细胞外基质硬度调控间充质干细胞分化

新近研究表叽细胞外基质（extracellularmatrix,ECM）的物理性质,特别是硬度或弹性,能对细胞的黏附、铺展、迁移、增殖、分化和凋亡等多种功能和行为产生重要影响。间充质干细胞（mesenchymalstemcells,MSCs）是组织工程和细胞治疗的理想种子细胞。ECM硬度可诱导MSCs向脂肪、软骨、神经、肌肉和骨等方向分化。该文综合论述了ECM硬度对干细胞分化的影响,涵盖了构建ECM硬度的测量、调控与表征等,不同培养条件下干细胞对硬度的响应和分化以及硬度和其他因素的联合作用;在此基础上,进一步论述了干细胞分化过程中细胞感应ECM硬度并转化为生物学信号的机制和信号通路。该文还总结了在ECM硬度调控干细胞分化行为领域最新的研究进展情况,较为系统地分析了材料学、细胞生物学、分子生物学水平的主要影响因素,并对本领域未来需要重点研究的问题进行了展望。     

Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC.     

microRNA499 慢病毒载体转染诱导大鼠骨髓间充质干细胞 向心肌样细胞分化*

目的:探讨microRNA 499(miR-499)慢病毒转染对诱导大鼠骨髓来源间充质干细胞(BM-MSCs)向心肌样细胞分化的作用。方法:取第四代Wistar大鼠骨髓来源间充质干细胞进行流式细胞检测,鉴定干细胞表面特异标记物。使用符合干细胞鉴定标准的细胞批次用于后续实验。实验设置miR499慢病毒转染、慢病毒空白转染2个处理组,分别于处理后即日、1d,3d,5d,7d收集细胞进行下列实验:实时荧光定量PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,western-blot检测心肌特异蛋白I(cTnI)的表达。结果:培养第四代Wistar大鼠骨髓来源间充质干细胞表达干细胞表面特异标记物,可用于实验。大鼠骨髓来源间充质干细胞microRNA 499慢病毒载体转染后microRNA 499表达明显升高,且转染后1d,3d,5d,7d,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。慢病毒空白转染组未见明显变化。western-blot检测自第3天开始可见cTnI阳性表达条带,慢病毒空白转染组未检测到明显阳性表达条带。结论:microRNA 499可诱导大鼠骨髓来源间充质干细胞向心肌样细胞分化。     

Secretome of Differentiated PC12 Cells Enhances Neuronal Differentiation in Human Mesenchymal Stem Cells Via NGF-Like Mechanism

The secretome-mediated responses over cellular physiology are well documented. Stem cells have been ruling the field of secretomics and its role in regenerative medicine since the past few years. However, the mechanistic aspects of secretome-mediated responses and the role of other cells in this area remain somewhat elusive. Here, we investigate the effects of secretome-enriched conditioned medium (CM) of neuronally differentiated PC12 cells on the neuronal differentiation of human mesenchymal stem cells (hMSCs). The exposure to CM at a ratio of 1:1 (CM: conditioned medium of PC12 cells) led to neuronal induction in hMSCs. This neuronal induction was compared with a parallel group of cells exposed to nerve growth factor (NGF). There was a marked increase in neurite length and expression of neuronal markers (β-III tubulin, neurofilament-M (NF-M), synaptophysin, NeuN in exposed hMSCs). Experimental group co-exposed to NGF and CM showed an additive response via MAPK signaling and directed the cells particularly towards cholinergic lineage. The ability of CM to enhance the neuronal properties of stem cells could aid in their rapid differentiation into neuronal subtypes in case of stem cell transplantation for neuronal injuries, thus broadening the scope of non-stem cell-based applications in the area of secretomics.     

机械力对间充质干细胞向成骨细胞分化的力学响应机制研究

间充质干细胞的分化受多种因素的影响,其中力学是重要因素之一.为了探讨力学信号在间充质干细胞分化中的传导机制,利用力学加载装置在成骨细胞诱导体系条件下,对小鼠骨髓间充质干细胞系(D1细胞)加载不同拉伸应变,运用RT-PCR方法、Flou-3-AMCa2 染色技术、激光共聚焦显微镜技术及5种信号阻断剂,SB203580(p38MAPK特异抑制剂)、PD98059(MEK-1/2MAPK特异抑制剂)、LY294002(PI3Ks特异抑制剂)、细胞松弛素B(微丝结构阻断剂)、EGTA(Ca2 螯合剂),探讨力学信号的基本传导途径.结果显示:3%的拉伸应变能明显提高细胞内Ca2 水平;细胞微丝结构破坏后,延迟了3%拉伸应变对细胞内Ca2 水平增加的影响;细胞外Ca2 螯合后,拉伸应变不能促进细胞内Ca2 水平的升高,该结果提示,拉伸应变对细胞内Ca2 水平的影响主要通过细胞外Ca2 的内流实现.5种信号阻断剂能完全阻断干细胞向成骨细胞分化过程中关键基因骨钙蛋白(osteocalcin,OCN)和OSX mRNA的表达.p38MAPK途径、MEK-1/2MAPK途径被阻断后,拉伸应变激活了OCN和Osterix(OSX,与成骨细胞分化相关的关键基因)mRNA的表达;PI3Ks途径阻断后,拉伸应变部分激活了OCN和OSX mRNA的表达;细胞微丝破坏及胞外Ca2 螯合后,拉伸应变不能促进OCN和OSXmRNA的表达.上述结果表明:力学信号通过Ca2 信号、细胞微丝结构以及PI3Ks信号途径引起细胞的应答反应和生物学效应.     

