多能干细胞分化来源视网膜色素上皮细胞移植治疗视网膜变性研究进展

视网膜色素上皮（RPE）对视觉功能的维持起着至关重要的作用。视网膜变性是全球不可治愈性致盲疾病的重要原因,它由视网膜色素上皮功能失常所引起。因此,视网膜色素上皮移植是视网膜变性患者恢复视力的一种最有前景的手段之一。随着干细胞技术的快速发展,从多能干细胞（PSC）到有功能的视网膜色素上皮细胞的体外分化诱导技术已经成熟,其中包括胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）等。此外,从患者特异性iPSCs分化而来的RPE更能用于阐明发病机理并有针对性地个体治疗。更值得一提的是,经诱导得到RPE的移植不论在动物模型中,还是在临床试验里都已经得到了可喜的治疗效果。本文回顾PSC来源RPE干预治疗视网膜变性的最新研究进展。     

胚胎干细胞治疗心肌梗死的研究进展

胚胎干细胞 (ES细胞 )是一种多能细胞 ,来源于囊胚期胚胎 ,具有很强的自我更新能力 ,并能分化成很多细胞类型。体外 ,ES细胞能自发聚集形成胚胎体 (EB) ,分化成许多种细胞类型 ;ES细胞注射到免疫缺陷的小鼠体内 ,产生畸胎瘤 ,其中包含有三个胚层的细胞。添加生长因子或与其它细胞共培养等方法可以促进ES细胞体外分化为心肌细胞 ,筛选后移植到梗死的心肌 ,可以提高心脏功能 ,是治疗心肌梗死的一种很有潜力的方法     

人羊膜上皮细胞具有胚胎干细胞和移植免疫耐受等优良特性

干细胞具有自身复制和多向分化的潜能,在体内能够维持组织器官形态和生理功能的稳态,并具有修复外伤和病理引起的损伤的作用。故而,近年来在细胞生物学以及再生医学研究领域倍受重视。可以预见,干细胞在细胞治疗、组织工程、器官修复等方面的临床应用,将有广泛的前景和市场潜力;同时在遗传、发育、分化、调亡等生物学问题研究以及新药开发、药效学和毒性评估等方面,也有着广泛的科学探索和实际应用价值。干细胞研究必将成为生命科学研究的主战场和生物经济最为活跃的     

胚胎干细胞移植治疗帕金森病

胚胎干细胞起源于植入前胚胎的内细胞团(inner cell mass,ICM),能够在体外进行增殖并能分化成三个胚层的所有细胞类型.如果能有效地将胚胎干细胞(embryonic stem cell,ES细胞)诱导分化为特定的细胞类型,ES细胞将成为细胞替代治疗中供体细胞的一个理想来源,从而可以应用于帕金森病(Parkinson's disease,PD)、糖尿病、心脓死等的退行性病变疾病的治疗.近年来,将ES细胞来源的多巴胺能神经元用于PD治疗的研究越来越广泛.现对这些研究中有关基因表达调控、实验研究进展、临床应用以及所遇到的问题作一综述.虽然这些研究还没能最终大面积应用于临床.但为PD的治疗提供了一个广阔的应用前景.     

人羊膜上皮细胞和胚胎干细胞

人胚胎干细胞有着巨大的医学应用前景,但人胚胎干细胞要求的生长条件很高,体外很难模拟其生长的体内环境,因此控制人胚胎干细胞的生长常不理想,而使用鼠胚胎成纤维细胞(MEF)作为滋养层则存在动物源性污染的问题。该文阐述人羊膜上皮细胞(HAEC)的特点及其作为滋养层培养胚胎干细胞的现状,并探讨基因组DNA甲基化修饰在胚胎干细胞分化过程中的作用,为建立更优化的培养系统提供依据。     

胚胎干细胞移植治疗糖尿病的研究进展

糖尿病是一种严重的免疫缺陷性疾病,目前的治疗方法很难从根本上治愈。近年来的研究表明,通过诱导胚胎干细胞定向分化为胰岛β细胞,并进行移植治疗糖尿病,是一种有希望的根治方案。本文就利用胚胎干细胞移植治疗糖尿病的最新进展作一综述。     

