miR-424负向调节人脂肪源间充质干细胞向成脂细胞分化

目的研究miR-424对人脂肪源间充质干细胞(hAMSCs)向成脂分化的影响。方法用real-time PCR检测hAMSCs成脂分化前后细胞内miR-424水平的变化。在脂质体介导下,用人工合成的miR-424模拟物转染hAMSCs提高细胞内miR-424的含量,随后对其进行成脂诱导。用油红O染色法观察细胞内脂滴形成情况,用real-time PCR和Western blot检测成脂关键转录因子PPARγ及相关标记物mRNA的表达。结果 hAMSCs成脂分化后,miR-424表达下调。与对照组细胞相比,转染miR-424模拟物的实验组细胞中miR-424含量明显升高。成脂诱导第8天油红O染色结果显示,实验组脂滴形成显著少于对照组。PPARγ和成脂相关标记物的表达量也出现下调。结论 miR-424可负向调节hAMSCs向脂肪细胞分化。     

miR-424 negatively regulates adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells

目的 研究miR-424对人脂肪源间充质干细胞(hAMSCs)向成脂分化的影响.方法 用real-time PCR检测hAMSCs成脂分化前后细胞内miR-424水平的变化.在脂质体介导下,用人工合成的miR-424模拟物转染hAMSCs提高细胞内miR-424的含量,随后对其进行成脂诱导.用油红O染色法观察细胞内脂滴形成情况,用real-time PCR和Western blot检测成脂关键转录因子PPARγ及相关标记物mRNA的表达.结果 hAMSCs成脂分化后,miR-424表达下调.与对照组细胞相比,转染miR-424模拟物的实验组细胞中miR-424含量明显升高.成脂诱导第8天油红O染色结果显示,实验组脂滴形成显著少于对照组.PPARγ和成脂相关标记物的表达量也出现下调.结论 miR-424可负向调节hAMSCs向脂肪细胞分化.     

Collagen I gel promotes homogenous osteogenic differentiation of adipose tissue-derived mesenchymal stem cells in serum-derived albumin scaffold

Repair of bone defects is a difficult clinical problem for reconstructive surgeons. Bone tissue engineering using an appropriate scaffold with cells is a new therapy for the repair of bone defects. The aim of this study was to evaluate the in vitro osteogenesis of canine adipose tissue-derived mesenchymal stem cells (Ad-MSCs) cultured in a combination of collagen I gel and a porous serum-derived albumin scaffold. A serum-derived albumin scaffold was prepared with canine serum by cross-linking and freeze-drying procedures. Ad-MSCs were seeded into serum-derived albumin scaffolds with or without collagen I gel, and were exposed to osteogenic differentiation conditions in vitro. After 28?days of in vitro culture, the distribution and osteogenic differentiation of Ad-MSCs cultured in the scaffold were evaluated by scanning electron microscopy, histology, immunohistochemistry, alkaline phosphatase (ALP) activity assay, and calcium colorimetric assay. Ad-MSCs showed more homogeneous distribution and osteogenic differentiation in the scaffold with collagen I gel than without collagen I gel. ALP activity and extracellular matrix mineralization in the construct with type I collagen were significantly higher than in the construct without type I collagen (p?<?0.05). In conclusion, the combination of collagen I gel and the serum-derived albumin scaffold enhanced osteogenic differentiation and homogenous distribution of Ad-MSCs.     

脂肪间充质干细胞向髓核样细胞定向分化研究

目的探讨SD大鼠来源的髓核(nucleus pulposus,NP)细胞促使脂肪间充质干细胞(adipose tissue-derived mesenchymal stem cells,AMSCs)向NP样细胞定向分化的分子机制。方法采用酶消化法取脂肪细胞,极限稀释法纯化细胞;采用组织块培养法培养NP细胞。利用流式细胞技术,免疫荧光及RT-PCR检测对AMSCs及NP细胞进行鉴定。结果 AMSCs中Sca-1和CD44的阳性率较高,而CD45和CD11b阴性,共培养组荧光强度明显亮于单纯AMSCs组,AMSCs在NP细胞的诱导下聚焦蛋白聚糖(Aggrecan)、Ⅱ型胶原蛋白(CollagenⅡ)、Sox-9等表达水平较对照组高。结论共培养环境中髓核细胞分泌的可溶性因子TGF-1能促使AMSCs向NP样细胞定向分化。     

Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells

Cell-based therapies have become a promising approach to promote tissue regeneration and the treatment of musculoskeletal disorders. Bone regeneration maintains bone homeostasis, mechanical stability and physical performance. Mechanical stimulation showed to induce stem cell differentiation into the osteogenic fate. However, the effect of various osteogenic protocols on the osteogenic commitment of equine adipose-derived stem cells is not fully elucidated. Here we examined the influence of fluid-based shear stress (FSS) via mechanical rocking to assess whether mechanical stimulation promotes osteogenic differentiation of equine adipose-derived stem cells (ASCs). ASCs were induced into osteogenic fate using osteogenic differentiation medium (ODM) protocol or additional supplementation of 5?mM CaCl₂ and 7.5?mM CaCl₂ protocol compared to cells cultivated in basal medium (BM) up to 21 day. The ASCs proliferation pattern was evaluated using the sulforhodamine B (SRB) protein assay. Osteogenic differentiation examined via semi-quantification of alizarin red staining (ARS) and alkaline phosphatase activity (ALP) as well as, via quantification of osteocalcin (OC), alkaline phosphatase (ALP), osteopontin (OP), and collagen type-1 (COL1) gene expression using RT-qPCR. We show that mechanical FSS increased the proliferation pattern of ASCs compared to the static conditions. Mechanical FSS together with 5?mM CaCl₂ and 7.5?mM CaCl₂ promoted osteogenic nodule formation and increased ARS intensity compared to the standard osteogenic protocols. We observed that combined mechanical FSS with ODM protocol increase ALP activity compared to static culture conditions. We report that ALP and OC osteogenic markers expression were upregulated under mechanical FSS culture condition particularly with the ODM protocol. Taken together, it can be assumed that mechanical stress using FSS promotes the efficiency of the osteogenic differentiation protocols of ASCs through independent mechanisms.     

神经型钙黏蛋白对小鼠脂肪间充质干细胞的成神经作用

目的:探讨神经型钙黏蛋白(N-cadherin)在小鼠脂肪间充质干细胞(ADMSCs)成神经分化中的作用。方法;用差速培养法获取小鼠ADMSCs,传至第5代,经成神经诱导液诱导ADMSCs 7 d后,免疫荧光检测胶质纤维酸性蛋白(GFAP)、神经元特异性烯醇化酶(NSE)和微管相关蛋白2(MAP2)的表达,半定量PCR检测细胞中N-cadherin mRNA的表达;采用N-cadherin对细胞进行基因修饰,免疫荧光检测神经丝蛋白(NF)和GFAP的表达。结果:ADMSCs经成神经诱导7 d后表达GFAP、NSE和MAP2,半定量PCR结果显示,与对照组相比成神经诱导后的细胞表达N-cadherin显著增高。转染N-cadherin的N-cadherin细胞培养24 h后发出细长的突起,与相邻细胞之间形成网状连接。免疫荧光结果显示,转染后的细胞NF和GFAP表达阳性。结论:ADMSCs在体外多种作用下,具有向神经细胞分化的潜能。N-cadherin转染后能改变ADMSCs的形态,并且在ADMSCs向神经分化中发挥一定的作用。     

Titania-hydroxyapatite nanocomposite coatings support human mesenchymal stem cells osteogenic differentiation