GABA Effects During Neuronal Differentiation of Stem Cells

Gamma-amino butyrate (GABA) is the most prevalent inhibitory neurotransmitter in the adult brain. In this review, we summarize the pharmacology and regulation of GABAergic transmission components (biosynthetic enzymes, receptors and transporters) in adult non-neurogenic brain regions. The effects of targeted mutations in genes relevant for GABAergic functions and how they influence specific neuronal circuits and pathological states are presented. We then review GABA actions on neuronal differentiation. During brain development, GABA has depolarizing activity in cerebrocortical neural precursors, controlling cell division and contributing to neuronal migration and maturation. In the adult forebrain there are two neurogenic regions exposed to synaptic and non-synaptic GABA release. Neural stem cells and neuronal progenitors express GABA receptors in subventricular and subgranular zones. GABA effects in these cells are very similar to those found in embryonic cortical precursor cells, and therefore it is possible that this amino acid has important roles during adult brain plasticity. Special issue article in honor of Dr. Ricardo Tapia.     

间充质干细胞诱导分化为胰岛β细胞的研究进展

间充质干细胞是一类具有多向分化潜能的成体干细胞,在体内外不仅可以被诱导分化为中胚层细胞,而且可以分化为内胚层和神经外胚层细胞。间充质干细胞易分离,体外可大量扩增,异体移植不引起免疫排斥反应,在细胞治疗和组织工程中具有广阔的应用前景。经过适当诱导,间充质干细胞可能成为胰岛β细胞的来源之一。就间充质干细胞的生物学性状和优势,以及诱导分化为胰岛β细胞的技术方法和发展趋势进行了综述。     

MicroRNA-27a Modulates HCV Infection in Differentiated Hepatocyte-Like Cells from Adipose Tissue-Derived Mesenchymal Stem Cells

Background and Aims

Despite the discovery of hepatitis C virus (HCV) entry factor, the mechanism by which it is regulated by miRNAs remains unclear. Adipose tissue-derived human mesenchymal stem cells (AT-hMSCs) have been widely used for differentiated hepatocyte-like cells (DHCs). Here, we established an in vitro HCV infection model using DHCs from AT-hMSCs and identified miRNAs that modulate HCV infectivity.

Methods

AT-hMSCs were differentiated into DHCs using the conditional media, and evaluated for hepatocyte characteristics using RT-PCR, immunocytochemistry, periodic acid-Schiff staining, and a urea synthesis assay. The expression of HCV candidate receptors was also verified using immunocytochemistry. The levels of candidate miRNAs targeting HCV receptors were then determined by relative quantitative RT-PCR (rqRT-PCR). Finally, DHCs were infected using HCVcc and serum from HCV-infected patients, and infectivity of the virus was measured by rqRT-PCR and transmission electron microscopy (TEM).

Results

The expected changes in morphology, function and hepatic gene expression were observed during hepatic differentiation. Moreover, the expression of candidate HCV entry factors and miR-27a were altered during hepatic differentiation. The infection and replication of HCV occurred efficiently in DHCs treated with HCVcc or infected with serum from HCV-infected patients. In addition, HCV infectivity was suppressed in miR-27a-transfected DHCs, due to the inhibition of LDLR expression by miR-27a.

Conclusions

Our results demonstrate that AT-hMSCs are a good source of DHCs, which are suitable for in vitro cultivation of HCV. Furthermore, these results suggest that miR-27a modulates HCV infectivity by regulating LDLR expression.     

