Limbal Basal Epithelial Cells Research Articles

Age-related Changes of Human Limbus on In Vivo Confocal Microscopy

To show age-related changes in the presence of Vogt palisades in human limbus and the quantity and size of the limbal basal epithelial cells (LBECs) by in vivo confocal microscopy (CM). One hundred sixty eyes of 160 healthy subjects were enrolled in this study after a routine slit-lamp examination. They were divided into 4 groups according to age: A (0-19 years), B (20-39 years), C (40-59 years), and D (>60 years). In vivo CM examination was performed on the inferior limbus, and the images were recorded. For each group, the positive rate of Vogt palisades was analyzed and the average size of LBECs was measured. The positive rate of Vogt palisades in groups A, B, C, and D was 97.5% (39/40), 95.0% (38/40), 77.5% (31/40), and 40.0% (16/40), respectively, showing a significant decreasing tendency with age. The average size of LBECs in groups A, B, C, and D was 9.89 +/- 1.12, 10.65 +/- 1.45, 10.70 +/- 1.39, and 12.36 +/- 1.68 microm. Significant differences between groups were obvious except for that between groups B and C. The presence of Vogt palisades in human limbus declined with age. Therefore, so did the qualities of LBECs, with an age-related cell size enlargement.

Air Exposure–Induced Squamous Metaplasia of Human Limbal Epithelium

Squamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency. Human limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBPalpha and C/EBPbeta, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins. The squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation-evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells-ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBPalpha and C/EBPbeta, downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580. Dryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness.

Nerve growth factor and its receptor TrkA serve as potential markers for human corneal epithelial progenitor cells

Nerve growth factor (NGF), a member of the neurotrophin family, has been identified as an essential growth factor supporting stem cell self-renewal outside the nervous system and was previously shown to stimulate corneal epithelial proliferation both in vivo and in vitro. In this study, we evaluated the expression of NGF and its corresponding receptors in the human corneal and limbal tissues, as well as in primary limbal epithelial cultures by immunofluorescent staining and relatively quantitative real-time polymerase chain reaction. We found that NGF was uniquely expressed in the human limbal basal epithelium, together with its two corresponding receptors: the high-affinity receptor TrkA and the low-affinity receptor p75NTR. TrkA was shown to preferentially localize to limbal basal epithelial cells. NGF and TrkA were also found co-localized with stem cell-associated molecular markers (drug-resistance transporter ABCG2 and p63), but not with the differentiation marker cytokeratin 3 in the human limbal basal epithelial layer. In cultured limbal epithelial cells, NGF and TrkA were found to be preferentially expressed by a small population of limbal epithelial cells. The NGF and TrkA immuno-positive subpopulations were enriched for certain properties (including ABCG2 and p63 expression) of putative limbal epithelial stem cells ( P < 0.01, compared with the entire cell population). Levels of NGF and TrkA transcripts were found to be much more abundant in limbal than in corneal tissues, and in young cultured cells in the proliferative stage than in airlifted stratified cultures containing differentiated cells. The co-expression of NGF with its two corresponding receptors in limbal basal epithelial cells, but not in the cornea, suggests that NGF may function as a critical autocrine or paracrine factor supporting stem cell self-renewal in the limbal stem cell niche. The spatial expression of NGF and TrkA by small clusters of basal cells interspersed between negative cell patches suggests that they are potential markers for human corneal epithelial progenitor cells.

The Phenotype of Limbal Epithelial Stem Cells

The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. 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. 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. 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.

UVB Irradiation-Induced Changes in the 27-kd Heat Shock Protein (HSP27) in Human Corneal Epithelial Cells

This study investigated the presence of the 27-kd heat shock protein (HSP27) and its responses to ultraviolet B (UVB) irradiation in human corneal epithelium and in cultured corneal epithelial cells. Human corneal epithelial cells including presumed corneal epithelial stem cells were cultured in vitro. HSP27 expression and intracellular localization in normal corneas or cultured corneal cells were examined using immunofluorescence staining. The expression of HSP27 in cultured corneal cells was also detected using western blotting, and the phosphorylated isoforms of HSP27 were identified using isoelectric focusing. In normal corneal tissue, HSP27 was present in limbal basal and suprabasilar epithelial cells. In cultured epithelial corneal cells, HSP27 expression was heterogeneous: Some cells expressed virtually no HSP27 and others showed relatively strong expression. HSP27 was localized to the cytoplasm in nonstressed cells and translocated to the perinuclear and nuclear areas after UVB irradiation. UVB irradiation also induced the phosphorylation of HSP27, resulting in the increase in monophosphorylated isoform and formation of biphosphorylated isoform. UV induced the phosphorylation of HSP27 apparently through activation of p38 mitogen-activated protein kinase. HSP27 is present mainly as a nonphosphorylated isoform in corneal epithelium and cultured corneal epithelial cells under nonstressed conditions. The constitutional expression of HSP27 suggests that it plays a physiologic role in the cornea. After UVB irradiation, HSP27 undergoes rapid phosphorylation and translocation. This stress response may be related to a protective role of HSP27 for survival of UVB-exposed corneal cells.