In addition to mechanical and chemical stability, the third design goal of the ideal bone-implant coating is the ability to support osteogenic differentiation of mesenchymal stem cells (MSCs). Plasma-sprayed TiO(2)-based bone-implant coatings exhibit excellent long-term mechanical properties, but their applications in bone implants are limited by their bioinertness. We have successfully produced a TiO(2) nanostructured (grain size <50 nm) based coating charged with 10% wt hydroxyapatite (TiO(2)-HA) sprayed by high-velocity oxy-fuel. On Ti64 substrates, the novel TiO(2)-HA coating bond 153× stronger and has a cohesive strength 4× higher than HA coatings. The HA micro- and nano-sized particles covering the TiO(2)-HA coating surface are chemically bound to the TiO(2) coating matrix, producing chemically stable coatings under high mechanical solicitations. In this study, we elucidated the TiO(2)-HA nanocomposite coating surface chemistry, and in vitro osteoinductive potential by culturing human MSCs (hMSCs) in basal and in osteogenic medium (hMSC-ob). We assessed the following hMSCs and hMSC-ob parameters over a 3-week period: (i) proliferation; (ii) cytoskeleton organization and cell-substrate adhesion; (iii) coating-cellular interaction morphology and growth; and (iv) cellular mineralization. The TiO(2) -HA nanocomposite coatings demonstrated 3× higher hydrophilicity than HA coatings, a TiO(2)-nanostructured surface in addition to the chemically bound HA micron- and nano-sized rod to the surface. hMSCs and hMSC-ob demonstrated increased proliferation and osteoblastic differentiation on the nanostructured TiO(2)-HA coatings, suggesting the TiO(2)-HA coatings nanostructure surface properties induce osteogenic differentiation of hMSC and support hMSC-ob osteogenic potential better than our current golden standard HA coating.     

Myeloma cells inhibit osteogenic differentiation of mesenchymal stem cells and kill osteoblasts via TRAIL-induced apoptosis

Introduction

Myeloma bone disease (MBD) is the result of the increased activity of osteoclasts (OCs), which is not accompanied by a comparable increase of osteoblast (OB) function, thus leading to enhanced bone resorption. Osteoblasts can also regulate osteoclast activity through expression of cytokines, such as receptor activator of nuclear factor-κB ligand (RANKL), which activates osteoclast differentiation, and osteoprotegerin (OPG), which inhibits RANKL by acting as a decoy receptor.

Material and methods

Based on a series of 21 patients with multiple myeloma (MM) and human osteoblast cell line HFOB1.19, we provide evidence that the bone marrow-derived mesenchymal stem cells (BMMSCs) of patients with MM exhibit normal phenotype, but showed reduced efficiency to differentiate into OBs as compared with normal controls.

Results

In vitro assays showed that MM cells inhibited the potential of osteogenic differentiation of BMMSCs from healthy controls and rendered the OBs sensitive to TRAIL-induced apoptosis. There was no evidence of the formation of tartrate-resistant acid phosphatase positive OCs. The osteogenic differentiation of HFOB1.19 was also inhibited in the presence of RPMI 8266 or XG7 MM cells, as confirmed by von Kossa and ALP staining. Osteoblast s induced from BMMSCs supported survival and proliferation of MM cells, especially when the MM cells were cultured in medium containing rhTRAIL and dexamethasone. Multiple myeloma cells proliferated and grew well in the presence of residual OBs.

Conclusions

Besides OCs, our results demonstrated that OBs and MM cells were dependent upon each other and made a microenvironment suitable for MM cells.     

Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.     

体外诱导脂肪源性干细胞向类肝细胞的定向分化

背景：用脂肪源性干细胞治疗肝脏疾病之前,如何建立有效稳定的肝细胞分化诱导方案,纯化并快速扩增性能稳定的类肝细胞等问题亟待解决。 目的：建立大鼠脂肪源性干细胞转化为类肝细胞的程序化诱导体系。 方法：分离纯化Lewis大鼠脂肪源性干细胞,流式细胞仪鉴定其表面标志,分3个阶段加入含有肝细胞生长因子、成纤维细胞生长因子4、酸性成纤维细胞生长因子、制瘤素M细胞因子的诱导培养体系,使脂肪源性干细胞向肝细胞转化。 结果与结论：大鼠脂肪源性干细胞诱导7,14,21 d后,细胞阳性表达 ALB、AFP、CK18mRNA,表达量随诱导时间延长而增强,类肝细胞具有白蛋白合成功能。氨代谢和尿素的合成功能在9~12 d出现并持续存在。结果表明脂肪源性干细胞体外分段诱导可成功转化为类肝细胞。     

