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.     

Developmental changes in patterns of expression of tenascin-C variants in the human cornea

PURPOSE: To study patterns of expression of alternatively spliced tenascin-C (TN-C) variants believed to mediate cellular activities in human corneal development. METHODS: Serial sections of preterm, neonatal, child, and adult globes with normal anterior segments were labeled with monoclonal antibodies to TN-C. The antibodies included BC-4 and BC-8, which recognize epitopes in conserved domains of TN-C and can thus detect all TN-C variants, and BC-2, alpha-A2, alpha-A3, alpha-IIIB, TN11, and alpha-D, which bind to epitopes in alternatively spliced fibronectin type III repeats of TN-C. Bound antibodies were localized and visualized using an avidin-biotin complex-alkaline phosphatase technique. RESULTS: BC-4 and BC-8 showed similar patterns of staining, widely observed in preterm corneas, less so in neonatal corneas, and restricted to the limbus in the child and adult. BC-2, alpha-A2, alpha-A3, alpha-IIIB, TN11, and alpha-D staining was largely localized in corneal epithelium (preterm and neonatal), limbal epithelium, mast cells, and matrix surrounding limbal vessels (preterm, neonatal, child, and adult). CONCLUSIONS: TN-C may play a role in corneal development and in growth and differentiation of stem cells because it is widely expressed in the preterm cornea, less so in the neonate, and is restricted to the limbus in the child and adult. The differential patterns of expression of TN-C variants in normal corneas (preterm and neonatal), and in the limbus (preterm, neonatal, child, and adult), suggest specific roles played by each variant, and cell type-specific expression of the different variants.     

Infant limbus: An immunohistological study

All published literature to date has identified the human corneo-scleral limbus as the site within which stem cells of the ocular surface reside. Recently we described a unique anatomical structure at the limbus, termed the Limbal Epithelial Crypt (LEC) that has features of a putative stem cell niche. In this study we examined infant limbus tissue (donor age 4 months) for evidence of LEC and performed immunohistological comparison between infant limbus and adult LEC. No defined LEC were detected in the infant limbus. However, the entire infant limbus has characteristics resembling adult LEC. Both infant limbus and LEC demonstrated negative expression for desmoglein 3. p63 and integrin β1 expressions were located to the distal region of the infant limbus and to the basal region of the LEC. ABCG2 expression was positive throughout most of the infant limbus as was connexin 43. Infant limbus and in particular the distal region, appeared to house cells that are more “stem-like” in nature. The LEC may be a result of normal physiological developmental in order to protect and maintain stem cells at the ocular surface.     

Localization of corneal epithelial stem cells in the developing rat.

A monoclonal antibody, 4G10.3, was developed that preferentially binds limbal basal cells in adult rat, rabbit, and human corneas. These cells were hypothesized to be the stem cells for the corneal epithelium. The antibody 4G10.3 was localized by immunofluorescence microscopy in rats 1 d and 1, 1.5, 2, 3, 4, and 6 wk of age. Until 1.5 wk, 4G10.3 bound intensely to all basal cells in the cornea and the limbus. At 2 wks, the basal cells at the central cornea abruptly changed their shape from flattened or ovoid to large and cuboidal and bound 4G10.3 with greatly reduced intensity. Increased stratification of epithelium also was seen. Cells binding 4G10.3 gradually became sequestered to the limbal area after 2 wk, concomitant with increased stratification. At 4 and 6 wk, 4G10.3 binding was identical to that in adult corneas with only limbal basal cells showing positive binding. Basal cells in the limbal epithelium did not decrease their intense binding of 4G10.3 or change their ovoid cellular shape from 1 d through adult life. These results suggest that, during development, stem or stem-like cells are localized throughout the basal layer of the corneal and limbal epithelium. As the cornea matures, these cells are sequestered in the limbus at the same time that stratification of the epithelium and shape changes occur in the basal cells.     

