microRNA在多能干细胞向心肌细胞分化中的作用

microRNAs(miRNAs)是长约22 nt的非编码RNAs,广泛参与细胞的增殖、分化、病变、修复和凋亡等多种生命活动．多能干细胞（pluripotent stem cells）是指体外具有自我更新和多向分化潜能的细胞,在一定条件下可被定向诱导分化为多种细胞类型．miRNAs在多能干细胞中表达丰富,并通过调控基因表达影响其自我更新及分化．由多能干细胞向心肌细胞分化的方法主要有3种,即拟胚体形成法、与内胚层细胞共培养法和特定诱导物添加法．虽然这3种方法均可成功诱导多能干细胞向心肌细胞分化,但重复率很低. 所以,人们把研究的视野逐渐转向miRNAs--这个广泛参与细胞生命活动的小分子物质．大量研究表明,在多能干细胞中,不同的miRNAs可通过打靶不同基因影响其向心肌细胞分化．在间充质干细胞中,miR-1、miR-133 和miR-499可分别打靶Hes-1、SRF和Pdcd4| 而在胚胎干细胞中,miR-1和miR-499分别打靶 Hand2和Pacs2促进其向心肌细胞分化．miRNAs在多能干细胞向心肌分化作用机制的研究必将促进再生医学在心脏疾病治疗上的应用．     

人类多能干细胞向脊髓前角运动神经元定向分化方法研究进展:从发育生物学到临床转化

人类多能干细胞(human pluripotent stem cell,hPSC)包括人胚胎干细胞(embryonic stem cell,ESC)及人诱导多能干细胞(induced pluripotent stem cell,iPSC),它们具有向人体多种类型细胞分化的潜能。近年来,其体外定向分化为脊髓前角运动神经元的研究取得了一定进展。该文基于对神经发育的理解,回顾总结了hPSC向脊髓前角运动神经元定向分化的研究进展,并介绍了它们在研究人类神经发育、对疾病进行体外建模和细胞替代疗法方面的应用。     

诱导性多能干细胞向神经细胞分化的研究进展

诱导性多能干细胞(induced pluripotent stem cell,iPS cell)是通过转染外源特定的基因组合来诱导成体细胞重编程为类似于胚胎干细胞的一种多潜能干细胞,iPS细胞与胚胎干细胞不仅在形态上相似,而且在功能方面几乎相同.另外,iPS细胞的诞生克服了胚胎干细胞在临床应用时涉及的移植免疫排斥与伦理道德问题,因此具有重要的临床应用价值.目前iPS在治疗中枢神经系统性疾病方面的研究已取得很大进展,包括iPS细胞向神经细胞诱导分化方法的改进、分化机理的探索以及iPS细胞分化来源神经细胞在神经系统疾病模型中治疗作用的研究等.从iPS细胞的创建及特点、iPS细胞向神经细胞分化的诱导方法及研究新进展方面予以综述.     

小分子物质对维持胚胎干细胞未分化状态的调控作用

胚胎干细胞作为一种具有多潜能和高度自我更新能力的种子细胞,己被广泛地应用于医学研究领域。在体外培养条件下,胚胎干细胞可被诱导分化为三个胚层来源的组织细胞,故被看作为最具有应用前景的种子细胞。近年来,对于在体外培养条件下如何维持胚胎干细胞的多能性即使其较长时期的处于未分化状态成为研究热点,其中一些天然存在或人工合成的小分子物质可通过作用于某些特定的靶信号通路,调控胚胎干细胞的分化命运。本文概述了几种小分子物质的最新研究进展,并对小分子物质在成体多分化潜能胚胎样干细胞分化调控方面的应用前景进行评述。     

胚胎干细胞诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ESC)因其具有自我更新能力和发育的多能性,成为当前医学研究的热点。ESC不但可以自发分化,而且在诱导因素作用下可以定向分化为某一种特定的成熟细胞。因此,ESC在移植医学、发育生物学等领域有着广阔的应用前景。本文对几种定向诱导ESC分化的策略进行了综述。     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

脂肪干细胞的共培养及其分化应用概述

干细胞在体外特定培养条件下可以被诱导分化成具有不同体细胞表型的细胞。除了通过不同培养条件进行体外诱导分化的方法外,用成熟体细胞与干细胞共培养同样可以诱导干细胞定向分化。以下首先简述了脂肪干细胞 (Adipose-derived stem cells,ADSCs) 的来源及其标志,然后重点就ADSCs的不同培养方法、诱导分化及最新的临床应用进行阐述,包括药物及化学诱导培养、体细胞与ADSCs二维、三维共培养等,最后提出ADSCs的问题所在并对此技术进行展望。     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

