Gene Expression Pattern of Human Chorion-Derived Mesenchymal Stem Cells during Adipogenic Differentiation

Purpose: The aim of this study was to identify the adipocyte-specific gene expression patterns in chorion-derived mesenchymal stem cells during adipogenic differentiation. Materials and Methods: Chorionic cells were isolated from the third trimester chorions from human placenta at birth and identified morphologically and by fluorescence-activated cell sorting analysis. After inducing adipogenic differentiation for 28 days, cells at days 3, 10, 21 and 28 were analyzed by Oil red O staining and RNA extraction in order to assess the expression levels of adipocyte marker genes, including CCAAT-enhancer binding protein α (C/EBPα), peroxisome proliferator- activated receptor γ (PPARγ), fatty acid binding protein 4 (FABP4) and Glycerol-3-phosphate dehydrogenase (GPD2). Cells not induced for differentiation were compared with the induced cells as a control group. Results: Chorion-derived cells showed the same pattern as fibroblasts, and expressed CD73, CD105, and CD166 antigens, but not CD45, CD34, and HLA-DR antigens. On day 3 after differentiation, cells began to stain positively upon Oil red O staining, and continuously increased in lipid granules for 4 weeks. The expression level of C/EBPα increased 4.6 fold on day 3 after induction, and continued to increase for 4 weeks. PPARγ was expressed at a maximum of 2.9 fold on day 21. FABP4 and GPD2 were significantly expressed at 4.7- and 3.0-fold, respectively, on day 21, compared to controls, and further increased thereafter. Conclusion: Human chorion-derived mesenchymal stem cells exhibited the sequential expression pattern of adipocyte marker genes during differentiation, corresponding to adipogenesis.     

Adhesion to Vitronectin and Collagen I Promotes Osteogenic Differentiation of Human Mesenchymal Stem Cells

The mechanisms controlling human mesenchymal stem cells (hMSC) differentiation are not entirely understood. We hypothesized that the contact with extracellular matrix (ECM) proteins normally found in bone marrow would promote osteogenic differentiation of hMSC in vitro. To test this hypothesis, we cultured hMSC on purified ECM proteins in the presence or absence of soluble osteogenic supplements, and assayed for the presence of well-established differentiation markers (production of mineralized matrix, osteopontin, osteocalcin, collagen I, and alkaline phosphatase expression) over a 16-day time course. We found that hMSC adhere to ECM proteins with varying affinity (fibronectin > collagen I >/= collagen IV >/= vitronectin > laminin-1) and through distinct integrin receptors. Importantly, the greatest osteogenic differentiation occurred in cells plated on vitronectin and collagen I and almost no differentiation took place on fibronectin or uncoated plates. We conclude that the contact with vitronectin and collagen I promotes the osteogenic differentiation of hMSC, and that ECM contact alone may be sufficient to induce differentiation in these cells.     

Phenol Red Inhibits Chondrogenic Differentiation and Affects Osteogenic Differentiation of Human Mesenchymal Stem Cells in Vitro

The purpose with this study was to investigate the effect of phenol red (PR) on chondrogenic and osteogenic differentiation of human mesenchymal stem cells (hMSCs). hMSCs were differentiated into chondrogenic and osteogenic directions in DMEM with and without PR for 2, 7, 14, 21, and 28 days. Gene expression of chondrogenic and osteogenic markers were analyzed by RT-qPCR. The presence of proteoglycans was visualized histologically. Osteogenic matrix deposition and mineralization were examined measuring the alkaline phophatase activity and calcium deposition. During chondrogenic differentiation PR decreased sox9, collagen type 2, aggrecan on day 14 and 21 (P?<?0.05), and proteoglycan synthesis on day 21 and 28. Collagen type 10 was decreased on day 21 (P?<?0.05). During osteogenic differentiation PR increased alkaline phosphatase on day 7 while decreased on day 21 (P?<?0.05). PR increased collagen type 1 on day 7, 14, and day 21 (P?<?0.05). The alkaline phosphatase activity was increased after 2, 7, and 14 days (P?<?0.05). The deposition of calcium was decreased on day 21 (P?<?0.05). Our results indicate that PR should be removed from the culture media when differentiating hMSCs into chondrogenic and osteogenic directions due to the effects on these differentiation pathways.     

