神经干细胞在视神经修复再生中的应用

神经干细胞可持续增生、分化产生神经元、星型胶质细胞和少突胶质细胞。神经干细胞的这种特性为进行视网膜病变和视神经损伤的治疗打下了基础。本文就神经干细胞的来源、分化调控机制及神经干细胞的移植在视神经修复再生中的应用进行综述。     

视神经损伤激活视网膜干细胞的初步研究

有研究表明,视神经损伤可以引起视网膜干细胞标志物表达增多[1,2].为进一步了解视神经损伤激活视网膜干细胞情况,我们应用液压冲击颅脑损伤仪(FPI)建立视神经损伤动物模型.观察胚胎视网膜十细胞同源转录因子(Chx10)和神经干细胞标志物巢蛋白(nestin)的表达,现将结果报道如下. 1 材料和方法     

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.     

干细胞治疗青光眼的研究进展

青光眼是一类不可逆性视神经变性和视野缺损的疾病,青光眼导致视神经损伤的机制目前尚未明确,临床上对青光眼的治疗一直未取得良好的效果.干细胞具有自我增殖和分化的能力.近年来随着对干细胞研究的深入,人类利用干细胞治疗青光眼成为可能.青光眼干细胞移植分两大类:一是基于小梁网的干细胞替代治疗,另一类是视网膜神经节细胞的替代治疗.本文就干细胞治疗青光眼的研究进展进行综述.     

干细胞对视神经损伤的修复作用

视网膜神经节细胞损伤或死亡往往会导致视功能不可逆性损害。目前尚无有效的治疗方法修复损伤的视神经,恢复受损的视功能。干细胞具有多向分化潜能,其在视神经保护及损伤修复中的作用日渐成为研究热点。本文旨在对干细胞在视神经损伤修复基础和临床研究方面的进展进行综述。     

脊椎动物视网膜干细胞研究进展

由于脊椎动物的视网膜干细胞在增生、分化潜能方面存在差异,因此将其视网膜干细胞统一称为"视网膜干细胞和(或)祖细胞".对脊椎动物视网膜干细胞和(或)祖细胞的研究,涉及范围从鱼类、两栖类到鸟类、哺乳类动物的胚胎和成年个体,特别是哺乳类动物成体视网膜干细胞和(或)祖细胞的发现,改变了视网膜不能再生的传统观点,有可能通过各种手段促进视网膜的再生而修复视网膜功能.视网膜干细胞主要来源包括睫状缘(CMZ)的干细胞和(或)祖细胞、睫状体色素上皮(PCM)细胞、视网膜MOiler细胞等.与鱼类、两柄类和鸟类等低等脊椎动物较强的视网膜再生能力相比,哺乳类动物视网膜中干细胞和(或)祖细胞的存在具有争议.进一步明确哺乳动物视网膜中的干细胞的存在和生物学特性,以及在视网膜损伤和变性过程中干细胞对视网膜可能的自修复作用和调控机制,对各种视网膜、视神经致盲性眼病的认识和治疗,将具有巨大的临床应用价值.     

视网膜神经胶质细胞与青光眼

青光眼视神经病变以进行性的视网膜神经节细胞丧失和相应的视功能损害为特征.导致青光眼视神经病变的病理生理机制尚不明确.在中枢神经系统,神经胶质细胞参与了神经元在受到机械、缺血、缺氧等损伤后的病理变化过程.胶质细胞参与青光眼视神经病变的机制复杂多样,但部分机制与中枢神经系统相似.本文就视网膜神经胶质细胞的生理功能、动物青光眼模型建立后胶质细胞的反应以及针对胶质细胞的青光眼视神经保护治疗手段做一综述.     

诱导多能干细胞在视网膜疾病治疗中的应用研究

近年来诱导多能干细胞(induced pluripotent stem cells,iPSC)作为一种有用的干细胞来源在基础研究和临床实验方面得到广泛应用.基于iPSC的视网膜细胞替换技术在治疗某些视网膜和视神经疾病方面取得进展,主要包括视网膜色素上皮、感光细胞和视网膜神经节细胞等.基于iP-SC的细胞替换技术通过多种不同技术方法实现iPSC向视网膜细胞诱导分化,进而将获得的目标细胞移植到视网膜受损动物模型,取得了一定的治疗效果.然而,在iPSC技术从实验室走向临床应用的道路上还有大量问题需要解决.     