APA微囊微环境影响胚胎干细胞增殖分化的体外研究

以小鼠胚胎T细胞（embryonic stem cell,ESC）为模型,在牛理条件F对ESC进行微囊化包封、培养,并利用免疫组织化学技术及RT-PCR方法检测其生长及未分化状态,以期建立微囊化ESC这一体外培养模型,同时明确海藻酸钠-聚赖氨酸-海藻酸钠（alginate-poly-lysine-alginate,APA）微囊微环境对ESC增殖及分化潜能的影响。结果表明：ESC能够在微囊（包括液化型及非液化型）或微球（海藻酸钙胶珠）内生长良好,但因生长环境存在差异,其表现的生长行为各具特征。比较其它类型,ESC在液化型APA微囊内的存活期限最长。经体外维持培养3周以上,仍能持续表达胚胎源未分化T细胞的标志性蛋白AP,SSEA-1及转录因子Oct-4。为进一步明确微囊内增殖的ESC是台仍具有多向分化的干细胞潜能,应用机械破囊法释放微囊内ESC团,并在体外进行定向诱导。经过近3周的条件诱导,其结果为：细胞团DTZ染色阳性：anti-insulin免疫荧光检测阳性;且特异性表达Pdx-1,Ins-1基因。上述结果证明：APA微囊为ESC维持未分化状态的增殖提供了特殊的微环境,APA微囊内所形成的ESC团仍具有多向分化的干细胞潜能。     

移植骨髓间充质干细胞治疗大鼠糖尿病的研究

目的 通过移植骨髓间充质干细胞（mesenchymal stem cell,MSC）的方法试治疗大鼠糖尿病。方法 贴壁生长的MSC与大鼠胰腺的细胞共培养以检测其向胰岛细胞分化的潜能。并将体外培养扩增的MSC移植入糖尿病大鼠体内,观测其能否改善糖尿病病情及其在大鼠体内微环境中的分化情况。结果 共培养法可使MSC分化为胰岛样细胞。对大鼠的MSC移植能明显缓解糖尿病病情。结论 MSC移植的方法对大鼠糖尿病有一定的治疗作用。     

诱导性多潜能干细胞(iPS cells)——现状及前景展望

主要从 iPS细胞发展历程、获得 iPS细胞的几个关键步骤 (如基因导入方式、诱导 iPS细胞所需因子组合与小分子化合物运用和体细胞种类选择等)、病人或疾病特异性 iPS细胞、iPS细胞体内外诱导分化与其衍生物的临床应用和制备无遗传修饰的(genetic modification-free) iPS细胞的可行性与前景等方面对 iPS细胞最新研究进展做评述．日本和美国研究小组先后用4种基因将小鼠(2006年8月)和人(2007年11～12月)的体细胞在体外重编程为诱导性多潜能干细胞(induced pluripotent stem cells,iPS cells),此后在短短两年多时间内,iPS 细胞的研究和关注度呈爆炸式增长．体细胞重编程、去分化和多潜能干细胞来源等一系列热点问题再次成为干细胞和发育生物学等研究的热点和焦点．与胚胎干细胞(embryonic stem cells,ES cells)一样,iPS细胞在体内可分化为3个胚层来源的所有细胞,进而参与形成机体所有组织和器官．迄今,在体外已由 iPS细胞定向诱导分化出功能性的多种成熟细胞．因此,iPS细胞研究不仅具有重要理论意义,而且在再生医学、组织工程和药物发现与评价等方面极具应用价值．     

从胚胎干细胞到视网膜

视网膜退行性病变影响着全世界数百万人。然而,视网膜是人体再生能力很差的一类组织,成年机体无法自我更新那些病变中丢失的视网膜细胞,导致视网膜退行性病变的不可逆性。因此,恢复患者视觉将依赖于引入外源细胞替代丢失的视网膜神经元。胚胎干细胞（ES细胞）具有无限的自我更新能力和形成机体所有类型细胞的巨大分化潜力。这两个特性使得ES细胞成为细胞替代疗法的理想供体细胞。近年来,人们在探索将ES和诱导多能干细胞（iPS细胞）体外定向诱导分化为视网膜神经元,甚至整个视网膜方面已取得多项进展,并且体外形成的视网膜细胞可以与宿主视网膜整合。在此篇综述中,首先简要概括哺乳动物视网膜的组织结构、发育过程和调控机制,然后,重点阐述近年来科研工作者探索ES/iPS细胞体外诱导分化为视网膜细胞和组织的研究进展。     

Pluripotent human stem cells for the treatment of retinal disease

Despite advancements made in our understanding of ocular biology, therapeutic options for many debilitating retinal diseases remain limited. Stem cell-based therapies are a potential avenue for treatment of retinal disease, and this mini-review will focus on current research in this area. Cellular therapies to replace retinal pigmented epithelium (RPE) and/or photoreceptors to treat age-related macular degeneration (AMD), Stargardt's macular dystrophy, and retinitis pigmentosa are currently being developed. Over the past decade, significant advancements have been made using different types of human stem cells with varying capacities to differentiate into these target retinal cell types. We review and evaluate pluripotent stem cells, both human embryonic stem cells and human induced pluripotent stem cells, as well as protocols for differentiation of ocular cells, and culture and transplant techniques that might be used to deliver cells to patients.     