兔骨髓间充质干细胞定向诱导分化为脂肪细胞及成骨细胞的研究

用密度梯度离心和贴壁法分离和纯化兔骨髓间充干细胞，建立诱导兔MSCs向脂肪细胞及成骨细胞表型转化的方法及条件。在成脂诱导剂或成骨诱导剂作用下，对原代和第2代兔MSCs进行成脂和成骨诱导培养，并鉴定成脂及成骨表型。结果表明：原代及第2代兔MSCs均有一定的成脂、成骨能力，且第2代细胞的分化能力较原代低。在诱导培养条件下，原代及第2代兔MSCs均能分化，成脂诱导21d，75％的兔MSCs转化为脂肪细胞；成骨诱导21d，75％的兔MSCs转化为成骨细胞。兔MSCs在适当的诱导条件下可快速分化为脂肪细胞或成骨细胞。     

A Novel Semisynthetic Molecule Icaritin Stimulates Osteogenic Differentiation and Inhibits Adipogenesis of Mesenchymal Stem Cells

Background We previously reported that the constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common nuclear stem) exerted beneficial effects on the bone, including promoting bone formation and inhibiting bone marrow fat deposition. Recent in vivo study showed that Icaritin was a common metabolite of these constitutional flavonoid glycosides, indicating that Icaritin is a bioactive compound. The present study was designed to investigate whether Icaritin could promote osteogenic differentiation and suppress adipogenic differentiation of marrow mesenchymal stem cells (MSCs).Methods Primary MSCs were harvested from adult mice and exposed to Icaritin to evaluate whether it could promote osteogenesis and suppress adipogenesis using the following assays: determination of alkaline phosphatase (ALP) activity and mineralization; mRNA expression of osteogenic differentiation marker Runx2; osteocalcin and bone sialoprotein (BSP) by RT-PCR; quantification of adipocyte-like cells by Oil Red O staining assay and mRNA expression for adipogenic differentiation markers peroxisome proliferator-activated receptor gamma (PPARγ); adipocyte fatty acid binding protein (aP2) and lipoprotein lipase (LPL) by RT-PCR. For the underlying mechanism, glycogen synthase kinase-3beta (GSK3β) and β-catenin were also explored by western blotting.Results Icaritin promoted osteogenic differentiation and maturation of MSCs as indicated by increased mRNA expression for Runx2, osteocalcin and BSP, and enhanced ALP activity and mineralization; Icaritin inhibited adipogenic differentiation, as indicated by decreased mRNA expression for PPARγ, LPL, aP2, and suppressed formation of adipocyte-like cells; Icaritin inactivated GSK3β and suppressed PPARγ expression when promoting osteogenesis and suppressing adipogenesis of MSCs.Conclusion This was the first study demonstrating that the novel semisynthetic molecule Icaritin could stimulate osteogenic differentiation and inhibit adipogenesis of MSCs, which was associated with the suppression of GSK3β and PPARγ.     

人血小板裂解液促进人羊膜来源间充质干细胞的成骨分化

目的:研究人血小板裂解液(HPL)对羊膜来源间充质干细胞(AMMSCs)成骨分化的作用。方法:分别以含7%HPL(HPL组)和10%胎牛血清(FBS,FBS组)的LG-DMEM培养介质扩增AMMSCs,比较两组扩增AMMSCs效率及其免疫表型SSEA-3和SSEA-4的表达差异。使用MSCs成骨分化培养液诱导AMMSCs成骨,对比观察两组钙盐沉积量、钙化结节、碱性磷酸酶(ALP)活性;提取两组成骨诱导后AMMSCs总RNA,采用RTPCR检测成骨分化调节因子RUNX-2和ALP mRNA相对表达量。结果:HPL组扩增速度快于FBS组;AMMSCs的SSEA-3和SSEA-4表达较FBS组明显下调(P0.05)。HPL组钙盐沉积量、钙化结节数量、ALP活性以及RUNX-2和ALP mRNA表达量均明显高于FBS组(P均0.05)。结论:含HPL培养介质可促进AMMSCs成骨分化。     