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     

干细胞核移植效率及核移植胚胎干细胞

干细胞为一类具有无限的或者永生的自我更新能力的细胞,包括胚胎性干细胞和成体干细胞.胚胎性干细胞有胚胎干细胞、畸胎瘤细胞和原始生殖细胞.成体干细胞主要有骨髓间充质干细胞,造血干细胞、神经干细胞、表皮干细胞、脂肪干细胞等.随着体细胞核移植技术与干细胞培养技术的成熟,两者相结合便产生了核移植来源胚胎干细胞(embryonic stem cells via nuclear transfer,ntES细胞),其不仅用于基础的研究,而且也用于临床医学的组织修复和移植的研究.现就干细胞作为核供体时的核移植效率,ntES细胞系的建立、其性质及诱导分化等的研究进展进行综述.     

体细胞起源的人胚胎干细胞

根据治疗性克隆假设,可以通过体细胞核移植技术获得与病人具同样基因型的细胞或组织。这样起源的细胞或组织植回病人将不会引起免疫排斥反应。本研究将5岁、42岁、52岁和60岁4个不同年龄的人体细胞核植入去核的兔卵母细胞中重新启动,发育至囊胚,并分离人胚胎干细胞。研究结果提示,年龄不影响体细胞被重新启动的效率。经过核型分析,同源染色体分析,原位杂交,PCR和免疫组化染色等多种鉴定,ntES细胞具有人染色体。ntES细胞可以长期增殖并保持不分化状态,也可以形成类胚体并分化出包括神经和肌肉在内的多种细胞类型。由类胚体诱导生成的混合细胞群体表达所有三个胚层(外、中、内胚层)细胞类型标记,说明ntES细胞具有分化成所有三个胚层的潜力。因此,从人体细胞核获得的ntES细胞与普通人胚胎干细胞一样具有向多种细胞类型分化的能力。     

人胚胎干细胞向神经上皮祖细胞的诱导分化

人胚胎干细胞具有自我更新和多向分化潜能,是研究早期胚胎发育和细胞替代治疗的重要细胞来源.采用一种与小鼠成纤维细胞共培养的方法进行人胚胎干细胞的神经诱导,可产生高纯度的神经上皮祖细胞,其神经上皮特异性基因的表达有一定的时空性;诱导生成的神经上皮祖细胞具有增殖潜能并可分化为神经元和星型胶质细胞,是潜在的神经干细胞.人胚胎干细胞来源的神经上皮祖细胞为研究神经发育和神经诱导提供了新材料,也为神经系统疾病的细胞替代治疗提供了新的细胞来源.     

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.     

Morphogenesis and nature of the pigment granules in the adult human retinal pigment epithelium

Summary The pigment epithelial cells of the retina are a layer of highly specialized melanocytes. Beginning in the early embryonic period they produce melanin throughout the entire life. The Golgi apparatus plays a key role in the biosynthesis of melanin. The following steps can be distinguished morphologically: (a) Golgi-vesicles, (b) intermediate vesicles, (c) melanosomes, (d) melanin granules. Structures with a ringlike appearance that are described as lipofuscin granules in the literature prove to be altered intermediate vesicles and melanosomes.This investigation was carried out in part at the Francis I. Proctor Foundation for Research in Ophthalmology, San Francisco, California, U.S.A., and supported by United States Public Health Service Program Project Grant EY 00310, and Deutsche Forschungsgemeinschaft, Training Grant Nr. Sp 102/1.     

The effect of clinical-grade retinal pigment epithelium derived from human embryonic stem cells using different transplantation strategies

Dear Editor, Many forms of sight-threatening diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD), are caused by the dysfunction, degeneration and loss of the retinal pigment epithelium (RPE)(Strauss, 2005). RPE cell transplantation may potentially recover or halt disease progression, in which human embryonic stem cells (hESCs) could serve as an unlimited donor source for RPE differentiation, and a few clinical trials have shown the safety and effective of transplantation of hESCs-derived RPE (hESC-RPE) for AMD patients (Schwartz et al., 2012;Schwartz etal., 2015;Song etal., 2015;da Cruz et al., 2018;Kashani et al., 2018;Liu et al., 2018).     