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.     

Fate of undifferentiated mouse embryonic stem cells within the rat heart: role of myocardial infarction and immune suppression

It has recently been suggested that the infarcted rat heart microenvironment could direct pluripotent mouse embryonic stem cells to differentiate into cardiomyocytes through an in situ paracrine action. To investigate whether the heart can function as a cardiogenic niche and confer an immune privilege to embryonic stem cells, we assessed the cardiac differentiation potential of undifferentiated mouse embryonic stem cells (mESC) injected into normal, acutely or chronically infarcted rat hearts. We found that mESC survival depended on immunosuppression both in normal and infarcted hearts. However, upon Cyclosporin A treatment, both normal and infarcted rat hearts failed to induce selective cardiac differentiation of implanted mESC. Instead, teratomas developed in normal and infarcted rat hearts 1 week and 4 weeks (50% and 100%, respectively) after cell injection. Tight control of ESC commitment into a specific cardiac lineage is mandatory to avoid the risk of uncontrolled growth and tumourigenesis following transplantation of highly plastic cells into a diseased myocardium.     

Induction of pluripotency in primordial germ cells

Primordial germ cells (PGCs) are the founder cells of all gametes. PGCs differentiate from pluripotent epiblasts cells by mesodermal induction signals during gastrulation. Although PGCs are unipotent cells that eventually differentiate into only sperm or oocytes, they dedifferentitate to pluripotent stem cells known as embryonic germ cells (EGCs) in vitro and give rise to testicular teratomas in vivo, which indicates a "metastable" differentiation state of PGCs. We have shown that an appropriate level of phosphoinositide-3 kinase (PI3K)/Akt signaling, balanced by positive and negative regulators, ensures the establishment of the male germ lineage by preventing its dedifferentiation. Specifically, hyper-activation of the signal leads to testicular teratomas and enhances EGC derivation efficiency. In addition, PI3K/Akt signaling promotes PGC dedifferentiation via inhibition of the tumor suppressor p53, a downstream molecule of the PI3K/Akt signal. On the other hand, Akt activation during mesodermal differentiation of embryonic stem cells (ESCs) generates PGC-like pluripotent cells, a process presumably induced through equilibrium between mesodermal differentiation signals and dedifferentiation-inducing activity of Akt. The transfer of these cells to ESC culture conditions results in reversion to an ESC-like state. The interconversion between ESC and PGC-like cells helps us to understand the metastability of PGCs. The regulatory mechanisms of PGC dedifferentiation are discussed in comparison with those involved in the dedifferentiation of testicular stem cells, ESC pluripotency, and somatic nuclear reprogramming.     

干细胞在妇产科若干疾病中研究及应用

干细胞是一类具有自我更新和增殖分化能力的细胞,按其发育阶段可分为胚胎干细胞(embryonic stem cell,ESC)和成体干细胞(adult stem cell,ASC)。由于干细胞这种特殊的增殖分化潜能,使其具备着多学科临床治疗的可塑性,研究意义巨大。随着子宫内膜干细胞的发现以及对其他类型干细胞提取手段的进步,干细胞为治疗子宫内膜相关性疾病带来了全新的思路。此外,宫内干细胞移植治疗胎儿疾病,干细胞介导损伤后血管内皮的修复以及改善生育功能、治疗不孕症等几个领域的研究也取得了显著的成果。本文参考近7年国内外文献,以干细胞治疗妇产科几种常见疾病的最新研究进展为主要内容进行综述。     

The prospect of pluripotent stem cell-based therapy

Human embryonic stem cells (hESC) are able to maintain pluripotency in culture, to proliferate indefinitely and to differentiate into all somatic cell types. Due to these unique properties, hESC may become an exceptional source of tissues for transplantation and have a great potential for the therapy of incurable diseases. Here, we review new developments in the area of embryonic stem cells and discuss major challenges — standardization of protocols for cell derivation and cultivation, identification of specific molecular markers, development of new approaches for directed differentiation, etc. — which remain to be settled, prior to safe and successful clinical application of stem cells. We appraise several potential approaches in hESC-based therapy including derivation of autologous cells via therapeutic cloning (1), generation of immune tolerance to allogenic donor cells via hematopoetic chimerism (2), and development of the banks of hESC lines compatible with the main antigens and exhibiting equivalent pluripotency (3). In addition, we discuss briefly induced pluripotent cells, which are derived via genetic modification of autologous somatic cells and are analogous to ESC. Our analysis demonstrates that uncontrollable differentiation in vivo and teratogenic potential of hESC are critical limitations of their application in clinical practice. Therefore, the major approach in hESC therapy is derivation of a specific differentiated progeny, which has lower proliferative potential and immune privilege, yet poses fewer risks for organism. The review demonstrates that cell therapy is far more complex and resource-consuming process as compared with drug-based medicine and consequently pluripotent stem cell biology and technology still requires further investigation and development before these cells can be used in clinical practice.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.