人脐带间充质干细胞成软骨诱导过程中软骨标志基因表达的研究

目的：观察脐带间充质干细胞（UCMSCs）体外生物学特征,分析成软骨诱导过程中软骨标志基因的表达,探索分化的最佳时间,为其以后应用于组织工程奠定基础。方法体外培养UCMSCs并使用特定的诱导培养基对第3代UCMSCs进行成脂、成骨、成软骨诱导培养并染色鉴定,流式细胞术检测细胞表面标志物,实时定量PCR检测成软骨诱导0、3、7、14、21 d后SOX9、COL2A1、ACAN的mRNA的表达水平。结果 UCMSCs经过体外培养后,细胞形态均一,呈梭形。UCMSCs经成脂、成骨、成软骨诱导后油红O、茜素红、阿利新蓝染色分别呈阳性反应。流式细胞术检测发现UCMSCs低表达CD34、CD45,高表达CD44、CD105。成软骨标志基因SOX9、COL2A1 mRNA的表达在诱导14 d达到最高水平,ACAN的mRNA的表达在诱导7 d达到最高水平。结论 UCMSCs体外传代培养3代后依然可以保持干细胞的特性,其体外诱导成软骨的最佳时间为14 d。     

Pancreatic islet differentiation of human embryonic stem cells by microRNA overexpression

Development of stem cell‐based therapies for the treatment of type 1 diabetes would provide a renewable supply of human β‐cells. Human embryonic stem cells (ESCs) are considered to be one of the stem cell populations with sufficient proliferative capacity to achieve this goal. Currently, differentiation protocols directing ESCs toward a pancreatic fate employ a variety of expensive cytokines and inhibitors. With the known significance of microRNAs in islet development, we present a novel and cost‐effective strategy in which miR‐375 overexpression promotes pancreatic endocrine differentiation in hESCs in the absence of any extrinsic factors. miR‐375 has been shown to be a key regulator of pancreatic development and function in zebrafish, mouse and human. In this study, hESCs were transduced with lentiviral vectors containing human miR‐375 precursor and aggregated to form human embryoid bodies (hEBs) for up to 21 days. Morphological assessment, immunocytochemistry and DTZ staining confirmed that miR‐375‐induced hEBs have similar characteristics to those of mature islets. In addition, the dynamic expression profile of endodermal marker Foxa2 and endocrine‐specific genes, including HNF4α, Pdx1, Pax6, Nkx6.1, Glut2 and insulin, were detected by quantitative real‐time PCR. Finally, insulin release upon glucose stimulation was detected in our differentiated clusters. The data presented here demonstrate the feasibility of using microRNAs to direct differentiation into the pancreatic lineage. Copyright © 2013 John Wiley & Sons, Ltd.     

血小板裂解液对脐带间充质干细胞增殖与分化的影响

背景：脐带间充质干细胞是近年来干细胞生物学研究中发现的重要细胞之一,在细胞治疗领域有着良好的应用前景。血小板裂解液对于干细胞的增殖及诱导分化都存在不同程度的有利作用,在一定程度上甚至可以代替甚至优于血清在培养基中的作用。目的：对血小板裂解液结合脐带间充质干细胞的研究作一综述。方法：于万方等中文数据库和PubMed等英文数据库中,以＂脐带,间充质干细胞,血小板裂解液,分化,增殖＂为关键词检索文献,总结脐带间充质干细胞及血小板裂解液的生物学特性和相关临床应用,并汇总有关血小板裂解液对脐带间充质干细胞增殖活性和诱导分化能力影响的研究结果。结果与结论：血小板裂解液对脐带间充质干细胞的增殖活性及诱导分化有正面影响,这使血小板裂解液在干细胞移植治疗中将会起到无可比拟的作用。     

