极小胚胎样干细胞研究现状

视网膜退行性疾病包括视网膜色素变性、增殖性糖尿病性视网膜病变、增殖性玻璃体视网膜病变和年龄相关性黄斑变性等,是一类由于视网膜感光细胞变性丢失而引起的不可逆性致盲性疾病.干细胞移植技术为此类疾病带来了一线希望,干细胞整合入视网膜,分化成为特定的感光细胞,可以重建视网膜功能.最近,科学家在骨髓和一些成人组织中发现了一种表达CXCR4受体的干细胞,称为极小胚胎样干细胞（VSELSCs）,这种细胞表达多种胚胎细胞及原生殖细胞的标志物,有实验证实其具有分化为三个胚层细胞的潜能.VSELSCs这种可以定向分化的特性预示了它在治疗退行性疾病中的广阔前景.本研究主要着眼于VSELSCs的研究现状,探讨其在视网膜退行性疾病中的应用前景.     

干细胞移植治疗视网膜变性疾病的研究进展

视网膜变性疾病是一类与遗传相关的变性疾病,导致患者渐进性视觉丢失,是主要的致盲性眼病之一,其共同病理基础是视网膜光感受器细胞的变性、坏死和凋亡,然而目前尚无有效的治疗方法。细胞移植治疗是目前实验研究治疗视网膜变性疾病的主要方法之一。近年研究表明将感光前体细胞或视网膜色素上皮细胞移植到视网膜下腔或玻璃体内,可以延缓宿主感光细胞的凋亡、替代凋亡的感光细胞、挽救残存的视觉功能和修复视网膜结构。细胞移植治疗的一个很重要的问题是移植细胞的来源问题,目前主要的移植细胞来源是视网膜祖／干细胞、胚胎干细胞和诱导多潜能干细胞等。本文就干细胞移植治疗视网膜变性疾病的研究进展进行综述。     

眼源性干细胞移植治疗视网膜疾病的研究进展

许多视网膜疾病,如视网膜色素变性、年龄相关性黄斑变性、视网膜脱离等都会引起视网膜光感受器的损伤,从而导致不可逆的视力丧失。目前对于此类疾病,临床上尚无有效的治疗方法。近年来随着对细胞工程研究的不断深入,干细胞移植被认为是治疗视网膜疾病最有前景的方法之一,将给不可逆性致盲眼病患者带来新的希望。本文就眼源性干细胞移植治疗视网膜疾病的最新研究进展综述如下。     

干细胞移植治疗视网膜神经节细胞损伤性疾病的研究进展

视网膜神经节细胞是视觉形成的重要参与者。视网膜神经节细胞损伤或死亡往往会导致视功能不可逆转的损害。青光眼、糖尿病视网膜病变、高血压、视网膜变性等致盲性疾病,均会引起视网膜神经节细胞损伤或进行性大量凋亡。目前此类疾病在临床上尚无明确的治疗方法。为了恢复患者视网膜神经节细胞功能,国内外学者将研究焦点集中在干细胞移植上。干细胞移植主要指两大类,一类是基于干细胞的替代治疗,另一类则是通过干细胞移植促进某些因子分泌来保护视网膜神经节细胞。我们旨在对干细胞移植治疗视网膜神经节细胞损伤疾病的潜力进行综述,并着重讨论不同种干细胞分化为视网膜神经节细胞的研究进展。     

干细胞治疗视网膜疾病的研究进展

目的 视网膜是视觉中光电感应的部位，其功能与解剖结构关系密切。外界光刺激、机械损伤或其他疾病的并发症均可导致光感受器的病理变化，进一步引起视网膜色素变性、视网膜脱离及其他致盲性疾病。对于这类疾病，国内外学者进行了大量的研究，随着干细胞研究的推广，越来越多的研究人员认识到干细胞作为细胞供体进行视网膜疾病治疗的潜能。本文对其中的一些工作进行简要综述。     

间充质干细胞在视网膜疾病中的临床应用展望

间充质干细胞(MSC)用于治疗视网膜疾病有两种移植方法:全身静脉注射和局部移植,后者具有更好的针对性。根据疾病不同,需要采用不同的MSC治疗策略:(1)MSC定向分化替代受损细胞,如光感受器细胞和视网膜色素上皮细胞;(2)利用其免疫抑制作用治疗自身免疫性葡萄膜视网膜炎;(3)利用其神经保护作用减轻或延缓视网膜组织损伤,如目前的视网膜色素变性、视网膜缺血再灌注损伤和青光眼动物模型;(4)转基因后的MSC移植靶向治疗,如脉络膜新生血管的治疗;(5)利用MSC抗炎和促进创伤愈合作用治疗多种视网膜疾病。迄今为止,其抑制葡萄膜视网膜炎进展以及视神经营养保护作用较为明确。但MSC在各种视网膜疾病中的疗效还需要严格评估,其发挥作用的机制尚未完全明了,不同研究结果差异较大。此外,MSC的移植途径和方法,治疗的远期效果及安全性也有待进一步评估。     

