markedly increased the kinetics of adipocyte differentiation, at low (less than 5,000 cells/cm2) or high cell density. Greater than 50% cell differentiation could be achieved in 2 days in low density cultures; 80-95% differentiation could be achieved in just 4 days, compared to 8-12 days in a typical culture. The maximum amount of differentiation in HP was potentiated by AD4743 to a greater degree in poor lots of HP; however, the kinetics were increased in all lots. Adipocytic differentiation was measured both morphologically and by Northern blot analyses of differentiation-specific genes. AD4743 at 1-10 micrograms/ml appeared to be most effective, depending on the cell density and other conditions. The mechanism of action of AD4743 remains to be elucidated, but the enhancement of adipocyte differentiation does not appear to occur via an insulin-dependent pathway.     

Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO)

Abstract. The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 nonterminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growtharrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.     

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells

BACKGROUND: Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS: A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS: In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS: These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.     

Transforming growth factor type beta is a specific inhibitor of 3T3 T mesenchymal stem cell differentiation

In the current studies we examined the effects of transforming growth factor type beta (TGF-beta) on the control of differentiation of BALB/c 3T3 T stem cells. We report that TGF-beta is a potent, reversible inhibitor of adipocyte differentiation (50% inhibition at approximately 0.06-0.08 ng/ml), while other biologically active polypeptides, such as epidermal growth factor (EGF), human growth hormone (hGH), and somatomedin C, have no specific effect on differentiation at even higher concentrations (200 ng/ml). We also report that TGF-beta inhibits differentiation in a cell cycle-dependent manner by its effect on a specific phase in the differentiation process. We therefore suggest that if TGF-beta is an important regulatory factor, one of its critical mechanisms of action may be its ability to inhibit the process of cell differentiation.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Transforming growth factor type β is a specific inhibitor of 3T3 T mesenchymal stem cell differentiation

In the current studies we examined the effects of transforming growth factor type β (TGF-β) on the control of differentiation of BALB/c 3T3 T stem cells. We report that TGF-β is a potent, reversible inhibitor of adipocyte differentiation (50% inhibition at ˜0.06–0.08 ng/ml), while other biologically active polypeptides, such as epidermal growth factor (EGF), human growth hormone (hGH), and somatomedin C, have no specific effect on differentiation at even higher concentrations (200 ng/ml). We also report that TGF-β inhibits differentiation in a cell cycle-dependent manner by its effect on a specific phase in the differentiation process. We therefore suggest that if TGF-β is an important regulatory factor, one of its critical mechanisms of action may be its ability to inhibit the process of cell differentiation.     

Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study, we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPARγ2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 μM) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 μM) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.     

生长因子对骨髓间充质干细胞增殖、分化的影响

骨髓间充质干细胞(bone mesenchymal stemcell,BMSC)是骨髓基质细胞的重要组成部分,由于其不但能与其他细胞一起支持造血干细胞造血,而且还具有较强的增殖功能及多向分化潜能,在一定诱导因素下可定向分化成骨细胞、软骨细胞和脂肪细胞等,近年来已成为生物学和医学的研究热点。本文简要介绍了不同生长因子如血管内皮生长因子、碱性成纤维细胞生长因子、转化生长因子-β等对BMSC增殖、分化的影响。     

Hepatogenic differentiation of mesenchymal stem cells induced by insulin like growth factor-I

AIM:To improve hepatic differentiation of human mesenchymal stem cell(MSC)using insulin growth factor 1(IGF-Ⅰ),which has important role in liver development,hepatocyte differentiation and function.METHODS:Bone marrow of healthy donors was aspirated from the iliac crest.The adherent cells expanded rapidly and were maintained with periodic passages until a relatively homogeneous population was established.The identification of these cells was carried out by immunophenotype analysis and differentiation potential into osteocytes and adipocytes.To effectively induce hepatic differentiation,we designed a protocol based on a combination of IGF-Ⅰ and liver specificfactors(hepatocyte growth factor,oncostatin M and dexamethasone).Morphological features,hepatic functions and cytological staining were assessed to evaluate transdifferentiation of human marrow-derived MSCs.RESULTS:Flow cytometric analysis and the differentiation potential into osteoblasts and adipocytes showed that more than 90% of human MSCs which were isolated and expanded were positive by specif ic markers and functional tests.Morphological assessment and evaluation of glycogen storage,albumin and α-feto protein expression,as well as albumin and urea secretion revealed a statistically signif icant difference between the experimental groups and control.CONCLUSION:In vitro differentiated MSCs using IGF-Ⅰwere able to display advanced liver metabolic functions,supporting the possibility of developing them as potential alternatives to primary hepatocytes.     

mTOR and the differentiation of mesenchymal stem cells

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase, belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells. mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.     