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.

Identification of human corneal epithelial stem cells

To investigate the molecular markers of corneal epithelial stem cells. The anatomy structure of corneal limbus was analyzed by histological examination. Fluorescence microscopy and laser scan confocal microscopy (LSCM) were used to evaluate the expression of un differentiation markers as p63, ABCG2, alpha6, alpha9 and beta1 integrin, alpha-enolase, EGFR, cytokeratin 19 (CK19) and CD71 in cornea-limbus sections and whole mount cornea tissue using immunohistochemistry staining. Semiquantitative RT-PCR and in situ hybridization (ISH) were used to evaluate gene expression of corneal and limbal epithelia. The limbal area in horizontal cut direction showed that limbal epithelial cells were as papilloma-form to correspond to the Palisades of Vogt environment. The expressions of beta1 integrin, K19, alpha-enolase and EGFR were much stronger in limbal basal cells than superficial cells. Un differentiation markers p63, ABCG2, alpha9 integrin were detected only in the basal limbal epithelial cells. LSCM and RT-PCR results showed that the protein and mRNA of p63, ABCG2 and alpha9 integrin were only detected in limbal epithelia. ISH showed that p63 mRNA only expressed in the limbal basal epithelial layer. The corneal limbal epithelial area is papilloma-form. Limbal stem cells have complex markers as p63 expressing in the nuclei, ABCG2 in the cytoplasm and alpha9 integrin expressing in the membrane. With these markers complex, it is easy to distinguish limbal stem cells from other epithelial cells in cornea.

Effect of immunosuppression on survival of allograft limbal stem cells.

To examine whether the application of immunosuppression enhances the survival of limbal allograft stem cells. Wistar (allograft) or Fischer 344 (isograft) rat limbal tissue was transplanted into superior lamellar excision sites in Fischer 344 rats. Allograft-recipient rats received an immunosuppressive agent or vehicle for 8 weeks. Graft-recipient rats were examined by slit-lamp microscopy for clinical signs of rejection, and some recipients were killed for immunohistochemical analysis during rejection. One month from the time of transplantation, other (five in each group) recipients received daily injections of 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks, followed by a 1-month BrdU-free period before death. After the rats were killed, limbal allograft eyes were removed for BrdU staining to identify label-retaining cells. The labeling index of transplanted limbal basal epithelial cells was determined. In nonimmunosuppressed allograft recipients, clinical rejection occurred between days 5 and 10 after transplantation (median, 7 days). In contrast, rejection was suppressed for more than 60 days (median value) when the immunosuppressive was administered. Numerous MHC class II(+), CD4(+), and CD8(+) T cells were identified with acute rejection. Immunosuppressive-treated allografts had significantly less inflammation compared with untreated controls. Furthermore, immunosuppressive-treated allografts showed more label-retaining basal cells in transplanted limbal epithelium compared with untreated allograft controls ( P < 0.01). The survival of limbal allograft stem cells can be improved by immunosuppression. The limbal allograft procedure described here provides a useful model for evaluating a suitable alternative means of sustaining the survival of corneal stem cells.

Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane

The aim of the study was to determine if human limbal epithelial cells (HLEC) do not form gap junctions (GJ) during ex vivo expansion on preserved and intact human amniotic membrane (AM). Thereby, we attempt to evaluate if characteristic features of the limbal epithelial progenitor cells are preserved on AM. Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 3–4 weeks, cell cultures were terminated and processed for immunofluorescence. In all cell cultures, formations of GJs were analyzed with a mouse monoclonal antibody to connexin 43 (Cx43) and a rabbit affinity purified antibody against connexin 26 (Cx26). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a GJ permeant dye was used to analyse functionality of GJ. Microinjection of LY into single cells was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. In vivo, a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells and labeling for Cx26 was observed in all cell layers of the human corneal epithelium, however, subpopulations of limbal basal epithelial cells lacked detectable fluorescence signals for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx43 (12·6%) was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index (LI) for Cx43 (42·7 and 52·3%, respectively). A significant lower immunostaining for Cx26 was observed in HLEC cultured on AM (LI: 35·16%) in comparison to HLEC cultured on plastic (68·4%), as well as, HPCEC cultured either on AM or plastic (61% and 79·3%, respectively; p<0·001). Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (51%, p<0·05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29·7% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52·9%; p<0·05) and HLEC on AM. Subpopulations of HLEC cultured on AM remain Cx43 and Cx26 negative and without functional GPs indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. These data provide support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.

Human limbal progenitor cells expanded on intact amniotic membrane ex vivo.