视网膜修复中干细胞的应用

视网膜疾病由于其不可逆转的致盲性而影响人类生存质量.随着对干细胞研究的深入,基于干细胞移植的细胞替代疗法为视网膜疾病的治疗开辟了新的途径.近年来眼组织来源的视网膜干细胞或祖细胞、非眼组织来源的间充质干细胞、造血干细胞、神经干细胞、胚胎干细胞以及诱导多能干细胞在视网膜损伤疾病中的应用取得了很多突破性进展,它们不仅可以被诱导分化为各种视网膜神经元细胞、胶质细胞,而且移植到体内可以整合到损伤视网膜,甚至可以发挥正常视网膜神经元细胞功能.     

诱导多能干细胞在眼科的研究进展

以视网膜神经元不可逆性损伤为病理基础的视网膜变性类疾病是目前主要的致盲性眼病之一,针对其神经损伤修复的药物治疗却十分有限.最新研究发现的诱导多能干细胞(iPSCs)是通过基因转染技术将转录因子导入动物或人的体细胞内,使体细胞直接重构成为胚胎干细胞样的多潜能细胞.由患者体细胞诱导的iPSCs不仅具有强大的自我更新和分化潜能,而且可避免以往胚胎干细胞存在的宿主排斥反应和伦理学限制等缺点,同时眼部屈光介质清晰的特点还使得自体干细胞移植治疗视网膜变性类疾病具有易于观察、便于操作的优势,相信会为这一难治性疾病的治疗开辟新的途径.对iPSCs的研究发展历程、iPSCs的特性和优势、iPSCs在眼科的诱导和定向分化能力进行综述.     

胚胎干细胞诱导分化视网膜细胞的方法新进展

视网膜变性疾病是引起视力丧失的重要原因,由于这类眼病的病因不明确、发病机制复杂,目前尚无有效的治疗方法.近年来,干细胞研究领域取得了突破性进展,干细胞具有分化为机体所有细胞的潜能,可以利用胚胎干细胞(ESCs)分化出各种视网膜细胞,这为视网膜变性疾病的治疗带来了新的曙光.ESCs治疗视网膜变性疾病至关重要的一步是将ESCs分化为视网膜光感受器细胞和视网膜色素上皮(RPE)样细胞.本文对自发诱导培养法、共培养法、细胞因子诱导法、单层贴壁诱导培养法和3D诱导培养法等ESCs分化为视网膜光感受器细胞和RPE细胞方法的最新进展进行综述.     

视网膜干细胞研究现状

干细胞是当今细胞生物学乃至整个生命科学研究的主要热点之一，视网膜干细胞的研究近年来颇有进展，这对于治疗眼科临床的一些疾病，如遗传性视网膜变性、原发或继发性视网膜变性疾病、老年性黄斑病变等，提供了具有较大发展潜力的新途径。现就视网膜干细胞的发现与鉴定，体外培养、增殖、分化以及移植方面的研究进展作一综述。(中华眼底病杂志,2005,21:58-60)     

The retinal pigment epithelium: Development,injury responses,and regenerative potential in mammalian and non-mammalian systems

Diseases that result in retinal pigment epithelium (RPE) degeneration, such as age-related macular degeneration (AMD), are among the leading causes of blindness worldwide. Atrophic (dry) AMD is the most prevalent form of AMD and there are currently no effective therapies to prevent RPE cell death or restore RPE cells lost from AMD. An intriguing approach to treat AMD and other RPE degenerative diseases is to develop therapies focused on stimulating endogenous RPE regeneration. For this to become feasible, a deeper understanding of the mechanisms underlying RPE development, injury responses and regenerative potential is needed. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but large lesions cannot be repaired as remaining RPE cells are unable to functionally replace lost RPE tissue. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer, while in others, proliferation is pathogenic and results in further disruption to the retina. This is in contrast to non-mammalian vertebrates, which possess tremendous RPE regenerative potential. Here, we discuss what is known about RPE formation during development in mammalian and non-mammalian vertebrates, we detail the processes by which RPE cells respond to injury, and we describe examples of RPE-to-retina and RPE-to-RPE regeneration in non-mammalian vertebrates. Finally, we outline barriers to RPE-dependent regeneration in mammals that could potentially be overcome to stimulate a regenerative response from the RPE.     