TRPC expression in mesenchymal stem cells

Transient receptor potential canonical (TRPC) channels are key players in calcium homeostasis and various regulatory processes in cell biology. Little is currently known about the TRPC subfamily members in mesenchymal stem cells (MSC), where they could play a role in cell proliferation. We report on the presence of TRPC1, 2, 4 and 6 mRNAs in MSC. Western blot and immunofluorescence staining indicate a membrane and intracellular distribution of TRPC1. Furthermore, the decrease in the level of TRPC1 protein caused by RNA interference is accompanied by the downregulation of cell proliferation. These results indicate that MSC express TRPC1, 2, 4 and 6 mRNA and that TRPC1 may play a role in stem cell proliferation.     

Prostanoid pattern and iNOS expression during chondrogenic differentiation of human mesenchymal stem cells

Availability of human chondrocytes is a major limiting factor regarding drug discovery projects and tissue replacement therapies. As an alternative human mesenchymal stem cells (hMSCs) from bone marrow are taken into consideration as they can differentiate along the chondrogenic lineage. However, it remains to be shown whether they could form a valid model for primary chondrocytes with regards to inflammatory mediator production, like nitric oxide (NO) and prostanoids. We therefore investigated the production of NO and prostanoids in hMSCs over the course of chondrogenic differentiation and in response to IL-1beta using primary OA chondrocytes as reference. Chondrogenic differentiation was monitored over 28 days using collagen I, collagen II, and collagen X expression levels. Expression levels of inducible nitric oxide synthase (iNOS), levels of NO, and prostanoids were assessed using PCR, Griess assay, and GC/MS/MS, respectively. The hMSCs collagen expression profile during course of differentiation was consistent with a chondrocytic phenotype. Contrary to undifferentiated cells, differentiated hMSCs expressed iNOS and produced NO following stimulation with IL-1beta. Moreover, this induction of iNOS expression was corticosteroid insensitive. The spectrum of prostanoid production in differentiated hMSCs showed similarities to that of OA chondrocytes, with PGE2 as predominant product. We provide the first detailed characterization of NO and prostanoid production in hMSCs in the course of chondrogenic differentiation. Our results suggest that differentiated hMSCs form a valid model for chondrocytes concerning inflammatory mediator production. Furthermore, we propose that IL-1beta stimulation, leading to corticosteroid-insensitive NO synthesis, can be used as a sensitive marker of chondrogenesis.     

脂肪干细胞与间充质干细胞体外诱导分化为心肌细胞的差别

本文旨在探讨脂肪干细胞(adipose-derived stem cells, ASCs)和骨髓间充质干细胞(mesenchymal stem cells, MSCs)在组织含量、体外培养和诱导分化为心肌细胞方面的差别.ASCs从新西兰白兔皮下脂肪组织提取,MSCs从大鼠四肢长骨骨髓提取,体外培养扩增,免疫细胞学方法鉴定.采用细胞集落形成法检测组织中干细胞的含量.将不同代的干细胞用不同浓度的5-氮胞苷诱导,观察其形态变化,免疫细胞化学方法检测诱导后细胞是否转化为心肌细胞.结果显示,体外培养的ASCs呈短梭形,分布均匀,生长迅速,细胞形态单一、稳定.MSCs原代生长非常缓慢,呈簇生长,细胞纯度偏低,容易混杂其它细胞类型,传代细胞容易分化和老化.脂肪组织中ASCs含量显著高于骨髓中MSCs含量,且前者含量受年龄影响小.5-氮胞苷诱导ASCs分化为心肌细胞的有效浓度为6～9μmol/L,而MSCs在3～15μmol/L 5-氮胞苷诱导下可见心肌细胞形成.ASCs诱导分化的心肌细胞呈球形细胞团,MSCs分化的心肌细胞呈条形或棒状,其心肌细胞分化率低于ASCs.幼年动物MSCs的组织含量和心肌细胞分化率均高于老年动物,而ASCs受动物年龄影响较小.结果表明,ASCs在组织含量、细胞纯度、生长速度和心肌细胞分化率等方面均明显优于骨髓MSCs,在心肌细胞再生方面较MSCs具有更大的优势.     

Induction of tenogenic differentiation of equine adipose-derived mesenchymal stem cells by platelet-derived growth factor-BB and growth differentiation factor-6

Molecular Biology Reports - Managing tendon healing process is complicated mainly due to the limited regeneration capacity of tendon tissue. Mesenchymal stem cells (MSCs) have...