Adipose Tissue-Derived Mesenchymal Stem Cells Facilitate Hematopoiesis in Vitro and in Vivo : Advantages Over Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.Hematopoiesis is a dynamic process that involves self-renewal of hematopoietic stem cells in the bone marrow, generation of lineage-committed cells, and mobilization of mature cells into the bloodstream. Mesenchymal stem cells (MSCs) present in bone marrow (BM) are thought to give rise to cells that constitute the hematopoietic microenvironment. MSCs produce a number of cytokines and extracellular matrix proteins and express cell adhesion molecules, all of which are involved in the regulation of hematopoiesis.¹ Human MSCs, when injected into the bone marrow cavity of nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, differentiate into pericytes, myofibroblasts, BM stromal cells, bone osteocytes, bone-lining osteoblasts, and endothelial cells, which constitute the functional components of the hematopoietic microenvironment.² In recent studies, cotransplantation of human MSCs and HSCs resulted in increased chimerism or accelerated hematopoietic recovery (or both) in animal models and in humans.^3,4,5,6All of the above studies used bone marrow-derived MSCs (BMSCs). However, there are several drawbacks in the use of BMSCs for clinical application, including the fact that they are only available in limited number even though large quantities of infused cells are required for treatment. In addition, there is a possibility that BMSCs might be contaminated with malignant cells when they are harvested from patients with a hematological malignancy (eg, leukemia). The discoveries that a large number of nonadipocyte stem cells exist in fat tissue (adipose tissue-derived MSCs [ADSCs]) and that these cells can be rapidly expanded ex vivo, suggested that ADSCs might be useful for clinical applications.⁷ Recent studies showed that ADSCs are a viable alternative to BMSCs for treatment of animal models of various kinds of diseases.^8,9,10,11,12 However, it has been reported that even though ADSCs and BMSCs are very similar, ADSCs are not completely identical to BMSCs.^13,14 To date, little is known concerning the ability of ADSCs to support hematopoiesis. We therefore extensively examined the hematopoiesis-supporting properties of ADSCs in vitro and in vivo and report that ADSCs possess several advantages over BMSCs.     

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins.As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin.Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.     

Distinct Stem Cells Subpopulations Isolated from Human Adipose Tissue Exhibit Different Chondrogenic and Osteogenic Differentiation Potential

Recently adipose tissue has become a research topic also for the searching for an alternative stem cells source to use in cell based therapies such as tissue engineer. In fact Adipose Stem Cells (ASCs) exhibit an important differentiation potential for several cell lineages such as chondrogenic, osteogenic, myogenic, adipogenic and endothelial cells. ASCs populations isolated using standard methodologies (i.e., based on their adherence ability) are very heterogeneous but very few studies have analysed this aspect. Consequently, several questions are still pending, as for example, on what regard the existence/ or not of distinct ASCs subpopulations. The present study is originally aimed at isolating selected ASCs subpopulations, and to analyse their behaviour towards the heterogeneous population regarding the expression of stem cell markers and also regarding their osteogenic and chondrogenic differentiation potential. Human Adipose derived Stem Cells (hASCs) subpopulations were isolated using immunomagnetic beads coated with several different antibodies (CD29, CD44, CD49d, CD73, CD90, CD 105, Stro-1 and p75) and were characterized by Real Time RT-PCR in order to assess the expression of mesenchymal stem cells markers (CD44, CD73, Stro-1, CD105 and CD90) as well as known markers of the chondrogenic (Sox 9, Collagen II) and osteogenic lineage (Osteopontin, Osteocalcin). The obtained results underline the complexity of the ASCs population demonstrating that it is composed of several subpopulations, which express different levels of ASCs markers and exhibit distinctive differentiation potentials. Furthermore, the results obtained clearly evidence of the advantages of using selected populations in cell-based therapies, such as bone and cartilage regenerative medicine approaches.     