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.     

Endothelial reconstitution by CD34+ progenitors derived from baboon embryonic stem cells

In this study, we used a large non‐human primate model, the baboon, to establish a step‐wise protocol to generate CD34+ endothelial progenitor cells (EPCs) from embryonic stem cells (ESCs) and to demonstrate their reparative effects. Baboon ESCs were sequentially differentiated from embryoid body cultures for 9 days and then were specified into EPCs by culturing them in monolayer for 12 days. The resulting EPCs expressed CD34, CXCR4 and UEA‐1, but neither CD31 nor CD117. The EPCs were able to form intact lumen structures when seeded on Matrigel, took up Dil‐LDL, and responded to TNF‐α. Angioblasts specified in EGM‐2 medium and ECGS medium had 6.41 ± 1.16% (n = 3) and 9.32 ± 3.73% CD34+ cells (n = 3). The efficiency of generating CD34+ EPCs did not differ significantly from ECGS to EGM‐2 culture media, however, angioblasts specified in ECGS medium expressed a higher percentage of CD34+/CXCR4+ cells (3.49 ± 1.32%, n = 3) than those specified in EGM‐2 medium (0.49 ± 0.52%, n = 3). To observe their reparative capacity, we purified CD34+ progenitors after specification by EGM‐2 medium; inoculated fluorescently labelled CD34+ EPCs into an arterial segment denuded of endothelium in an ex vivo system. After 14 days of ex vivo culture, the grafted cells had attached and integrated to the denuded surface; in addition, they had matured further and expressed terminally differentiated endothelial markers including CD31 and CD146. In conclusion, we have proved that specified CD34+ EPCs are promising therapeutic agents for repairing damaged vasculature.     

A novel in vitro retinal differentiation model by co-culturing adult human bone marrow stem cells with retinal pigmented epithelium cells

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.     

Connexin mutant embryonic stem cells and human diseases

Intercellular communication via gap junctions allows cells within multicellular organisms to share small molecules. The effect of such interactions has been elucidated using mouse gene knockout strategies. Although several mutations in human gap junction-encoding connexin (Cx) have been described, Cx mutants in mice do not always recapitulate the human disease. Among the 20 mouse Cxs, Cx26, Cx43, and Cx45 play roles in early cardiac or placental development, and disruption of the genes results in lethality that hampers further analyses. Embryonic stem cells (ESCs) that lack Cx43 or Cx45 have made analysis feasible in both in vitro differentiated cell cultures and in vivo chimeric tissues. The success of mouse ESCs studies is leading to the use of induced pluripotent stem cells to learn more about the pathogenesis of human Cx diseases. This review summarizes the current status of mouse Cx disruption models and ESC differentiation studies, and discusses their implication for understanding human Cx diseases.     

Transplantation of human embryonic stem cell‐derived endothelial cells for vascular diseases

Using endothelial cells for therapeutic angiogenesis/vasculogenesis of ischemia diseases has led to exploring human embryonic stem cells (hESCs) as a potentially unlimited source for endothelial progenitor cells. With their capacity for self‐renewal and pluripotency, hESCs and their derived endothelial cells (hESC‐ECs) may be more advantageous than other endothelial cells obtained from diseased populations. However, hESC‐ECs' poor differentiation efficiency and poorly characterized in vivo function after transplantation present significant challenges for their future clinical application. This review will focus on the differentiation pathways of hESCs and their therapeutic potential for vascular diseases, as well as the monitoring of transplanted cells' fate via molecular imaging. Finally, cell enhancement strategies to improve the engraftment efficiency of hESC‐ECs will be discussed. J. Cell. Biochem. 106: 194–199, 2009. © 2008 Wiley‐Liss, Inc.