The corneoscleral limbus in human corneal epithelial wound healing

We studied re-epithelialization of the ocular surface in 17 human eyes (14 patients) with large corneal and conjunctival abrasions. We focused on the healing of the limbal region. During re-epithelialization, cell movement was found to occur circumferentially along the corneoscleral limbus and centripetally from the corneoscleral limbus. In no patient did the central corneal defect close before the corneoscleral limbus had first re-epithelialized completely. Normal limbal healing was observed to occur by circumferentially migrating tongue-shaped corneal limbal epithelium. These tongue-shaped projections developed from either side of the remaining intact epithelium and advanced along the corneoscleral limbus until they met. A centripetal movement of cells from the corneoscleral limbus then completed the healing process. In three patients, however, the advancing conjunctival epithelium extended across the corneoscleral limbus before the tongue-shaped projections of corneal limbal epithelium had met. The surface of the cornea covered by conjunctival epithelium was thin and irregular, and later showed peripheral scarring, vascularization, and recurrent erosions.     

翼状胬肉的发生与角膜缘干细胞缺乏的病理研究

目的：研究翼状胬肉的发生与角膜缘干细胞缺乏的相关性。方法：通过对54例翼状胬肉手术病例切除的标本进行病理检查，在镜下观察翼状胬肉颈部组织的上皮细胞和正常角膜缘结膜上皮细胞以及干细胞的移行变化。结果：翼状胬肉头颈部表面上皮细胞层数减少，形态扁平，部分萎缩；翼状胬肉颈部与正常角膜缘球结膜的交界处上皮细胞排列层数逐渐移行减少，基底膜破坏，细胞排列不规则，缺乏基底的柱状细胞(角膜缘基底部柱状细胞包含角膜缘干细胞与短暂扩充细胞)。结论：角膜缘干细胞的缺乏和破坏可能是翼状胬肉发生的重要因素之一。     

The effectiveness of limbus epithelium transplantation

Background. The transplantation of the limbus is based on the theory that limbal stem cells are necessary for epithelialisation and maintenance of a clear cornea. We used a genetic analysis procedure to demonstrate the survival of transplanted limbus stem cells. Patient and methods. A lamellary corneal graft and limbus transplantation from donor material was carried out on a patient with severe alkali burns and destroyed Bowmann's membrane to prepare the cornea for perforating keratoplasty. Subsequently, perforating corneal graft was carried out and the epithelium and endothelium of the removed cornea were analysed with a gene analytical procedure. For the cellular typing we used the SGM kit, well known in forensic analysis, that allows simultaneous detection of alleles at 11 loci. The genetic analysis was performed using an ABI PRISM 310 Genetic Analyser (Perkin Elmer Applied Biosystems) after DNA amplification by the polymerase chain reaction. Results. The cornea-limbus transplant was integrated without vascularisation from the limbus under treatment with cyclosporin A. The epithelium cells of the corneal tissue gained by keratoplasty had the same genetic pattern as the primary donor in all loci, but no congruence with the recipient (except for the amelogenin marker because both the donor and the recipient were males). Therefore the epithelium cells must have originated from the transplanted stem cells of the donor. Conclusions. The identical genetic pattern found for the donor and the epithelium of the corneal-limbal transplant demonstrate the survival of the transplanted stem cells in a patient with adequate cytostatic therapy.     

新鲜羊膜移植对碱烧伤大鼠角膜缘干细胞增殖效应的影响

目的：观察新鲜羊膜移植对碱烧伤大鼠模型角膜缘干细胞增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达的影响。

方法：SD大鼠40只40眼制作眼碱烧伤模型; 随机选取20只20眼行新鲜羊膜移植为实验组,对照组为不处理的烧伤模型眼。于术后1,2,3,4wk取角膜缘组织,采用免疫组化技术观察PCNA在角膜缘干细胞的表达情况。

结果：PCNA在实验组和对照组的角膜缘干细胞中均有表达,位于角膜缘上皮细胞基底层的细胞核内,但羊膜移植组显著高于对照组,两者比较有统计学意义(P<0.05); 羊膜移植术后1,2,3,4wk时PCNA蛋白在角膜缘干细胞的表达不一致,呈线性趋势,1wk达到高峰,以后逐渐降低。

结论：眼碱烧伤后行羊膜移植术可促进角膜缘干细胞的增殖表达,利于角膜上皮的修复。     

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.     