力学因素对骨髓间充质干细胞成骨分化的影响

近年来,组织工程学领域发展突飞猛进,已被列为一种修复或再生许多组织和器官的方法。骨髓间充质干细胞具有良好的成骨向分化潜能,在骨组织工程领域中具有广阔的应用前景。骨髓间充质干细胞增殖和成骨向分化受多种力学因素的影响,且不同性质的力学刺激对其定向分化的调节作用不尽相同。目前,许多学者致力于深入探讨力学因素影响骨髓间充质干细胞成骨向分化的具体途径,但其调控机制尚未完全明确。本文综述及讨论力学因素对骨髓间充质干细胞所产生的增殖、定向成骨分化等生物学效应的影响及可能涉及的力化学信号转导通路作用机制,以期丰富研究思路     

人E 钙粘素融合蛋白基质对人骨髓间充质干细胞向肝细胞定向诱导分化的影响研究

人骨髓间充质干细胞（hBMSCs）源的肝细胞对于肝脏疾病的治疗和药物的开发具有巨大的应用潜力。利用基因工程技术构建了融合蛋白质粒,并通过真核细胞表达体系表达出了人E-钙粘素胞外域与免疫球蛋白Fc段的融合蛋白（hE-cadherin-Fc）,并将其用于疏水材料的表面修饰仿生构建细胞-细胞间相互作用的细胞外微环境,检测其对hBMSCs向肝样细胞定向诱导分化的影响。诱导分化培养4周后,与组织培养板（TC-PS）、明胶（Gelatin）基质相比,hE-cadherin-Fc基质显著促进细胞表达ALB、CK18、HNF-4等肝细胞分化基因,并且细胞的糖原合成和吲哚青绿（ICG）摄取功能均显著提高。在hE-cadherin-Fc基质表面分化培养4周时,白蛋白和尿素合成的量为2.7 pg/细胞/d和36.7 pg/细胞/d,相对于TC-PS和Gelatin分别提高了42.1%和28.6%、57.5%和25.7%。综上所述,利用hE-cadherin-Fc基质化构建细胞外微环境,有利于促进人骨髓间充质干细胞向功能化肝细胞的定向分化。     

Mesenchymal Stem Cells from Human Bone Marrow and Adipose Tissue: Isolation,Characterization, and Differentiation Potentialities

Comparative study of cultured human bone marrow and adipose tissue (lipoaspirate) mesenchymal stem cells was carried out. The main morphological parameters, proliferative activity, expression of surface and intracellular markers of these cells were characterized. Flow cytofluorometry and histological staining showed that both cell types exhibited similar expression of CD105, CD54, CD106, HLA-I markers, were positively stained for vimentin, ASMA, collagen-1, and fibronectin, but not HLA-DR, CD117, and hemopoietic cell markers. The cells underwent differentiation into adipocytes and osteoblasts under appropriate conditions of culturing. Incubation under neuroinductive conditions led to the appearance of a cell population positively stained for type III β-tubulin (neuronal differentiation marker). __________ Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 3, pp. 158–163, September, 2005     

Impact of Human Adipose Tissue-Derived Stem Cells on Malignant Melanoma Cells in An In Vitro Co-culture Model

This study focuses on the interactions of human adipose tissue-derived stem cells (ADSCs) and malignant melanoma cells (MMCs) with regard to future cell-based skin therapies. The aim was to identify potential oncological risks as ADSCs could unintentionally be sited within the proximity of the tumor microenvironment of MMCs. An indirect co-culture model was used to analyze interactions between ADSCs and four different established melanoma cell lines (G-361, SK-Mel-5, MeWo and A2058) as well as two low-passage primary melanoma cell cultures (M1 and M2). Doubling time, migration and invasion, angiogenesis, quantitative real-time PCR of 229 tumor-associated genes and multiplex protein assays of 20 chemokines and growth factors and eight matrix metalloproteinases (MMPs) were evaluated. Co-culture with ADSCs significantly increased migration capacity of G-361, SK-Mel-5, A2058, MeWo and M1 and invasion capacity of G-361, SK-Mel-5 and A2058 melanoma cells. Furthermore, conditioned media from all ADSC-MMC-co-cultures induced tube formation in an angiogenesis assay in vitro. Gene expression analysis of ADSCs and MMCs, especially of low-passage melanoma cell cultures, revealed an increased expression of various genes with tumor-promoting activities, such as CXCL12, PTGS2, IL-6, and HGF upon ADSC-MMC-co-culture. In this context, a significant increase (up to 5,145-fold) in the expression of numerous tumor-associated proteins could be observed, e.g. several pro-angiogenic factors, such as VEGF, IL-8, and CCL2, as well as different matrix metalloproteinases, especially MMP-2. In conclusion, the current report clearly demonstrates that a bi-directional crosstalk between ADSCs and melanoma cells can enhance different malignant properties of melanoma cells in vitro.     

