角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

人角膜缘干细胞体外培养的增殖与分化研究

目的了解角膜缘干细胞体外培养的增殖分化规律。方法组织块法培养细胞,测定细胞克隆形成率（ＣＦＥ）,免疫荧光染色检测干细胞表达角质蛋白Ｋ３的状况。结果原代培养２１天左右细胞生长达到饱和,传第１代７～１０天形成单层,ＣＦＥ为９．５２％±４．９７％;传第２代７天,ＣＦＥ为４．２５％±２．１０％（Ｐ＜０．０１）。正常角膜缘基底细胞不表达角质蛋白Ｋ３;原代培养的干细胞亦不表达Ｋ３,传第１代细胞有部分表达。结论人角膜干细胞位于角膜缘基底部,培养的角膜缘干细胞早期具有较高的增殖力并保持干细胞的分化特性。     

角膜缘干细胞的研究进展

健康的角膜上皮是维系完整的眼表以及良好视功能的前提条件,角膜上皮的稳定是由一群位于角膜缘基底部的干细胞实现的。一、角膜缘干细胞的概念Davanger和Evensen[1]于1971年首次观察到角膜缘色素样细胞作水平向心运动,推测角膜上皮细胞更替源于角膜缘,提出了角膜缘干细胞(limbalstem     

人角膜及角膜缘上皮细胞分化标记的检测

目的检测分化标记在人角膜及角膜缘上皮细胞的表达,以了解角膜及角膜缘细胞分化状态,旨在发现新的角膜上皮干细胞的阴性标记。方法获取人角膜及角膜缘组织,对冰冻切片及整个角膜组织行免疫荧光染色检测分化标记钙粘连素E、角蛋白3(CK3)、角蛋白12(CK12)、缝隙连接蛋白43、巢蛋白(nestin)和包壳蛋白(involucrin)的表达,经荧光显微镜及激光扫描共焦电镜观察,并行半定量RT-PCR以检测其相关分化标记基因的表达。结果分化标记CK3、CK12、缝隙连接蛋白43、巢蛋白和包壳蛋白在角膜和角膜缘上皮的表层细胞表达,角膜缘基底细胞不表达。激光扫描共焦电镜观察及RT-PCR结果显示角膜缘基底上皮细胞不表达细胞CK3、连接蛋白43和巢蛋白,而角膜上皮细胞则明显表达。结论角膜及角膜缘表层上皮较为成熟分化,而角膜缘基底细胞具有未分化细胞的特征,很可能是干细胞的部位。     

角膜缘组织定位培养和冷冻后培养的实验研究

目的验证角膜缘干细胞的组织学定位,探讨低温冷冻保存对其增殖活性的影响。方法取新鲜角膜缘上皮组织和相应部位浅层巩膜组织各10块进行体外细胞培养,对比观察细胞生长情况。取冷冻保存的角膜缘上皮组织14例,观察体外培养后细胞生长情况。通过免疫组化方法检测冷冻保存的角膜缘上皮细胞的增殖活性和培养后单层细胞K3角蛋白的表达。结果10例新鲜角膜缘上皮组织培养后,7例有上皮细胞生长,1周形成细胞单层;10例浅层巩膜组织培养后未见细胞生长。14例冷冻角膜缘上皮组织培养后,4例有上皮细胞生长,9d形成细胞单层。5例冷冻角巩膜环组织冰冻切片中,3例可见角膜缘上皮基底细胞PCNA表达阳性。培养细胞对K3角蛋白特异性的AE-5单克隆抗体免疫反应阳性。结论角膜缘干细胞定位于角膜缘上皮基底部,低温冷冻保存的角膜缘干细胞组织可以保持增殖活性,体外培养后生长分化成为角膜上皮。     

角膜缘干细胞及其临床意义

干细胞是一较新的概念,它具有细胞更新及组织再生功能。干细胞以分化度低、有丝分裂度低、DNA分离不对称等为特征。正常状态或上皮创伤修复过程中,角膜上皮细胞呈向心运动,角膜缘受损可影响结膜转向分化(transdifferentiation)。特异性角蛋白表达测定结果表明,干细胞位于角膜缘基底膜,上、下两侧角膜缘干细胞分布较多。创伤后异常角膜上皮愈合与角膜缘部干细胞缺乏或功能障碍有关。根据干细胞概念,本文还对有关角膜上皮疾病的发病机制作了新的认识。角膜缘部移植术已成功地用于临床;其疗效优于结膜移植术。     