成人骨髓间充质干细胞体外诱导分化为胰岛样细胞的研究进展

学术背景：近几年采用胰岛细胞或胰腺移植治疗糖尿病取得一些疗效，但仍存在供体匮乏和免疫排斥两大难题。骨髓间充质干细胞在体外可诱导成为胰岛样细胞。 目的：分析总结骨髓间充质细胞体外向胰岛样细胞的诱导分化研究进展。检索策略：由该论文的研究人员应用计算机检索Pubmed数据库1997—08／2007-08的相关文献，检索词“mesenchymal stem cells，insulin secreting cell”，并限定文章语言种类为English。同时计算机检索中国期刊全文数据库1997-08／2007-08的相关文献，检索词“骨髓间充质干细胞，胰岛素分泌细胞”，并限定文章语言种类为中文。共检索到108篇文献，对资料进行初审，纳入标准：①文章所述内容应与骨髓间充质细胞体外诱导分化为胰岛样细胞密切相关。②同一领域选择近期发表或在权威杂志上发表的文章。排除标准：①重复性研究。②Meta分析。 文献评价：文献的来源主要是通过对骨髓间充质细胞体外向胰岛样细胞的诱导分化方面内容进行汇总分析。所选用的30篇文献中，2篇为综述，其余均为临床或基础实验研究。 资料综合：①由于胰腺和神经系统具有相似的发育控制机制，目前认为神经生长因子可能是胰腺发育的关键信号。利用人骨髓间充质干细胞多向分化能力，用表皮生长因子、碱性成纤维细胞生长凶子将其诱导成为nestin阳性细胞，该细胞在添加含有胰岛细胞赖以生存的条件培养液中为适宜微环境，将被进一步诱导为胰腺样细胞。②目前针对胰岛素样细胞的鉴定与功能学研究有以下几方面：细胞形态学观察是否有类似胰岛样细胞团的聚集;免疫组织化学检测诱导后细胞胰岛素、胰高血糖素和生长抑素的表达;用RT—PCR法对诱导后细胞的胰岛素及胰高血糖素基因表达测定;葡萄糖刺激的胰岛索释放试验来研究诱导后?     

Differential bone‐forming capacity of osteogenic cells from either embryonic stem cells or bone marrow‐derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Perivascular cells as mesenchymal stem cells

Importance of the field: Mesenchymal stem cells are multipotent adult stem cell populations that have broad differentiation plasticity and immunosuppressive potential that render them of great importance in cell-based therapies. They are identified by in vitro characteristics based on their differentiation potential for clinical approaches while their biological properties and in vivo identities are often less understood.

Areas covered in this review: Recent research carried out in the last decade on mesenchymal stem cell biology suggests that mesenchymal stem cells from various tissues reside in a perivascular location and these can be identified as pericytes that function as mural cells in microvessels.

What the reader will gain: This review covers recent progress on understanding the link between pericytes and mesenchymal stem cells discussing specific points such as response to injury and tissue-specific functions.

Take home message: Despite a long and controversial history, there is a growing acceptance that perivascular cells are connected with mesenchymal stem cells, all that is really lacking is genetic evidence to show differentiation of pericytes into different cells types.     

Epigenetic approaches to regeneration of bone and cartilage from stem cells

Introduction: Embryonic stem cells (ESCs) or adult stem cells, especially mesenchymal stem cells (MSCs), have been intensively studied for skeletal tissue regeneration including bone and cartilage. Epigenetic mechanisms play essential roles in stem cell maintenance and differentiation. However, little is known about the epigenetic regulation of osteogenesis and chondrogenesis of stem cells.

Areas covered: In this review, features of ESCs and adult stem cells, epigenetics and chromatin structure, as well as epigenetic mechanisms, such as chromatin remodeling, DNA methylation and histone modifications, polycomb group (PcG) proteins and microRNAs are described. Epigenetic researches of stem cell are introduced.

Expert opinion: Epigenetic alterations of stem cell during the in vitro differentiation can be controlled for clinical applications. MSCs are effective resources for skeletal tissue regeneration in both undifferentiated and differentiated states. Understanding epigenetic signatures of MSC is crucial to maintain the stemness. In addition, investigation of epigenetic changes in the differentiation of MSCs is very important to develop methods or chemicals to promote efficient differentiation of MSCs. Inhibition of PcG protein enhancer of zeste (Ezh2) a chromatin modifier, could be a promising candidate to improve MSC differentiation by decreasing Ezh2-mediated H3K27me3.     

脐带间充质干细胞促进骨愈合的体外实验及临床应用1例

背景:华通胶来源脐带间充质干细胞比成人间充质干细胞更原始,研究表明其端粒酶活性更高、培养倍增时间更短、分化的细胞谱系更广.目的:观察脐带间充质干细胞在体外分化为成骨细胞的能力及治疗骨折不愈合的效果.方法:从脐带Wharton胶获取细胞培养、扩增,取传代细胞行免疫表型测定和成骨细胞诱导分化,并以脐带间充质细胞移植治疗1例骨折感染外露后长期不愈病例.结果与结论:培养细胞形态类成纤维细胞,可长期稳定培养,传代细胞表达间充质干细胞免疫表型,成骨诱导分化的细胞茜素红染色胞浆中有大量的钙沉积,Von Kossa染色有钙结节形成.患者采用脐带间充质干细胞混悬液外用4次,肉芽组织迅速增生填满窦道并上皮化,12 d创面愈合.说明,脐带间充质干细胞具有高度自我更新能力和分化潜能,能够向成骨细胞分化,将其移植治疗骨不连可以显著改善局部微环境.     