电磁场促进骨髓间充质干细胞成骨分化的研究进展

目的:探讨电磁场促进骨髓间充质干细胞成骨分化作用的研究现状。方法:检索电磁场和间充质干细胞相关论文,比对寻找电磁场促进成骨的相关证据,总结电磁场促进间充质干细胞成骨分化的相关研究。结果:电磁场对细胞的作用是复杂多样的,可影响干细胞的基因表达,也能对细胞缝隙通讯产生作用,实验室证明电磁场在体外培养的干细胞增殖和分化方面扮演了一定的角色,临床研究同样证明活体的骨细胞会受到电磁场的作用。结论:电磁场能促进骨髓间充质干细胞的成骨分化,近年来国内外在这个领域的研究方兴未艾,进一步深入研究非常必要尤其通讯频段电磁场更值得学术界的探讨。     

Jmjd3和Ezh2酶活性抑制剂对人间充质干细胞成脂分化的影响及机制研究

目的 研究EZH2和JMJD3对人脂肪间充质干细胞成脂分化的影响及可能机制。方法 体外培养人脂肪来源间充质干细胞并进行诱导分化。分化过程中分别加入EZH2酶活性抑制剂GSK126（6 μM）和JMJD3酶活性抑制剂GSKJ4（20 μM）,对照组加入DMSO。采用Realtime PCR方法检测脂肪细胞分化基因PPARγ、FABP4和ADIPOQ;棕色化标志基因UCP1、PRDM16和CIDEA,以及米色脂肪独有的标志基因CD137、TMEM26和TBX1,并计算各组与对照组的相对表达量。采用West     

骨髓间质干细胞体外分化为成骨细胞的实验研究

建立猪骨髓间质干细胞 (mesenchymalstemcells ,MSCs)体外分离培养方法。对猪MSCs体外分化为成骨细胞的能力进行研究。抽取猪骨髓 ,体外培养MSCs。取第二代MSCs ,以含有不同浓度的抗坏血酸、β -磷酸甘油、地塞米松及碱性成纤维细胞生长因子等条件培养基进行成骨细胞诱导分化。通过细胞形态变化 ,碱性磷酸酶染色及钙盐沉积对成骨细胞进行鉴定。结果表明MSC细胞形态由长梭形向多边形转变 ,ALP染色阳性 ,VonKossa染色阳性 ,经体外诱导分化后呈典型的成骨细胞样改变。猪骨髓MSCs可在体外长期、稳定培养 ,具有向成骨细胞分化的潜能 ,可以为骨组织工程研究提供较理想的细胞来源和动物模型。     

Electrospun Poly(L-Lactic Acid) Nanofibres Loaded with Dexamethasone to Induce Osteogenic Differentiation of Human Mesenchymal Stem Cells

Abstract

Dexamethasone (Dex), a synthetic corticosteroid, was loaded into poly(L-lactic acid) (PLLA) nanofibrous scaffolds with a concentration of 0.333 wt% by electrospinning. The Dex-loaded PLLA nanofibres increased the mechanical strength in comparison with pure PLLA nanofibres. A sustained release profile for over 2 months with an initial burst release after 12 h of 17% was shown. Importantly, the amounts of Dex released from the PLLA nanofibres every 3 days were close to the ones used for the standard osteogenic medium. The sustained osteoinductive environment created by released Dex strongly differentiated human mesenchymal stem cells (hMSCs) cultured in the Ost^–Dex medium. ALP activity, BSP expression and calcium deposition were significantly higher than those of the cells cultured on the PLLA scaffolds without Dex. A large amount of hydroxyapatite-like minerals was observed on the Dex-loaded PLLA scaffolds after 21 days culture. The cells on these scaffolds also indicated an osteoblastic morphology on the 14th day. Besides, these scaffolds slightly increased the cell proliferation comparing to the scaffolds without Dex. As such, the PLLA nanofibres loaded with 0.333 wt% Dex was an effective osteoinductive scaffold which acts as a promising strategy for bone treatment.