干细胞治疗视网膜血管损伤相关疾病的研究进展

视网膜血管损伤涉及许多眼部疾病,目前缺乏有效的治疗办法.有研究表明,胚胎干细胞和多种成体干细胞具有修复视网膜血管损伤的作用.胚胎干细胞可在体外诱导分化为成血管细胞,将后者应用于视网膜血管损伤动物模型,可有效修复损伤的血管;骨髓造血干细胞、骨髓间充质干细胞及脐血干细胞均具有内皮前体细胞活性,可在体外分化为血管内皮细胞,应用于一些血管损伤动物模型中,均具有修复损伤血管的作用.视网膜血管与视网膜神经组织联系紧密,视网膜损伤血管的修复,很有可能促进视网膜神经组织的保护和修复.     

玻璃体内细胞移植或细胞衍生物注入在视神经损伤中的作用

视网膜神经节细胞绝大部分位于神经节细胞层,可通过视神经通路将光感受器接收的视觉信息传递至高级视觉中枢,产生视觉。视网膜神经节细胞损伤和视神经轴突的中断,往往会导致视力下降甚至失明。近年来研究证实,玻璃体内细胞移植对损伤的视神经具有保护作用,为视神经损伤的修复治疗提供了新的方向。本文就玻璃体内细胞移植或细胞衍生物注入在视神经损伤中的作用展开综述。     

Partial enrichment of a population of human limbal epithelial cells with putative stem cell properties based on collagen type IV adhesiveness

The concept that corneal epithelium stem cells reside in limbus has been recognized for more than a decade, but isolation of these stem cells has not been accomplished. This study was an initial attempt to isolate a population of human limbal epithelial cells enriched for certain putative stem cell properties based on their phenotype. Epithelial cells harvested from fresh human limbal rings and their primary cultures were allowed to adhere to collagen IV-coated dishes for 20 min and 2 hr, sequentially. The rapidly adherent cells (RAC), slowly adherent cells and non-adherent cells were evaluated for certain stem cell properties: (a) BrdU-label retention, (b) expression of basal cell (integrin beta1, p63, ABCG2) and differentiation (involucrin, keratin 12) markers, and (c) colony forming efficiency (CFE) and growth capacity on a 3T3 fibroblast feeder layer. Among unfractionated cells and the three selected populations, the RAC, accounting for about 10% of whole population, were enriched 5-fold in BrdU label-retaining cells, displayed the highest number of integrin beta1 and p63 positive and involucrin negative cells, expressed high levels of DeltaNp63 and ABCG2 mRNA, and lacked involucrin and K12 expression, and possessed the greatest CFE and growth capacity. These findings demonstrated for the first time that human limbal epithelial cells with stem cell properties can be partially enriched by their adhesiveness to collagen IV. The RAC population enriched for certain putative stem cell properties may prove useful in the future for transplantation to diseased and damaged corneas with limbal stem cell deficiency.     

干细胞诱导分化为视网膜色素上皮细胞的途径探讨

近年来,干细胞在眼科领域的研究及应用受到高度关注,胚胎干细胞(ES)、成体干细胞能够被定向诱导分化成视网膜色素上皮细胞,由此可获得转分化的大量的视网膜色素上皮细胞源,通过体内干细胞及视网膜色素上皮细胞移植有望应用于各种视网膜退行性疾病的细胞替代治疗。本文就各种干细胞诱导分化为视网膜色素上皮细胞的途径及应用进行探讨。     

Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies

The self renewal capability of limbal epithelial stem (LEST) cells is fundamental to the maintenance and healing of corneal epithelium. Limbal stem cell deficiency (LSCD), due to dysfunction or loss of LEST cells, therefore presents as persistent epithelial defects, corneal vascularization, conjunctivalization etc. Stem cell-based therapy, in its simplest form – limbal autograft, has been used successfully for more than a decade. For bilateral LSCD, similar approaches with limbal allografts have been unsuccessful largely due to strong immune rejection. Therefore, as an alternate strategy for treating bilateral LSCD, ex vivo expansion of the remaining LEST cells or autologous stem cells sourced from other potential sites is being explored. Different culture systems (with and without xenobiotic supplements) using substrates like amniotic membrane or fibrin gels have been used successfully for ex vivo LEST cell maintenance and reproduction by imitating the stem cell niche. This paper is organized into sections reviewing the LEST cells, LSCD and various stem cell-based approaches for treating LSCD and discussing future direction and challenges.     