分阶段添加细胞诱导因子诱导豚鼠脂肪间充质干细胞定向分化内耳毛细胞

背景：感音神经性耳聋主要是由内耳毛细胞的缺失或受损造成,用脂肪间充质干细胞来再生修复内耳毛细胞是治疗听力损失的有效方法。目的：探讨体外定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞样细胞分化的可行性。方法：体外分离培养豚鼠脂肪间充质干细胞至第3代,流式细胞仪检测细胞免疫表型,分阶段加入表皮生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1、全反式维甲酸、脑源性神经营养因子、神经营养因子3,观察诱导分化的效果。结果与结论：豚鼠脂肪间充质干细胞体外培养呈梭形,漩涡状贴壁生长,第3代细胞表面标记CD29与CD44表达呈现阳性,CD34与CD45表达呈现阴性。应用细胞因子诱导后细胞早期nestin和GFAP表达阳性,继续诱导10 d后表达毛细胞特异性标记物MyosinⅦa和Math1,说明细胞因子可定向诱导豚鼠脂肪间充质干细胞向内耳毛细胞分化。     

DNA methyltransferases inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid‐derived mesenchymal stem cells via chromatin remodeling

Human amniotic fluid‐derived mesenchymal stem cells (AF‐MSCs) are a new potential stem cell source for cell therapy and regenerative medicine. These are fetal mesenchymal stem cells with multilineage differentiation potential found in amniotic fluid. The aim of the present study was to evaluate in vitro differentiation initiation of AF‐MSCs into cardiac progenitors upon application of inhibitors of DNA methyltransferases (DNMT), such as Decitabine (DEC; 5‐aza‐2′‐deoxycytidine) and Zebularine (ZEB). We assessed epigenetic changes and explored patterns of genes, enriched in association with hyperacetylated H4 after induced differentiation. Upregulation of cardiomyogenesis‐related genes (TNNT2, MYH6, ACTN2, and DES) and cardiac ion channels genes, downregulation of pluripotency genes markers as well as increase in Connexin43 expression indicated cardiomyogenic commitment. Evaluation of global epigenetic changes showed that levels of chromatin modifying enzymes, such as Polycomb repressive complex 2 proteins (EZH2, SUZ12), DNMT1, histone deacetylases 1 and 2 were reduced to the similar extent by both differentiation agents. Levels of specific histone marks keeping active state of chromatin (H3K4me3, H3K9Ac, and H4hyperAc) increased and marks of repressed chromatin state (H3K27me3 and H3K9me3) decreased after DEC or ZEB treatment. Chip‐Seq analysis after chromatin immunoprecipitation with H4hyperAc demonstrated enrichment of around 100 functionally annotated genes, related to chromatin reorganization and cardiomyogenesis and confirmed relation between H4 hyperacetylation and gene expression. Our results demonstrate that both DEC and ZEB can be potentially used as cardiomyogenic differentiation inducers in AF‐MSCs, and they cause various genetic and epigenetic changes resulting in global chromatin remodeling.     

携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞及其成脂分化

背景：胰岛素是成脂诱导剂的重要组成成分。然而胰岛素存在半衰期,会随着体内代谢不断灭活及降解,植入体内的细胞或材料无法像体外那样以更换培养液来达到目的。实验拟通过转染胰岛素基因,使转染后的干细胞稳定分泌胰岛素,促进其成脂分化。目的：探讨携带人胰岛素基因腺病毒载体转染人脐带间充质=F细胞后对其成脂分化能力的影响。方法：取第3代人脐带间充质干细胞,以感染复数为20转染腺病毒载体。实验分为4组：对照组为第4代人脐带间充质干细胞;实验组1为腺病毒转染的第4代人脐带间充质干细胞;实验组2为第4代人脐带间充质干细胞＋成脂诱导液;实验组3为腺病毒转染的第4代人脐带间充质干细胞＋成脂诱导液。结果与结论：转染48h后实验组1和实验组3的人脐带间充质干细胞在荧光显微镜下显现弱荧光,72h后荧光较强。经脂肪诱导培养液培养14d后,实验组1,2,3油红O染色后显微镜下呈红色,对照组未见脂滴形成,实验组1可见一些细小脂滴形成;实验组3与实验组2相比,脂滴大且多,定量分析显示,实验组3油红0染色阳性的面积和吸光度大于实验组2（P〈0．05）。由此说明携带人胰岛素基因腺病毒载体转染人脐带间充质干细胞后可促进其成脂能力。     

