胚胎干细胞衍生的视网膜色素上皮细胞移植治疗眼病

Schwartz等报告的用从人胚胎干细胞分化成的视网膜色素上皮细胞（RPE）移植治疗视网膜病,已观察4月,尚属成功。这是首次用从人胚胎干细胞（hESC）定向分化而成的细胞移植至患者取得成功。本文复习RPE移植的历史与现况;hESC分化而成的RPE（hESC-RPE）的实验研究以及临床移植的意义、方法、效果及存在问题,并展望了应用干细胞分化的RPE移植的前景。     

小分子化合物诱导干细胞向视网膜感光细胞分化的研究进展

干细胞是一类具有多向分化潜能的细胞群,如胚胎干细胞(embryonic stem cell,ESC)、诱导多潜能干细胞(induced pluripotent stem cell,i PSC)等,可在特定的条件下向包括视网膜感光细胞在内的多种细胞分化。小分子化合物是一类由组织细胞合成、分泌的小分子多肽类因子,特定的小分子化合物可作用于干细胞诱导其向视网膜感光细胞分化。目前,对干细胞体外培养,通过使用不同的诱导培养方案,探索干细胞向视网膜感光细胞分化的研究成为热点。早期,研究者们主要在共培养条件下采用小分子化合物诱导ESC向视网膜感光细胞分化,随着研究的进展,逐渐开始探索在无共培养条件下小分子化合物诱导ESC向视网膜感光细胞的分化以及小分子化合物诱导i PSC向视网膜感光细胞的分化。本文主要就小分子化合物促进ESC和i PSC向视网膜感光细胞分化的研究进展进行综述。     

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。     

绿色荧光蛋白基因修饰的人视网膜色素上皮细胞异种移植的长期观察

探讨利用视网膜色素上皮 (retinalpigmentepithelium ,RPE)细胞移植介导目的基因转移至视网膜的可行性。人的RPE细胞在体外经携带绿色荧光蛋白 (greenfluorescentprotein ,GFP)基因的逆转录病毒感染及G4 1 8筛选后 ,手术显微镜直视下经睫状体平坦部注射到兔眼视网膜下间隙。术后通过活体荧光眼底照相 ,荧光显微镜、共聚焦显微镜及透射电镜等观察眼球铺片及切片 ,发现经 gfp基因修饰的人RPE细胞在兔眼视网膜下间隙可存活一年以上。移植的RPE gfp细胞不仅仅局限于移植部位 ,大多扩散到超过 2～ 3个象限的眼底 ,镶嵌于宿主RPE细胞之间或呈单层排列在宿主色素上皮与神经视网膜之间 ,并持续高水平表达GFP。移植后早期玻璃体内每周注射免疫抑制剂普乐可复 (FK5 0 6 )可明显改善移植细胞的存活状态     

猪诱导性多能干细胞技术的研究进展及应用前景

猪作为实验材料,具有由于来源方便、基因序列与人类的相近及其在畜牧业中的重要地位等优势,成为国内外研究的热点,但是猪的胚胎干细胞(Embryonic stem cells,ESC)建系方面的研究进展缓慢。诱导性多能干细胞(induced pluripotent stem cells,iPSC)技术的诞生,开创了体细胞重编程的全新方法。猪iPSC体系的建立将为家畜ESC体系的建立奠定基础,同时也对提高猪转基因克隆的效率,高效育种和保种,乃至生物医学领域均产生深远的影响。文章综述了iPSC技术的主要进展,重点阐述了猪iPSC技术的现状及其在生物医学和畜牧业中的应用前景,以期为从事该领域研究的科研人员提供参考。     

视网膜干细胞的研究进展

干细胞研究是近年生命科学研究的热点之一,近来在干细胞研究领域的进步已经给治疗甚至是治愈某些变性疾病和不能治疗的人类疾病带来了希望.在体外,睫状缘色素上皮细胞来源的视网膜干细胞能增殖形成克隆球,并能分化成视网膜各种细胞类型,包括光感受器细胞,双极细胞及神经胶质细胞等等.视网膜干细胞的移植可以作为治疗一些致盲性眼病潜在治疗方式.因此,视网膜干细胞的研究对于视网膜变性疾病,如遗传性视网膜变性、青光眼、年龄相关性黄斑变性等,提供了新的治疗途径.现就视网膜干细胞的培养与鉴定,增殖、分化、移植以及信号机制方面的研究进展作一综述.     