The transplantation of human limbal epithelium on amniotic membrane as a substrate is a new treatment for limbal stem cell deficiency. Limbal epithelial stem cells are characterized by a slow cell cycle and the lack of K3 keratin and connexin 43 (Cx43), a gap junction protein. We investigated Cx43 expression, gap junction intercellular communication (GJIC), and proliferative activity of limbal epithelium expanded on amniotic membrane. Connexin 43 expression and bromodeoxyuridine (BrdU) incorporation were determined by immunohistology. The GJIC was investigated by a scrape-loading dye transfer assay. Expression of Cx43 and K3 keratin as well as BrdU-retaining nuclei were also analyzed after xenotransplantation in nude mice. Limbal epithelium showed mean +/- SD 12.4% +/- 14.5% positive units of Cx43 expression and a low BrdU labeling index of 2.4% +/- 0.9% (n = 5), of which the latter was due to slow cycling, as proved by its increase to 62.0% +/- 9.5% after continuous BrdU labeling for 5 days. Most of the expanded epithelium did not show GJIC (83%), significantly more than that grown on plastic (6%; P<.002). Basal cells of the stratified epithelium after xenotransplantation did not express Cx43 and K3 keratin, but their nuclei retained BrdU. These results support the hypothesis that intact amniotic membrane preferentially preserves and expands Cx43-negative, keratin K3-negative, and GJIC-deficient limbal epithelium, a phenotype resembling that of stem cell-containing limbal basal epithelial cells in vivo. Intact amniotic membrane is a suitable substrate for bioengineering limbal epithelia for ocular surface reconstruction.

Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane for treating corneal diseases with total limbal stem cell deficiency.

Stem cells (SC) for the corneal epithelium are located exclusively at the limbus, i.e., the anatomic junction between the cornea and conjunctiva. Limbal epithelial SC are the ultimate source of regeneration of the entire corneal epithelium under normal and injured states. The epithelial phenotype of the limbal basal epithelium does not express corneal epithelial-specific keratin3, keratin 12 and connexin 43. As shown in cell-cycle kinetic studies, some portions of the limbal basal epithelial cells are slow-cycling and label-retaining. Other studies further confirmed limbal epithelial SC have greater growth potential in explant cultures and higher clonogenicity when cocultured on 3T3 fibroblast-feeder layers than corneal epithelial cells, and their proliferative potential is resistant to tumor-promoting phorbol esters.

Phenotypic analyses of limbal epithelial cell cultures derived from donor corneoscleral rims.

Grafted cultures of limbal epithelial cells aid repair of the corneal epithelium, but their phenotype is unclear. In this study, the phenotype of cultures that were similar in age to those used clinically were analysed. Limbal epithelial cells were isolated from donor corneoscleral rims and grown in various media, including those designed for keratinocytes. Successful cultures in each medium developed predominantly small (10 microm) tightly packed cells. Immunocytochemistry and western blotting revealed expression of keratins 3, 14 and 19. Expression of these keratins in situ was confirmed by immunohistochemistry. Basal limbal epithelial cells were positive for keratins 14 and 19, and suprabasal cells were positive for keratin 3. However intense staining for keratin 14 was also observed at the inner cut edge of corneoscleral rims. These findings demonstrate the potential importance of keratins 14 and 19 as markers of epithelial cell differentiation in the human cornea.

Expression of proliferating cell nuclear antigen in corneas kept in long term culture.

To investigate the proliferative activity of the donor corneal cells and to examine how this property changed during long term culture. Fourteen human corneas from donors (ages from 50-91) were cultured in the medium (MEM+8% FBS with or without dextran). The proliferating status of corneal cells was evaluated by immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in the cells. Three corneas at each time point were fixed in paraformalin at day 0, day 3 and after 3 weeks cultured in medium as well as 3 weeks plus 2 or 5 days in fresh medium with 8% dextran. Paraffin-embedded corneas were sectioned to 4 microm and stained with antibody PC 10 against PCNA. The number of PCNA positive cells was identified under light microscope. Prior to organ culture only basal limbal epithelial cells stained positive for PCNA. After 3 days in culture 50 percent of the epithelial cells were positive as were several keratocytes and some endothelial cells in the peripheral corneas. After 21 days no cells showed proliferative activity. After 21 days in culture and 5 days in fresh deswelling medium the essentially monolayered epithelium stained positively in the limbal area. The proliferative activity of the keratocytes in the anterior stroma was extensive. Endothelial cells stained positive in the peripheral cornea. Limbal epithelial cells appear to survive in the organ culture. The corneas may be worth evaluating as sources of stem cells for grafting. Likewise, the keratocytes survive organ culture and can be induced to proliferate after a change to fresh medium. The endothelium is stimulated to proliferate in organ culture and in fresh medium after long term storage.

Concept and application of limbal stem cells.

Cumulative reported evidence indicates that some fraction of limbal basal epithelial cells are the stem cells for corneal epithelial cell proliferation and differentiation. Limbal epithelium is therefore crucial in maintaining the cell mass of corneal epithelium under normal conditions and plays an important role in corneal epithelial wound healing. Deficiency or absence of limbal stem cells explains well the pathogenesis of several ocular surface disorders characterised by defective conjunctival transdifferentiation or conjunctivalisation of cornea. This paper reviews and updates the basic concept of stem cells, the reported findings of limbal stem cells for corneal epithelium, and their therapeutic applications. Through this review, one hopes to gain a more complete understanding and increase proficiency in treating these diseases.