Recapitulating developmental mechanisms for retinal regeneration

Degeneration of specific retinal neurons in diseases like glaucoma, age-related macular degeneration, and retinitis pigmentosa is the leading cause of irreversible blindness. Currently, there is no therapy to modify the disease-associated degenerative changes. With the advancement in our knowledge about the mechanisms that regulate the development of the vertebrate retina, the approach to treat blinding diseases through regenerative medicine appears a near possibility. Recapitulation of developmental mechanisms is critical for reproducibly generating cells in either 2D or 3D culture of pluripotent stem cells for retinal repair and disease modeling. It is the key for unlocking the neurogenic potential of Müller glia in the adult retina for therapeutic regeneration. Here, we examine the current status and potential of the regenerative medicine approach for the retina in the backdrop of developmental mechanisms.     

Prospects for the application of Müller glia and their derivatives in retinal regenerative therapies

Neural cell death is the main feature of all retinal degenerative disorders that lead to blindness. Despite therapeutic advances, progression of retinal disease cannot always be prevented, and once neuronal cell damage occurs, visual loss cannot be reversed. Recent research in the stem cell field, and the identification of Müller glia with stem cell characteristics in the human eye, have provided hope for the use of these cells in retinal therapies to restore vision. Müller glial cells, which are the major structural cells of the retina, play a very important role in retinal homeostasis during health and disease. They are responsible for the spontaneous retinal regeneration observed in zebrafish and lower vertebrates during early postnatal life, and despite the presence of Müller glia with stem cell characteristics in the adult mammalian retina, there is no evidence that they promote regeneration in humans. Like many other stem cells and neurons derived from pluripotent stem cells, Müller glia with stem cell potential do not differentiate into retinal neurons or integrate into the retina when transplanted into the vitreous of experimental animals with retinal degeneration. However, despite their lack of integration, grafted Müller glia have been shown to induce partial restoration of visual function in spontaneous or induced experimental models of photoreceptor or retinal ganglion cell damage. This improvement in visual function observed after Müller cell transplantation has been ascribed to the release of neuroprotective factors that promote the repair and survival of damaged neurons. Due to the development and availability of pluripotent stem cell lines for therapeutic uses, derivation of Müller cells from retinal organoids formed by iPSC and ESC has provided more realistic prospects for the application of these cells to retinal therapies. Several opportunities for research in the regenerative field have also been unlocked in recent years due to a better understanding of the genomic and proteomic profiles of the developing and regenerating retina in zebrafish, providing the basis for further studies of the human retina. In addition, the increased interest on the nature and function of cellular organelle release and the characterization of molecular components of exosomes released by Müller glia, may help us to design new approaches that could be applied to the development of more effective treatments for retinal degenerative diseases.     

诱导多功能干细胞在视网膜再生医学中的应用

诱导多功能干细胞（induced pluripotent stem cell,iPSC）是由4个转录因子与成体细胞基因组进行重组诱导出的一种全能分化型干细胞.多种疾病会导致视网膜的结构或功能受损,可利用iPSC的增生分化能力使视网膜再生.在视网膜再生医学领域,iPSC的研究热点集中在：供体细胞、转录因子及转录载体的选择、诱导分化机制的研究、iPSC向视网膜不同类型细胞分化的诱导途径以及诱导出的视网膜色素上皮细胞和光感受器细胞的移植方法.但在目前关于iPSC的研究中仍存在定向诱导分化效率较低、眼内移植后存活率低、临床应用安全性难保障等一系列问题.唯有解决上述问题,iPSC研究才能在视网膜再生医学领域发挥更大的作用.     

Clonogenic analysis of ciliary epithelial derived retinal progenitor cells in rabbits

In lower vertebrates, multipotential retinal stem cells reside in a far peripheral retinal zone known as the ciliary marginal zone while more fate-restricted progenitor cells are located immediately adjacent to this retinal margin. To determine whether mammalian ciliary epithelium contains heterogenous stem cell and progenitor cell populations similar to lower vertebrates, we investigated the heterogeneity of the retinal progenitor (or stem) cells isolated from adult rabbit eyes, which are large and suited for precise identification of the region. Under clonogenic culture conditions for a neurosphere assay, spheres were generated from approximately 0.1% of the ciliary epithelial cells and expressed retinal selective markers when plated under conditions producing differentiation. Subsequently, we found that 13.7% of the primary spheres gave rise to a secondary sphere and that 77% of the primary sphere colonies were unipotential progenitors that generated either neurons or glial cells exclusively, while 23% were at least bipotential that could give rise to both cell types using double-labelled immunocytochemistry. Taken together, our results demonstrate that the ciliary epithelium of adult rabbits contains both (at least) bipotential retinal progenitor (or stem) cells and unipotential fate-restricted progenitor cells, similar to lower vertebrates.     