哺乳动物体细胞核移植研究进展

体细胞核移植技术已经在基础研究领域与产业化应用领域体现出了重要的价值,因而体细胞核移植技术及其相关研究已经成为了生物领域的持续性研究热点,但是围绕体细胞核移植技术仍然存在许多质疑,其中最主要的就是体细胞核移植的效率较低。尽管如此,体细胞核移植研究仍然在近年来取得了令人瞩目的成就,包括小鼠与恒河猴核移植胚胎干细胞系的建立。该文就体细胞核移植的研究历史与进展进行简要的论述,同时针对体细胞核移植研究中的细胞重编程与治疗性克隆研究中的发展与问题进行剖析,希望能够积极推动治疗性克隆的研究进展,加速核移植与干细胞技术在产业化领域中的应用。     

多能成体祖细胞培养条件促进人脂肪干细胞的视网膜神经保护功能

目的：检测多能成体祖细胞（MAPC）的培养条件对猴骨髓间充质细胞（BMMSCs）和人脂肪干细胞（hADSCs）生长的影响,旨在获得更适合治疗视网膜变性疾病的供体细胞。方法通过细胞形态观察、MTT实验、克隆形成率、PCR检测、以及成脂、成骨、成软骨分化潜能检测等,研究MAPC培养条件下猴BMMSCs和hADSCs的特征,并用DMEM/LG和MAPC培养条件培养的hADSCs进行RCS大鼠视网膜下腔移植,通过视网膜电图（ERG）和TUNEL检测,判断细胞移植治疗对视功能及视网膜细胞凋亡的影响。结果与常规培养基相比,MAPC培养条件能促进猴BMMSCs增殖,细胞变小,但传2代后,细胞变得宽大扁平,出现衰老征象;然而,MAPC培养条件下的hADSCs细胞增殖能力及克隆形成率均增强,形成的克隆较大可稳定传10代以上,且具有成脂、成骨、成软骨的多向分化潜能,细胞表面标记物及细胞因子出现差异表达：CD140b、CD90、CD47、HGF和PEDF显著上调,CD73、CD105和IL-6显著下调。与对照组相比,移植DMEM/LG和MAPC培养条件培养的hADSCs（P4）3周后,RCS大鼠的B波波幅明显升高,外核层细胞凋亡明显减少。结论 MAPC培养条件培养的hADSCs显示出更好的视网膜神经保护作用,适合用于治疗视网膜退行性疾病。     

诱导性多潜能干细胞技术进展

诱导性多潜能干细胞(induced pluripotent stem cells,iPSCs)可以通过在分化的成纤维细胞中导入特定的转录因子获得。IPS细胞与胚胎干细胞(embryonic stem cell,ESCs)在形态,增殖能力,基因表达谱和畸胎瘤形成上没有区别,因此在研究疾病机制,药物筛选和毒理学上有重要的应用价值。一旦解决了安全性和效率问题,iPS细胞将在再生医学上有重要的应用价值。主要从提高转化效率、制备无遗传修饰的iPS细胞和疾病特异性的iPS细胞这3个近来在iPS领域飞速进展的方向做一综述。     

Pluripotent stem cell-derived neural stem cells: From basic research to applications

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.     

Differentiation of primate ES cells into retinal cells induced by ES cell-derived pigmented cells