Adipogenesis induced by human adipose tissue-derived stem cells

Adipose tissue-derived stem cells (ASCs), including preadipocytes, may play an important role in de novo adipogenesis and are expected to be a useful external source of cells for adipose tissue engineering. In this study, we examined in vivo adipogenesis up to 24 weeks after implantation, induced by human ASCs that were isolated from adipose tissues and expanded in vitro. ASCs proliferated in vitro in the presence of basic fibroblast growth factor (bFGF), and the number of cells increased by more than 1000-fold at the fourth passage. The ability to differentiate into mature adipocytes was maintained up to the third passage. We incorporated designated numbers of third-passage-expanded cells into a type I collagen scaffold and implanted them into the back of nude mice with or without controlled-release bFGF. After the implantation of 2 x 10(6) ASCs with controlled-release bFGF, the greatest cross-sectional surface area of adipose tissue in the scaffold was 1.19 mm(2) at 12 weeks and 2.14 mm(2) at 24 weeks. About 2 x 10(6) ASCs with controlled-release bFGF was the best condition for total adipogenesis. Immunohistochemical analysis with antihuman vimentin antibody showed that the area of human-origin adipose tissue was maximum in the group with 8 x 10(6) ASCs incorporated in a scaffold at both 12 and 24 weeks. The amount of human-origin adipose tissue increased in all groups with implanted ASCs from 12 to 24 weeks. Only trace of human-origin adipose tissue was observed in other groups implanted ASCs. Our results show that human ASCs not only function as progenitor cells for in vivo adipogenesis, but also induce de novo adipogenesis for long period.     

Donor age negatively impacts adipose tissue-derived mesenchymal stem cell expansion and differentiation

Background

Human adipose tissue is an ideal autologous source of mesenchymal stem cells (MSCs) for various regenerative medicine and tissue engineering strategies. Aged patients are one of the primary target populations for many promising applications. It has long been known that advanced age is negatively correlated with an organism’s reparative and regenerative potential, but little and conflicting information is available about the effects of age on the quality of human adipose tissue derived MSCs (hAT-MSCs).

Methods

To study the influence of age, the expansion and in vitro differentiation potential of hAT-MSCs from young (<30 years), adult (35-50 years) and aged (>60 years) individuals were investigated. MSCs were characterized for expression of the genes p16^INK4a and p21 along with measurements of population doublings (PD), superoxide dismutase (SOD) activity, cellular senescence and differentiation potential.

Results

Aged MSCs displayed senescent features when compared with cells isolated from young donors, concomitant with reduced viability and proliferation. These features were also associated with significantly reduced differentiation potential in aged MSCs compared to young MSCs.

Conclusions

In conclusion, advancing age negatively impacts stem cell function and such age related alterations may be detrimental for successful stem cell therapies.     

Inflammatory conditions affect gene expression and function of human adipose tissue-derived mesenchymal stem cells

There is emerging interest in the application of mesenchymal stem cells (MSC) for the prevention and treatment of autoimmune diseases, graft‐versus‐host disease and allograft rejection. It is, however, unknown how inflammatory conditions affect phenotype and function of MSC. Adipose tissue‐derived mesenchymal stem cells (ASC) were cultured with alloactivated peripheral blood mononuclear cells (PBMC) (mixed lymphocyte reaction: MLR), with proinflammatory cytokines [interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐6] or under control conditions, and their full genome expression and function examined. Proinflammatory cytokines mainly increased indoleamine‐2,3‐dioxygenase expression, whereas ASC cultured with MLR showed increased expression of COX‐2, involved in prostaglandin E₂ production. Both conditions had a stimulatory, but differential, effect on the expression of proinflammatory cytokines and chemokines, while the expression of fibrotic factors was decreased only in response to proinflammatory cytokines. Functional analysis demonstrated that inflammatory conditions affected morphology and proliferation of ASC, while their differentiation capacity and production of trophic factors was unaffected. The immunosuppressive capacity of ASC was enhanced strongly under inflammatory conditions. In conclusion, ASC showed enhanced immunosuppressive capacity under inflammatory conditions, while their differentiation capacity was preserved. Therefore, in vitro preconditioning provides ASC with improved properties for immediate clinical immune therapy.     