Conditioned medium as a strategy for human stem cells chondrogenic differentiation

Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow‐derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly‐derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three‐dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage‐related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.     

Pdx1 and controlled culture conditions induced differentiation of human amniotic fluid‐derived stem cells to insulin‐producing clusters

This study investigated the differentiation of human amniotic fluid‐derived stem cells (hAFSCs) into insulin‐producing clusters in vitro. Adenovirally‐delivered mouse Pdx1 (Ad‐Pdx1) induced human Pdx1 expression in hAFSCs and enhanced the coordinated expression of downstream β‐cell markers. When Ad‐Pdx1‐transduced hAFSCs were sequentially treated with activin A, bFGF and nicotinamide and the culture plate surface coated with poly‐l ‐ornithine, the expression of islet‐associated human mRNAs for Pdx1, Pax6, Ngn3 and insulin was increased. C‐peptide ELISA confirmed that Ad‐Pdx1‐transduced hAFSCs processed and secreted insulin in a manner consistent with that pathway in pancreatic β‐cells. To sustain the β‐cell‐like phenotype and investigate the effect of three‐dimensional (3D) conformation on the differentiation of hAFSCs, Pdx1‐transduced cells were encapsulated in alginate and cultured long‐term under serum‐free conditions. Over 2 weeks, partially differentiated hAFSC clusters increased in size and increased insulin secretion. Taken together, these data demonstrate that ectopic Pdx1 expression initiates pancreatic differentiation in hAFSCs and that a β‐cell‐like phenotype can be augmented by culture conditions that mimic the stromal components and 3D geometry associated with pancreatic islets. Copyright © 2012 John Wiley & Sons, Ltd.     

脐带间充质干细胞促进骨愈合的体外实验及临床应用1例

背景：华通胶来源脐带间充质干细胞比成人间充质干细胞更原始,研究表明其端粒酶活性更高、培养倍增时间更短、分化的细胞谱系更广。目的：观察脐带间充质干细胞在体外分化为成骨细胞的能力及治疗骨折不愈合的效果。方法：从脐带Wharton胶获取细胞培养、扩增,取传代细胞行免疫表型测定和成骨细胞诱导分化,并以脐带间充质细胞移植治疗1例骨折感染外露后长期不愈病例。结果与结论：培养细胞形态类成纤维细胞,可长期稳定培养,传代细胞表达间充质干细胞免疫表型,成骨诱导分化的细胞茜素红染色胞浆中有大量的钙沉积,VonKossa染色有钙结节形成。患者采用脐带间充质干细胞混悬液外用4次,肉芽组织迅速增生填满窦道并上皮化,12d创面愈合。说明,脐带间充质干细胞具有高度自我更新能力和分化潜能,能够向成骨细胞分化,将其移植治疗骨不连可以显著改善局部微环境。     

血小板裂解液对脐带间充质干细胞增殖与分化的影响

背景:脐带间充质干细胞是近年来干细胞生物学研究中发现的重要细胞之一,在细胞治疗领域有着良好的应用前景.血小板裂解液对于干细胞的增殖及诱导分化都存在不同程度的有利作用,在一定程度上甚至可以代替甚至优于血清在培养基中的作用.目的:对血小板裂解液结合脐带间充质干细胞的研究作一综述.方法:于万方等中文数据库和PubMed 等英文数据库中,以"脐带,间充质干细胞,血小板裂解液,分化,增殖"为关键词检索文献,总结脐带间充质干细胞及血小板裂解液的生物学特性和相关临床应用,并汇总有关血小板裂解液对脐带间充质干细胞增殖活性和诱导分化能力影响的研究结果.结果与结论:血小板裂解液对脐带间充质干细胞的增殖活性及诱导分化有正面影响,这使血小板裂解液在干细胞移植治疗中将会起到无可比拟的作用.