Purpose: Photoreceptors cannot regenerate and recover their functions once disordered. Transplantation of retinal pigment epithelium (RPE) has recently become a possible therapeutic approach for retinal degeneration. In the present study, we investigated the induction of photoreceptors by coculturing primate embryonic stem cells (ESCs) with ESC-derived RPE cells. Methods: RPE cells were derived by coculturing ESCs and Sertoli cells. Photoreceptors were then induced by using ESC-derived RPE cells and retinoic acid (RA) Results: RPE cell generation was confirmed by morphological analysis, which revealed highly pigmented polygonal cells with a compact cell-cell arrangement. After coculturing ESCs and RPE cells, some ESC derivatives became immunopositive for rhodopsin. RT-PCR analysis demonstrated the expression of retina-related gene markers such as Pax6, CRX, IRBP, rhodopsin, rhodopsin kinase, and Muschx10A. When RA was added, a distinct increase in the expression of photoreceptor-specific proteins and genes was found. In addition, the differentiation of bipolar horizontal cells was demonstrated by protein and gene expression. The ESCs that were cocultured with RPE cells and treated with RA were transplanted into the renal capsule or intra-vitreal space of nude mice. Grafted ESC derivatives demonstrated extensive rhodopsin expression, and they survived and organized into recipient tissues, although they formed teratomas. Conclusion: These results indicate that coculturing ESCs with ESC-derived RPE cells is a useful and efficient method for inducing photoreceptors and providing an insight into the use of ESCs for retina regeneration.     

Microfluidic systems: A new toolbox for pluripotent stem cells

Conventional culture systems are often limited in their ability to regulate the growth and differentiation of pluripotent stem cells. Microfluidic systems can overcome some of these limitations by providing defined growth conditions with user-controlled spatiotemporal cues. Microfluidic systems allow researchers to modulate pluripotent stem cell renewal and differentiation through biochemical and mechanical stimulation, as well as through microscale patterning and organization of cells and extracellular materials. Essentially, microfluidic tools are reducing the gap between in vitro cell culture environments and the complex and dynamic features of the in vivo stem cell niche. These microfluidic culture systems can also be integrated with microanalytical tools to assess the health and molecular status of pluripotent stem cells. The ability to control biochemical and mechanical input to cells, as well as rapidly and efficiently analyze the biological output from cells, will further our understanding of stem cells and help translate them into clinical use. This review provides a comprehensive insignt into the implications of microfluidics on pluripotent stem cell research.     

Advances in genetic modification of pluripotent stem cells

Genetically engineered stem cells aid in dissecting basic cell function and are valuable tools for drug discovery, in vivo cell tracking, and gene therapy. Gene transfer into pluripotent stem cells has been a challenge due to their intrinsic feature of growing in clusters and hence not amenable to common gene delivery methods. Several advances have been made in the rapid assembly of DNA elements, optimization of culture conditions, and DNA delivery methods. This has lead to the development of viral and non-viral methods for transient or stable modification of cells, albeit with varying efficiencies. Most methods require selection and clonal expansion that demand prolonged culture and are not suited for cells with limited proliferative potential.     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

The new paradigm: retinal pigment epithelium cells generated from embryonic or induced pluripotent stem cells

Compared with neural crest‐derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light‐absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11‐cis‐retinal by re‐isomerizing all‐trans‐retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors’ outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age‐related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE‐like cells can be obtained relatively easily when stem cells are subjected to a two‐step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.     

多能干细胞向雄性生殖细胞定向分化的研究进展

生殖健康是生命科学领域关注的核心之一,各种原因所致男性不育亟待解决,然而由于伦理限制等原因,缺少合适的具有人类遗传背景的研究模型开展研究。胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）均属于多能干细胞,具有多向分化潜能。一方面,可利用ESCs?/?iPSCs向生殖细胞分化的模型研究人类生殖细胞的发育规律,另一方面,在此基础上,可建立带有人类疾病遗传背景的iPSCs模型,体外诱导其向雄性生殖细胞分化,利用该模型研究男性不育的发病机制。由于精子在体内的形成遵循一定规律,体外环境中不同发育阶段的生殖细胞在不同诱导因子作用下才可稳定地往下一阶段定向分化,因此,诱导ESCs?/?iPSCs向雄性生殖细胞方向分化时,诱导因子的种类和加入时间的选择应根据生殖细胞的体内发育特征而定,并且在诱导的不同阶段循序加入,以此模拟精子在体内的形成过程,进而更好地研究男性不育的发病机制。本文将对多能干细胞向雄性生殖细胞定向分化的常用诱导因子及存在问题和展望进行综述,为相关研究的开展提供借鉴。