人角膜上皮干细胞的识别

目的 探讨人角膜上皮干细胞的分子标记。方法 对人角膜和角膜缘部位行组织学检查以分析角膜缘解剖结构。对人角膜切片和整个角膜组织行免疫组织化学染色以检测中央角膜和角膜缘部位未分化标记,如核蛋白p63、乳腺癌抵抗蛋白（ABCG2,BCRP1）、烯醇化酶α、整合素拍、胡及β1、表皮生长因子受体（EGFR）、细胞角蛋白19（CK19）、14（CK14）及转铁蛋白受体（CDT1）的表达,经荧光显微镜和激光扫描共焦显微镜观察。对角膜中部和角膜缘上皮细胞的mRNA进行半定量逆转录聚合酶链反应（RT—PCR）和原位杂交以检测其相关基因的表达。结果 角膜缘部位横向切片显示角膜缘上皮细胞为乳头放射状排列,对应于Vogt栅栏环境。未分化标记整合素β1、EGFR、烯醇化酶α及CK19在角膜缘基底细胞胞质染色较表层细胞更强;p63、ABCG2、整合素胡蛋白仅见于角膜缘基底部上皮细胞。激光扫描共焦显微镜观察和RT—PCR结果显示角膜缘表达p63、ABCG2、整合素胡蛋白及mRNA。原位杂交显示p63仅表达于角膜缘基底层细胞。结论 角膜缘上皮呈乳头放射状排列,角膜缘干细胞群具有复合标记：p63表达于细胞核、ABCG2表达于胞质、整合素胡表达于胞膜。采用这些标记复合体,可将角膜缘干细胞群与其他上皮细胞区分。     

体外不同区域角膜上皮细胞对5—FU耐受性的研究

为体外培养角膜上皮移植选取细胞来源，本文应用溴代脱氧尿嘧啶（Ｂｒｄｕ，胸腺嘧啶的类似物）渗入ＤＮＡ合成，抗—Ｂｒｄｕ单克隆荧光抗体标记细胞法，在流式细胞仪（ＦＡＣＳｔａｒｐｌｕｓ）上检测离体情况下兔角膜不同区域上皮细胞对５－ＦＵ耐受情况，以确定长周期细胞的部位。结果显示：经５－ＦＵ处理后的角膜周边及角膜中央部上皮细胞增殖明显受到抑制，而角膜缘细胞对５－ＦＵ毒性则有相对耐受性。由此得出：角膜缘上皮细胞群中有长周期的角膜干细胞存在。因而，行体外培养角膜上皮时，应选取角膜缘部的细胞。     

上皮性钙黏附蛋白在人角膜上皮中的表达研究

目的 研究上皮性钙黏附蛋白（E-cadherin）在人角膜上皮中的分布。方法低钙培养基培养人角膜缘干细胞．并取人角膜中央上皮及角膜边缘上皮组织，分别使用免疫组织化学及免疫细胞化学方法检测E-cadherin在人角膜中央上皮、角膜边缘上皮及低钙培养的人角膜缘干细胞中的分布情况。结果E-cadherin在角膜边缘上皮全层细胞即处于分化阶段的短暂扩充细胞中大量表达，在抑制干细胞分化、促进增殖的低钙培养的人角膜缘干细胞中表达减少，在终末分化细胞即角膜中央上皮细胞中没有表达。结论E-cadherin在处于分化阶段的角膜上皮细胞中有大量的表达。     

大鼠角膜缘上皮细胞培养与同体移植的实验研究

目的：观察以明胶为载体培养的角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症的疗效。 方法：大鼠角膜缘上皮细胞在铺有明胶载体的细胞培养板上进行培养5d后,角膜上皮细胞移植术前24h用3H胸腺嘧啶核苷标记培养的角膜缘上皮细胞,培养标记的角膜缘上皮细胞对角膜缘干细胞缺乏的大鼠动物模型行角膜缘上皮细胞同体移植术,对移植术后角膜进行观察、病理学检查及同位素检测。 结果：大鼠角膜缘上皮细胞可以在明胶载体上培养、增殖、分化为密集角膜上皮细胞层;角膜缘上皮细胞移植术后角膜上皮完整、基质细胞浸润减轻、新生血管减少。病理学检查角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失及基质中炎性细胞浸润减轻。角膜缘上皮细胞移植术后4wk受眼角膜仍可测到3H胸腺嘧啶核苷。 结论：角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。     

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.     