角膜缘干细胞体外培养的研究进展

角膜缘干细胞是位于角膜缘Vogt栅栏区的一种成体单能干细胞,其可通过自我更新以补充角膜上皮的损伤修复,在维持正常角膜上皮完整性、角膜透明度以及维持正常视力中发挥着重要作用。各种原因造成的角膜缘干细胞缺失将导致患者角膜混浊、眼表血管化,最终失明,目前治疗相关疾病的主要方法为体外培养角膜缘干细胞后再行移植治疗,其中如何高效地体外扩增角膜缘干细胞成为治疗成功与否的关键,本文就角膜缘干细胞定位、获取方法以及体外扩增方式等进行综述。     

The phenotype of limbal epithelial stem cells

PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.     

Ex-vivo expansion of cornea stem cells. Experimental principles and initial clinical results

The ocular surface epithelium is composed of conjunctiva, limbus and cornea. These three compartments and an stable preocular tear film are crucial factors determining ocular surface health. Stem cells of the corneal epithelium are exclusively located at the limbus. Limbal stem cells are the ultimate source of regeneration of the entire corneal epithelium and in a variety of ocular surface diseases limbal stem cell deficiency has been observed to be a characteristic feature. Therefore renewal of the limbal stem cell population using different surgical techniques has been described as the only possible therapeutic strategy. Recent advancements in cell biology have enabled the development of new models of tissue engineering as a tool for tissue replacement. The aim of this review is to summarize recently reported clinical and experimental studies showing new developments in tissue engineering for the reconstitution of the ocular surface epithelium.     

羊膜移植及丝裂霉素对碱烧伤大鼠角膜缘基质微环境的影响

目的观察羊膜移植及丝裂霉素C（MMC）对碱烧伤大鼠角膜缘基质微环境的影响。方法采用SD大鼠角膜缘碱烧伤模型。将大鼠随机分为5个组：A组为正常组,B组为羊膜移植组,C组为羊膜移植联合MMC组,D组、E组为未干预组（D组为碱烧伤1周组,E组为碱烧伤2周组）。通过苏木精-伊红染色观察病理学改变,免疫组织化学技术观察髓过氧化物酶（MPO）、肝细胞生长因子（HGF）免疫染色阳性细胞数的变化。结果干预组（B组、C组）较未干预组（D组、E组）角膜上皮完整、基质细胞浸润减轻、新生血管减少,组织病理学染色证实角膜缘及其周边上皮细胞为多层结构,角膜新生血管减少及基质中炎性细胞浸润减轻。免疫组织化学结果显示MPO及HGF含量明显减少,B组、C组之间MPO含量差别不明显,C组中的HGF含量明显减少,上皮细胞层数轻微减少。结论羊膜移植和羊膜移植联合MMC可以改善角膜缘碱烧伤大鼠模型中的角膜缘基质微环境,MMC可能通过抑制成纤维细胞分泌HGF而影响上皮细胞的增生和移行。     

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的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。     

Melanocytes in the corneal limbus interact with K19-positive basal epithelial cells