Limbal Stem Cell Transplantation and Complications

ABSTRACT

Corneal epithelial stem cells are adult somatic stem cells located at the limbus and represent the ultimate source of transparent corneal epithelium. When these limbal stem cells become dysfunctional or deficient, limbal stem cell deficiency (LSCD) develops. LSCD is a major cause of corneal scarring and is particularly prevalent in chemical and thermal burns of the ocular surface. LSCD leads to conjunctivalization of the corneal surface, neovascularization, recurrent or persistent epithelial defects, ocular surface inflammation, and scarring that, in turn, lead to decreased vision, pain, and impaired quality of life. Several techniques have been reported for limbal stem cell transplantation (LSCT). We introduce the surgical techniques, examine the success rate, and discuss the postoperative complications of conjunctival limbal autograft (CLAU), cultivated limbal stem cell transplantation (CLET), simple limbal epithelial transplantation (SLET), and limbal allograft, including keratolimbal allografts (KLAL) and living-related conjunctival allograft (LR-CLAL).     

Stem cell differentiation and the effects of deficiency

Stem cells have several unique attributes, the key features being their potency and plasticity. They have the ability to give rise to multiple cell lineages and to transdifferentiate into totally different cell type(s) when relocated to a novel stem cell niche. Most self-renewing tissues are served by stem cells. At the ocular surface, the corneo-scleral limbus is believed to provide the niche for corneal epithelial stem cells. A large body of circumstantial evidence, both clinical and basic, supports this view. However, specific identification of limbal stem cells has proved elusive. Cytokeratin markers, vimentin, epidermal growth factor receptors, p63, and others have been used to identify epithelial cell populations at the limbus, which could harbour putative stem cells. In contrast, none of the known haematopoietic stem cell markers namely, CD34 and CD133, stain any specific subset of corneal or limbal epithelial cells. Singly or collectively, none of these markers point to any unique cell(s) that could be regarded as stem cells, supporting the notion that the corneal epithelium is served by 'committed progenitors' rather than by stem cells. Disease or destruction of the corneo-scleral limbus is associated with consequential events that eventually lead to visual impairment or blindness. Conjunctivalisation and vascularisation of the corneal surface and persistent or recurring epithelial defects are hallmarks of limbal deficiency.     

Molecular and cellular characterization of expanded and cryopreserved human limbal epithelial stem cells reveal unique immunological properties

Transplantation of ex vivo expanded autologous limbal stem cells into the diseased eye of patients with limbal stem cell deficiency (LSCD) has been in practice worldwide. However, isolation of limbal tissue from the normal eye of the patient with unilateral LSCD still remains a major concern for the donor. More importantly, autologous cell transplantation is not a viable option for patients with bilateral LSCD. The objective of the current study was to determine the expansion potential of human limbal epithelial stem cells (hLESCs) for their possible use in allo-transplantation. A total of six limbal biopsy samples were cultured and expanded in vitro up to passage level 1 (P-1), at which point the hLESCs were cryopreserved. Semi-quantitative RT-PCR and immunophenotypic analysis revealed that hLESCs obtained before and after cryopreservation retained the expression of major limbal epithelial stem cell markers such as p63, SSEA-4, ABCG2, cytokeratin 19 (CK19), integrin β1 and vimentin. One notable difference was that while P-0 hLESCs expressed HLA-DR mRNA, no HLA-DR gene expression was observed with the expanded and cryopreserved samples. Human LESCs did not express costimulatory proteins CD80 or B7-DC but expressed significant levels of CD86, B7-H1 and HLA-ABC molecules on the cell surface. Treatment of hLESCs with IFN-γ induced the expression of HLA-DR, indoleamine 2,3-dioxygenase (IDO) and HLA-G on these cells. Cultured hLESCs were unable to stimulate allogeneic T cell proliferation in vitro even in the presence of pro-inflammatory cytokine, IFN-γ. These results indicate that cryopreserved hLESCs are non-immunogenic in nature and express negative immunoregulatory molecules which may be critical for their survival in an allogeneic environment.     

Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle

Corneal epithelium is traditionally thought to be a self-sufficient, self-renewing tissue implying that its stem cells are located in its basal cell layer. Recent studies indicate however that corneal epithelial stem cells reside in the basal layer of peripheral cornea in the limbal zone, and that corneal and conjunctival epithelia represent distinct cell lineages. These ideas are supported by the unique limbal/corneal expression pattern of the K3 keratin marker for corneal-type differentiation; the restriction of the slow-cycling (label-retaining) cells in the limbus; the distinct keratin expression patterns of corneal and conjunctival epithelial cells even when they are provided with identical in vivo and in vitro growth environments; and the limbal cells' superior ability as compared with central corneal epithelial cells in undergoing in vitro proliferation and in reconstituting in vivo an intact corneal epithelium. The realization that corneal epithelial stem cells reside in the limbal zone provides explanations for several paradoxical properties of corneal epithelium including its 'mature-looking' basal cells, the preponderance of tumor formation in the limbal zone, and the centripetal cellular migration. The limbal stem cell concept has led to a better understanding of the strategies of corneal epithelial repair, to a new classification of various anterior surface epithelial diseases, to the use of limbal stem cells for the reconstruction of corneal epithelium damaged or lost as a consequence of trauma or disease ('limbal stem cell transplantation'), and to the rejection of the traditional notion of 'conjunctival transdifferentiation'. The fact that corneal epithelial stem cells reside outside of the cornea proper suggests that studying corneal epithelium per se without taking into account its limbal zone will yield partial pictures. Future studies need to address the signals that constitute the limbal stem cell niche, the mechanism by which amniotic membrane facilitates limbal stem cell transplantation and ex vivo expansion, and the lineage flexibility of limbal stem cells.     

Myogel supports the ex-vivo amplification of corneal epithelial cells

Limbal stem cell deficiency leads to conjunctivalisation of the cornea and subsequent loss of vision. The recent development of transplantation of ex-vivo amplified corneal epithelium, derived from limbal stem cells, has shown promise in treating this challenging condition. The purpose of this research was to compare a variety of cell sheet carriers for their suitability in creating a confluent corneal epithelium from amplified limbal stem cells. Cadaveric donor limbal cells were cultured using an explant technique, free of 3T3 feeder cells, on a variety of cell sheet carriers, including denuded amniotic membrane, Matrigel, Myogel and stromal extract. Comparisons in rate of growth and degree of differentiation were made, using immunocytochemistry (CK3, CK19 and ABCG2). The most rapid growth was observed on Myogel and denuded amniotic membrane, these two cell carriers also provided the most reliable substrata for achieving confluence. The putative limbal stem cell marker, ABCG2, stained positively on cells grown over Myogel and Matrigel but not for those propagated on denuded amniotic membrane. In the clinical setting amniotic membrane has been demonstrated to provide a suitable carrier for limbal stem cells and the resultant epithelium has been shown to be successful in treating limbal stem cell deficiency. Myogel may provide an alternative cell carrier with a further reduction in risk as it is has the potential to be derived from an autologous muscle biopsy in the clinical setting.     

干细胞与角膜眼表重建研究进展

角膜是眼表的重要防护屏障和光学系统的重要组成部分,其完整性和透明性是实现有效视功能的重要前提。各种原因造成的眼表疾病未经及时有效治疗都有可能发展到终末阶段———角膜缘干细胞功能障碍,包括角膜缘干细胞数目的减少和微环境的病理改变。自体角膜移植无疑是治疗角膜缘干细胞功能障碍最有效的方法。但是供体角膜来源十分有限,且切除健眼组织行角膜移植可能会对健眼造成长期损害。随着干细胞研究的不断深入和组织工程技术的兴起,组织工程角膜应运而生,并且迅速发展。其基本原理为选用生物性能良好的支架材料,体外模拟角膜缘干细胞微环境,诱导各类种子干细胞分化为角膜类上皮,然后再行角膜眼表重建。常用种子细胞包括角膜缘干细胞、胚胎干细胞、诱导多能干细胞、骨髓间充质干细胞、皮肤干细胞、口腔黏膜干细胞。本文就上述干细胞在角膜眼表重建中的应用作一综述。