Regional heterogeneity in human corneal and limbal epithelia: an immunohistochemical evaluation.

The authors studied the distribution of specific keratins within the superior, inferior, medial, and lateral regions of human limbus and cornea to determine whether the limbal epithelium exhibits regional heterogeneity in its microstructure. A corneal epithelial basic keratin (K3), recognized by monoclonal antibody AE5, was immunohistochemically undetectable in the basal layers of the limbus in these four regions, but was seen in all layers in the central cornea. The pattern of immunostaining with another monoclonal antibody, AE1, which recognizes several acidic keratins, was complementary to AE5 staining in that AE1 recognized a similar heterogeneity in the limbal epithelial cells. AE1 immunoreacted with the basal cells of the limbus, but not those of the central corneal epithelium. Limbal characteristics, as defined by AE1-positive and AE5-negative staining, extended deeply into peripheral cornea in the superior and inferior regions, but to a lesser extent in the lateral and medial regions. The broader regions of epithelium with limbal characteristics in the superior and inferior regions raises the possibility that these regions play an important role in corneal epithelial maintenance and wound healing.     

Expression of the 55-kD/64-kD corneal keratins in ocular surface epithelium.

According to the concept of keratin pairing defined by tissue coexpression, a 55-kD/64-kD keratin pair is a marker of "corneal-type" differentiation. Intermediate filament (IF)-enriched preparations from guinea pig and bovine corneal epithelium were analyzed, and a rabbit antiserum was generated against a 55-kD polypeptide enriched in these preparations. This antiserum generated a typical IF-like pattern in cultured bovine corneal epithelial cells. Immunofluorescence microscopic analysis of frozen sections of guinea pig and bovine tissue revealed that the 55-kD antiserum labeled corneal and limbal epithelium. In addition, the antiserum stained a subpopulation of peripheral limbal cells that were distributed in both basal and suprabasal layers of the epithelium. The monoclonal antibody AE5 was used to investigate the distribution of the 64-kD polypeptide in guinea pig and bovine tissue. Immunoblotting analysis revealed that AE5 antibodies recognized a 64-kD polypeptide in guinea pig cornea, but recognized a 66-kD polypeptide in bovine cornea. Immunofluorescence microscopic analysis of guinea pig tissue revealed that AE5 antibodies labeled suprabasal layers of corneal epithelium, in suprabasal layers of limbal epithelium, and in groups of cells in the peripheral limbal epithelium. We discuss the possibility that the ocular epithelial cells recognized by either the 55-kD or the 64-kD antibodies in the peripheral limbus may play a role in the reepithelialization of the cornea after wounding.     

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures

BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. METHODS: The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. RESULTS: Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. CONCLUSIONS: These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.     

Differentiation in cultured limbal epithelium as defined by keratin expression.

The authors investigated differentiation of cultured corneal and limbal epithelial cells by immunochemically evaluating the changes in the profiles of keratins recognized by two monoclonal antibodies: AE5, which recognizes K3, and AE1, which recognizes a group of acidic keratins including K16, which is present in the hyperproliferative cells. After 1 and 2 weeks in culture, the human epithelial cells did not react with AE5 but did react strongly with AE1. At 3 weeks, only suprabasal cells exhibited a moderate reactivity with AE5, whereas AE1 binding was seen in all of the cells. After 5 to 6 weeks in culture, all of the cells reacted moderately with AE5 and AE1. Treatment of 2-week-old limbally derived cultures with mitomycin C (mitosis inhibitor) did not inhibit subsequent K3 expression. Thus, K3 expression was associated with maturation or a later stage of differentiation that did not require an additional cell division. Unlike human epithelial cells, rabbit suprabasal epithelial cells expressed K3 (reactivity to AE5) after only ten days in culture. The epithelium derived from central human cornea lost K3 by 1 week in tissue culture but expressed keratin(s) recognized by AE1. Even after 4-6 weeks, cells derived from the central cornea did not become confluent and did not react with AE5. Thus, limbally derived human and rabbit epithelial cells undergo chronological changes in K3 expression similar to that seen in rabbit epithelial cells derived from central cornea. However, cultured human limbal epithelial cells take a significantly longer time to express K3 (a phenotypic characteristic of differentiated corneal epithelium) than do rabbit epithelial cells.     

Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2

Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2+ cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II+ cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2+ cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2+ cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC class II antigen. These findings may have important implications for our understanding of the characteristics of limbal slow-cycling cells.     

Phenotypic study of a case with successful transplantation of ex vivo expanded human limbal epithelium for unilateral total limbal stem cell deficiency

OBJECTIVE: To minimize the risk to the donor eye when a conjunctival limbal autograft is performed for unilateral total limbal stem cell deficiency (LSCD), a new approach has been reported of expanding limbal epithelial progenitor cells from a small limbal biopsy cultured on amniotic membrane (AM). Herein, we present for the first time the morphologic and phenotypic outcome of one such patient. DESIGN: Interventional case report. METHODS: A 31-year-old male with a severe acid burn to his left eye received AM transplantation at the acute stage and a keratolimbal allograft (KLAL) at the chronic stage for total LSCD. As an alternative to combat the failed KLAL, the above-mentioned new surgical procedure was performed. The corneal button, obtained after a penetrating keratoplasty performed 5.5 months later, and a normal corneal button as a control were submitted to hematoxylin-eosin and immunofluorescence staining for keratin K3, connexin 43, goblet-cell mucin MUC 5AC, laminin 5, and integrins alpha3beta1 and alpha6beta4. MAIN OUTCOME MEASURES: Clinical and immunohistologic features. RESULTS: The resultant epithelium was stratified with five to six cell layers and anchored to laminin 5 of the amniotic basement membrane via integrins alpha3beta1 and alpha6beta4 in a manner similar to the normal corneal epithelium. Intriguingly, the epithelial phenotype was limbal and not corneal, based on the negative expression of keratin K3 and connexin 43 of the basal epithelium. CONCLUSIONS: The technique described ensures the preservation of amniotic basement membrane, which allows formation of adhesion complexes and maintains normal corneal architecture. The preservation of a limbal epithelial phenotype on the reconstructed corneal surface indicates that AM provides a unique stromal environment conducive to the preservation and expansion of limbal epithelial progenitor cells.     

Identification and characterization of limbal stem cells

The maintenance of a healthy corneal epithelium under both normal and wound healing conditions is achieved by a population of stem cells (SC) located in the basal epithelium at the corneoscleral limbus. In the light of the development of strategies for reconstruction of the ocular surface in patients with limbal stem cell deficiency, a major challenge in corneal SC biology remains the ability to identify stem cells in situ and in vitro. Until recently, the identification of limbal stem cells mainly has been based on general properties of stem cells, e.g. lack of differentiation, prolonged label-retaining, indefinite capacity of proliferation exemplified by the clonogenic assay as well as their special role in corneal wound healing. During the last years, a number of molecular markers for the limbal SC compartment has been proposed, however, their role in distinguishing limbal SC from their early progeny is still under debate. Data reported from the literature combined with our own recent observations suggest, that the basal epithelial cells of the human limbus contain ABCG2, K19, vimentin, KGF-R, metallothionein, and integrin alpha9, but do not stain for K3/K12, Cx43, involucrin, P-cadherin, integrins alpha2, alpha6, and beta4, and nestin, when compared to the basal cells of the corneal epithelium. A relatively higher expression level in basal limbal cells was observed for p63, alpha-enolase, K5/14, and HGF-R, whereas there were no significant differences in staining intensity for beta-catenin, integrins alphav, beta1, beta2, and beta5, CD71, EGF-R, TGF-beta-RI, TGF-beta-RII, and TrkA between limbal and corneal basal epithelial cells. Therefore, a combination of differentiation-associated markers (e.g. K3/K12, Cx43, or involucrin) and putative SC-associated markers (e.g. ABCG2, K19, vimentin, or integrin alpha9) may provide a suitable tool for identification of human limbal SC. While most putative SC markers label the majority of limbal basal cells and, therefore, may not distinguish SC from progenitor cells, only ABCG2 was strictly confined to small clusters of basal cells in the limbal epithelium. At present, ABCG2 therefore appears to be the most useful cell surface marker for the identification and isolation of corneal epithelial SC. Moreover, the characteristics of the specific microenvironment of corneal SC, as provided by growth factor activity and basement membrane heterogeneity in the limbal area, could serve as additional tools for their selective enrichment and in vitro expansion for the purpose of ocular surface reconstruction.