The human corneal limbus is identified by the distinct features of the palisades of Vogt (POV), which contain pigment granules that are aligned with the microplicae of the epithelium. Although it is presumed that pigments are produced by melanocytes, the characterization of melanocytes in the limbus has not been clearly documented. We examined human limbal tissues by whole mounts and serial histological sections to localize epithelial cells containing melanin granules. Most of the pigmented cells observed by immunohistochemistry were K19 (+) cells in the basal limbal epithelium. A superimposed image revealed that melanin granules were oriented towards the apex of each K19 (+) cell, acting as a pigmented cap facing the ocular surface. Melanocytes were identified by MART1, an antigen specific to melanocyte-lineage cells. Melanocytes were shown to exist as sporadic cells with dendritic processes that extend to surrounding epithelial cells. Melanocytes were also found in light-pigmented donor tissue when visualized by the tyrosinase assay using the enzyme substrate DOPA. Since tyrosinase activity was not found in epithelial cells, the production of melanin is exclusively the role of melanocytes that comprised 5.3+/-2.7% of the total cells in cytospin samples (N=3). Melanocytes and K19 (+) epithelial cells may form a functional network similar to the melanin unit of the skin.     

Impression cytology-proven corneal stem cell deficiency in patients after surgeries involving the limbus

PURPOSE: To report three cases of limbal stem cell deficiency (confirmed by impression cytology) that followed multiple pterygium surgeries and therapeutic penetrating keratoplasty. METHODS: The first case, after multiple pterygium surgeries, presented with corneal scarring and thickened epithelium with vascularization sparing the central cornea in the right eye and involving the entire cornea in the left eye. The second case presented with superficial scarring and extensive vascularization after failed therapeutic graft performed for a large perforated corneal ulcer. The third case was a clear graft performed for a progressing fungal ulcer with signs of conjunctivalization inferotemporally. Limbal stem cell deficiency was clinically suspected in all of these cases. RESULTS: Goblet cells with mucin globules were found on the corneal surface by impression cytology in all three cases. CONCLUSIONS: We report three cases of limbal stem cell deficiency (proven by impression cytology) that followed multiple pterygium surgeries and therapeutic penetrating keratoplasty. Surgical insult to the limbus is the predisposing factor for stem cell damage in these cases. Involvement of the limbus by infection and use of intensive medications are probable contributing factors for stem cell damage in cases of therapeutic penetrating keratoplasty.     

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).     

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。     

角膜缘移植联合羊膜移植治疗角膜缘缺陷症

目的 探讨角膜缘移植联合羊膜移植术在治疗角膜缘缺陷症中的作用。方法 以自体或异体角膜缘移植手术治疗复发性翼状胬肉、眼化学烧伤、热烧伤等角膜缘缺陷症患者52例59眼，术中随访5－24月。结果 随访期内植片存活，角膜上皮重建，角膜透明性增另，新生血管减退。仅2眼翼状胬肉复发。结论 角膜缘移植联合羊膜移植是一种较好的治疗角膜缘缺陷症的手术方法。     

Limbal stem cells of the corneal epithelium

Stem cells have certain unique characteristics, which include longevity, high capacity of self-renewal with a long cell cycle time and a short S-phase duration, increased potential for error-free proliferation, and poor differentiation. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. Stem cells for the cornea reside at the corneoscleral limbus. The limbal palisades of Vogt and the interpalisade rete ridges are believed to be repositories of stem cells. The microenvironment of the limbus is considered to be important in maintaining the stemness of stem cells. Limbal stem cells also act as a "barrier" to conjunctival epithelial cells and normally prevent them from migrating on to the corneal surface. Under certain conditions, however, the limbal stem cells may be partially or totally depleted, resulting in varying degrees of stem cell deficiency with resulting abnormalities in the corneal surface. Such deficiency of limbal stem cells leads to "conjunctivalization" of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. This results in ocular discomfort and reduced vision. Partial stem cell deficiency can be managed by removing the abnormal epithelium and allowing the denuded cornea, especially the visual axis, to resurface with cells derived from the remaining intact limbal epithelium. In total stem cell deficiency, autologous limbus from the opposite normal eye or homologous limbus from living related or cadaveric donors can be transplanted on to the affected eye. With the latter option, systemic immunosuppression is required. Amniotic membrane transplantation is a useful adjunct to the above procedures in some instances.