Wnts Enhance Neurotrophin-Induced Neuronal Differentiation in Adult Bone-Marrow-Derived Mesenchymal Stem Cells via Canonical and Noncanonical Signaling Pathways

Wnts were previously shown to regulate the neurogenesis of neural stem or progenitor cells. Here, we explored the underlying molecular mechanisms through which Wnt signaling regulates neurotrophins (NTs) in the NT-induced neuronal differentiation of human mesenchymal stem cells (hMSCs). NTs can increase the expression of Wnt1 and Wnt7a in hMSCs. However, only Wnt7a enables the expression of synapsin-1, a synaptic marker in mature neurons, to be induced and triggers the formation of cholinergic and dopaminergic neurons. Human recombinant (hr)Wnt7a and general neuron makers were positively correlated in a dose- and time-dependent manner. In addition, the expression of synaptic markers and neurites was induced by Wnt7a and lithium, a glycogen synthase kinase-3β inhibitor, in the NT-induced hMSCs via the canonical/β-catenin pathway, but was inhibited by Wnt inhibitors and frizzled-5 (Frz5) blocking antibodies. In addition, hrWnt7a triggered the formation of cholinergic and dopaminergic neurons via the non-canonical/c-jun N-terminal kinase (JNK) pathway, and the formation of these neurons was inhibited by a JNK inhibitor and Frz9 blocking antibodies. In conclusion, hrWnt7a enhances the synthesis of synapse and facilitates neuronal differentiation in hMSCS through various Frz receptors. These mechanisms may be employed widely in the transdifferentiation of other adult stem cells.     

大戟苷抑制大鼠骨髓间充质干细胞成骨分化

目的:观察泽漆主要活性成分大戟苷(euphornin)对大鼠骨髓间充质干细胞(rMSC)成骨分化的影响。方法:从大鼠股骨中分离培养rMSC,并诱导其成骨分化。用MTT法检测细胞增殖情况,通过茜素红染色,碱性磷酸酶(ALP)活性检测和钙含量测定分别定性、定量地判断其在成骨分化中的效果。实时定量PCR(Q-PCR)检测主要成骨标志因子骨桥蛋白(OPN)和一型胶原蛋白(COL-Ⅰ)及主要转录因子骨形成蛋白2(BMP2)、Runt相关转录因子2(Runx2)和Osterix(Osx)mRNA的表达。结果:大戟苷能剂量依赖性地抑制rMSC成骨分化,并一定程度地抑制其细胞增殖。COL-Ⅰ和OPN的表达在第4、8天分别有显著下降。BMP2、Runx2和Osx等关键转录因子的表达也被明显抑制。结论:大戟苷能抑制rMSC成骨分化,其作用主要是通过抑制BMP通路相关因子的表达而实现的。     

间充质干细胞向成骨细胞分化的信号通路

间充质干细胞具有向成骨细胞分化的潜能,可体外分离、培养和扩增,是骨组织工程中理想的种子细胞。近年的研究表明间充质干细胞的成骨分化受到多种信号通路的调控,现就其中研究较为深入的MAPK和Notch通路的情况作一简要综述。     

间充质干细胞分化为心肌细胞的诱导方法研究进展

间充质干细胞作为一种取材方便、易于分离培养、体外扩增快、免疫原性低的成体干细胞,具有自我更新和多向分化潜能,可在体内外不同的诱导条件下分化为心肌细胞,是理想的心肌再生治疗的种子细胞。本文综述了间充质干细胞分化为心肌细胞的诱导方法,包括化学试剂、中药制剂、机械力和电磁刺激、心肌环境因子、损伤组织条件培养、组织工程方法等,为其在心肌损伤性疾病尤其是心肌梗死治疗中的应用提供基础。     

Reversible Modulation of Myofibroblast Differentiation in Adipose-Derived Mesenchymal Stem Cells

Unregulated activity of myofibroblasts, highly contractile cells that deposit abundant extracellular matrix (ECM), leads to fibrosis. To study the modulation of myofibroblast activity, we used human adipose-derived mesenchymal stem cells (ADSCs), which have much potential in regenerative medicine. We found that ADSCs treated with TGF-β developed a myofibroblastic phenotype with increases in α-smooth muscle actin (α-SMA), a myofibroblast marker, and ECM proteins type I collagen and fibronectin. In contrast, treatment with bFGF had the opposite effect. bFGF-differentiated ADSCs showed marked down-regulation of α-SMA expression, collagen I, and fibronectin, and loss of focal adhesions and stress fibers. Functionally, bFGF-differentiated ADSCs were significantly more migratory, which correlated with up-regulation of tenascin-C, an anti-adhesive ECM protein, and vimentin, a pro-migratory cytoskeletal protein. On the other hand, TGF-β-differentiated ADSCs were significantly more contractile than bFGF-differentiated cells. Interestingly, cells completely reversed their morphologies, marker expression, signaling pathways, and contractility versus migratory profiles when switched from culture with one growth factor to the other, demonstrating that the myofibroblast differentiation process is not terminal. Cell differentiation was associated with activation of Smad2 downstream of TGF-β and of ERK/MAP kinase downstream of bFGF. Reversibility of the TGF-β-induced myofibroblastic phenotype depends, in part, on bFGF-induced ERK/MAP kinase signaling. These findings show that ADSC differentiation into myofibroblasts and re-differentiation into fibroblast-like cells can be manipulated with growth factors, which may have implications in the development of novel therapeutic strategies to reduce the risk of fibrosis.