糖皮质激素促间充质干细胞的成骨效应

背景：糖皮质激素是有效的免疫抑制和抗炎症药物而广泛应用于临床,然而长期服用后也会引起一些不良反应如骨质疏松症。糖皮质激素通过促进成骨细胞与骨细胞的凋亡,同时抑制间充质干细胞向成骨细胞分化,导致成骨细胞数量减少;然而在一定的条件下,糖皮质激素又能够促进间充质干细胞表达成骨细胞标志基因诱导其向成骨方向分化。 目的：对以地塞米松为代表的糖皮质激素在间充质干细胞成骨分化中发挥的作用以及可能的分子机制进行综述。 方法：由第一作者检索PubMed数据库1978至2012年有关地塞米松和间充质干细胞成骨分化的文献,英文关键词“Mesenchymal stem cell,dexamethasone,osteogenesis,osteoporosis,Runx2,BMP,Noggin,GILZ,glucocorticoid receptor”以不同的组合方式查找,排除重复性的研究,最终保留55篇进行归纳综述。 结果与结论：地塞米松能够调节Runx2,Noggin以及亮氨酸拉链蛋白等基因调控间充质干细胞成骨分化。当糖皮质激素作用于间充质干细胞时,可能由糖皮质激素受体介导其生理作用,也可能在糖皮质激素与其受体结合之前就受到11β-羟甾类脱氢酶的调节。另外,生理剂量(10^-8 mol/L)的地塞米松能促进间充质干细胞成骨分化,药理剂量(≥10^-7 mol/L)有抑制作用。     

微重力对骨髓间充质干细胞成骨分化的影响

骨髓间充质干细胞（BMSCs）是一种多能成体于细胞,是组织工程重要的种子细胞来源之一.微重力对BMSCs成骨分化具有抑制作用,可使骨量减少和骨微结构改变,从而导致骨质疏松症.这一过程受到多条信号通路的调控,如MAPK信号通路、Notch信号通路和Wnt/β-catenin信号通路等,它们协同调节微重力下BMSCs向成骨细胞方向的分化.研究微重力对BMSCs成骨分化的影响,可以阐明骨质流失机理,为相关疾病的治疗提供新的靶点,促进我国太空宇航事业的发展.     

骨髓间充质干细胞诱导分化成骨方法的研究与进展

背景：骨髓间充质干细胞具有良好的成骨分化潜能,移植入体内后可分化为骨、软骨、肌肉、脂肪、肝脏和神经等多种细胞。 目的：综述大鼠骨髓间充质干细胞诱导分化成骨最新方法进展。 方法：分别以“骨髓间充质干细胞,成骨细胞,诱导分化”、 “bone mesenchymal stem cells ,osteoblast cells,osteogenic differentiation”为检索词,应用计算机检索CHKD期刊全文数据库,PubMed数据库和万方数据库1995至2011年有关文章。纳入有关骨髓间充质干细胞的文献。排除内容重复和缺乏原创性文献。保留34篇文献做进一步分析。 结果与结论：骨髓间充质干细胞在适当的生长因子诱导下可以快速地向成骨细胞分化和增殖。在体外诱导骨髓间充质干细胞向成骨细胞分化需要特定的诱导条件、优良的诱导剂以及合适的剂量。骨髓间充质干细胞向成骨细胞分化受许多自身调控因素(控制基因表达的转录因子)和环境调控因素(分泌因素、细胞外基质,化学因素、细胞与细胞间的相互作用)的影响,这些调控因素并不是孤立存在的,而是相互联系、相互作用,共同调控骨髓间充质干细胞向成骨细胞分化。     

体外定向诱导人骨髓间质干细胞分化为成骨细胞的研究

目的:建立体外定向诱导成人骨髓间质干细胞(MSC)分化为成骨细胞的模型。方法:采用Ficoll-Paque淋巴细胞分离液分离成人MSC,体外扩增,流式细胞仪检测细胞表面抗原的表达,应用地塞米松、β-甘油磷酸钠、vitaminC定向诱导MSC分化为成骨细胞,检测碱性磷酸酶(AP)活性、钙沉积、骨桥蛋白的表达鉴定成骨细胞,比较P3和P10代MSC成骨分化的能力。结果:MSC在体外扩增原代可获得(5-6)×10⁵个细胞,10代可获得2×10¹⁰个细胞。流式细胞仪检测结果显示CD29、CD44、CD59、CD105、CD166表达阳性,CD11a、CD14、CD33、CD34、CD45、CD38、CD80、CD86、CD117表达为阴性。加入成骨诱导剂,细胞形态发生变化,AP活性增强,骨桥蛋白表达阳性,钙沉积逐渐出现。P3和P10代的MSC均有良好的分化为成骨细胞的能力。结论:成人骨髓间质干细胞在体外可分化为成骨细胞。