大鼠视神经损伤后移植人胚胎神经干细胞的实验研究

目的 探讨异种神经干细胞注射到玻璃体腔后迁移到视网膜的存活及分化，为进一步研究重建视觉传导通路奠定基础。方法在近SD大鼠眼球后壁夹住视神经根部5min，造成视神经损伤，将起源于人胚胎大脑海马的神经干细胞悬浮液注射到SD大鼠的玻璃体腔中。移植的神经干细胞预先用荧光染料Hoechst标记。分别于手术后不同的时间处死，剥离出视网膜，进行免疫组织化学染色，在荧光显微镜下观察实验结果。结果宿主视网膜在荧光显微镜下可见Hoechst标记阳性的移植细胞，移植前移植细胞神经胶质原纤维酸蛋白（GFAP）阳性表达的，移植后为阴性表达。结论视神经损伤大鼠的玻璃体腔内注射起源于人胚胎大脑海马的神经干细胞，移植细胞可以迁移到视网膜组织中存活。     

从胚胎干细胞诱导视网膜细胞的分子生物学

近年来诱导人多能干细胞的研究激发了人们对再生医学和组织工程的极大热情,干细胞在眼科疾病治疗中的可能应用也得到了更多的重视。在这里我们着重讨论视网膜发育中的细胞分子生物学过程,以及从胚胎干细胞诱导分化的一些考虑,以期对进一步的研究工作及实现其临床应用有所帮助。     

Distribution of Müller stem cells within the neural retina: Evidence for the existence of a ciliary margin-like zone in the adult human eye

Much interest has been generated by the identification of neural stem cells in the human neural retina and ciliary body. However, it is not clear whether stem cells identified in these ocular compartments are of the same origin or whether they ontogenically derive from different cell populations. This study examined the in situ anatomical distribution of these cells within the neural retina and ciliary body, as well as their ability to proliferate in response to EGF. Human retinae and ciliary body were examined for co-expression of Nestin, cellular retinaldehyde binding (CRALBP) or Vimentin, and the stem cell markers SOX2, CHX10, NOTCH1 and SHH. Retinal explants were cultured with epidermal growth factor (EGF) to assess retinal cell proliferation. Intense Nestin and CRALBP staining was observed in the neural retinal margin, where cells formed bundles of spindle cells (resembling glial cells) that lacked lamination and co-stained for SOX2, CHX10 and SHH. This staining differentiated the neural retina from the ciliary epithelium, which expressed SOX2, CHX10 and NOTCH1 but not Nestin or CRALBP. Nestin and CRALBP expression decreased towards the posterior retina, where it anatomically identified a population of Müller glia. All Vimentin positive Müller glia co-stained for SOX2, but only few Vimentin positive cells expressed Nestin and SOX2. Cells of the retinal margin and the inner nuclear layer (INL), where the soma of Müller glia predominate, re-entered the cell cycle upon retinal explant culture with EGF. Lack of lamination and abundance of Müller glia expressing stem cell markers in the marginal region of the adult human retina resemble the ciliary marginal zone (CMZ) of fish and amphibians. The findings that cells in this CM-like zone, as well in the inner nuclear layer proliferate in response to EGF suggest that the adult human retina has regenerative potential. Identification of factors that may promote retinal regeneration in the adult human eye would provide efficient treatments for retinal degenerative conditions for which treatments are not yet available.     

干细胞移植治疗视网膜和视神经变性疾病的现状和进展

不可逆性的视网膜和视神经退行性变会导致视功能严重损伤甚至致盲.近年来,干细胞移植治疗视网膜和视神经损伤的研究进展为组织的修复和再生带来了希望.干细胞既可以用于细胞替代治疗,也可以通过分泌细胞因子发挥神经营养保护作用.胚胎干细胞、间充质干细胞、诱导多能干细胞和Müller细胞等各种来源的干细胞都极具发展前景,为眼科此类难治性疾病提供了丰富的资源.     

Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish

Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells. During adult growth, Müller glial nuclei undergo sporadic, asymmetric, self-renewing mitotic divisions in the inner nuclear layer to generate a rod progenitor that migrates along the radial fiber of the Müller glia into the outer nuclear layer, proliferates, and differentiates exclusively into rod photoreceptors. When retinal neurons are destroyed, Müller glia in the immediate vicinity of the damage partially and transiently dedifferentiate, re-express retinal progenitor and stem cell markers, re-enter the cell cycle, undergo interkinetic nuclear migration (characteristic of neuroepithelial cells), and divide once in an asymmetric, self-renewing division to generate a retinal progenitor. This daughter cell proliferates rapidly to form a compact neurogenic cluster surrounding the Müller glia; these multipotent retinal progenitors then migrate along the radial fiber to the appropriate lamina to replace missing retinal neurons. Some aspects of the injury-response in fish Müller glia resemble gliosis as observed in mammals, and mammalian Müller glia exhibit some neurogenic properties, indicative of a latent ability to regenerate retinal neurons. Understanding the specific properties of fish Müller glia that facilitate their robust capacity to generate retinal neurons will inform and inspire new clinical approaches for treating blindness and visual loss with regenerative medicine.