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INTRODUCTION

Limbal stem cell transplantation, either fresh limbal tissue or cultured cells, has shown to improve the cor-neal conditions of patients suffering from severe limbal deficiency.1–7 The limbal epithelial basal layer contains cells with a phenotype suggesting undifferentiated pluripotential cells, such as expression of the α isoform of Delta N p63α.8Nevertheless, it has been reported that the mouse central cornea contained epithelial progenitors involved in steady-state corneal renewal.9

The limbal epithelial progenitors are currently thought to be the source of corneal epithelial cells.10,11

Culture of limbal stem cells have been developed using Green media, which consists of Dulbecco’s Modified Eagle’s Medium and Ham F12 medium with fetal bovine serum, cholera toxin, insulin, hydrocorti-sone, l-glutamine, and antibiotics.12Further modifica-tions added adenine, tri-iodo-thyronin, HEPES buffer, and amphotericin B.13,14

Culture medium seems to meet an appropriate level of safety (i.e. chemicals controlled and animal

Current Eye Research, 37(7), 644–653, 2012Copyright © 2012 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/1460-2202 onlineDOI: 10.3109/02713683.2012.669510

Received 09 September 2011; revised 07 February 2012; accepted 10 February 2012

Correspondence: Djida Ghoubay, Institut de la Vision, UPMC Université Paris 06, UMR_S 968/INSERM, U968/CHNO des XV-XX/CNRS, UMR_7210, Paris, France; Laboratoire du Centre Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France; Banque de Tissus, Établissement Français du Sang, Site Saint Antoine Paris, France. E-mail: djida.ghoubay@inserm.fr

09 September 2011

07 February 2012

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ORIGINAL ARTICLE

Effects of Isoproterenol and Cholera Toxin on Human Limbal Epithelial Cell Cultures

Djida Ghoubay-Benallaoua1,2,3, Florence Pécha1,3, Pablo Goldschmidt1,2, Anne Fialaire-Legendre3, Christine Chaumeil2, Laurent Laroche1, and Vincent M. Borderie1,3

1Institut de la Vision, UPMC Université Paris 06, UMR_S 968/INSERM, U968/CHNO des XV-XX/CNRS, UMR_7210, Paris, France, 2Laboratoire du Centre Hospitalier

National d’Ophtalmologie des Quinze-Vingts, Paris, and 3Banque de Tissus, Établissement Français du Sang, Site Saint Antoine Paris, France

ABSTRACT

Purpose: Cholera toxin and isoproterenol (β-adrenergic receptor agonist) are largely used to enhance cell proliferation. The aim of the study was to assess the effects of cholera toxin and isoproterenol on growth and differentiation of cells cultured from human superficial limbal explants.

Methods: Limbal epithelial cells were cultured from superficial limbal explantsin basal medium either supple-mented with cholera toxin or isoproterenol for 3 weeks. Growth kinetics and morphometry were studied by light and confocal microscopy. Progenitor and differentiated epithelial cell markers were studied by immu-nocytochemistry, flow cytometry, Colony Formation Assay, and reverse transcription and polymerase chain reaction.

Results: Cell proliferation was significantly higher with 0.5 µg/ml (p = 0.049), 1 µg/ml (p = 0.005), and 2 µg/ml (p = 0.008) isoproterenol whereas, cholera toxin and 4 µg/ml isoproterenol did not significantly increase cell proliferation. Multilayered epithelial cell sheets were obtained in all culture conditions. Addition of isopro-terenol resulted in smaller cell size (p < 0.05) 14 days after cells were cultured, whereas cholera toxin had no effects. Strong expression of cytokeratins 3 and 4/5/6/8/10/13/18 and lower expression of cytokeratin 19, vimentin, and Delta N p63α were observed after 3 weeks of culture with no significant differences in the percentage of positive cells according to culture medium. Colony-forming efficiencies were observed after 2 weeks in all culture condition but not after 3 weeks.

Conclusion: Isoproterenol was more efficient than cholera toxin for enhancing cell proliferation and resulted in smaller cell size. It appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency.

Keywords: Isoproterenol, Cholera toxin, Epithelial cell culture, Immunocytochemistry, Stem cell

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derivatives traced for their origin) for all components but not for the cholera toxin that is obtained from bacte-ria cultured on bovine brain broth and fetal calf serum. To minimize the risks of transmission of prions to human, we currently grow human limbal epithelial cells for clinical use in a cholera toxin-free medium with no animal feeder cells. This medium has been approved by the French National Regulation Agency (AFSSaPS).15

Cholera toxin is a bacterial toxin secreted by Vibrio cholerae. Effects of the cholera toxin result from ADP-ribosylation of regulatory guanine nucleotide-binding (G) proteins, which leads to an accumulation of cellular cyclic AMP.16 This intracellular second signaling mes-senger regulates a variety of biological events, including cell proliferation and cell differentiation. Cholera toxin has been shown to be mitogenic for rabbit limbal and corneal epithelial cells.17 It enhances growth of murine corneal epithelial cells cultured in serum-free, low cal-cium medium.18 Isoproterenol is a β-adrenergic agonist that increases intracellular calcium concentration of bovine corneal epithelial cells and it induces stimula-tion of cyclic AMP.19,20 Isoproterenol has been shown to decrease the migratory rates of cultured adult murine corneal epithelial cells.21,22 It modifies the circadian mitotic rhythm in rat corneal epithelium, including an initial suppression of cell division, followed by two dis-tinct rises in cell proliferation.23 Isoproterenol is a chemi-cal product with well-established effects in humans. It could then be a safe and interesting alternative to chol-era toxin use for growing human limbal stem cells.

The objective of the present work was to assess the effects of cholera toxin and isoproterenol on growth kinetics and differentiation of cells cultured from human superficial limbal explants.

MATERIALS AND METHODS

This study was carried out according to the tenets of the Declaration of Helsinki and it followed international ethic requirements for human tissues.

Donor Corneal Tissue

Human limbal tissue was obtained from corneoscleral rims after trephination of donor corneas (8-mm trephi-nation) that had been organ-cultured before transplan-tation as previously described.24 All corneoscleral rims were processed for culture less than 24 h after surgery. They were stored at 37°C in organ culture medium con-taining dextran.

Preparation of Explants

Limbal tissue was prepared under an operative micro-scope. A 15° knife was used to perform a stromal

dissection at 1/3 of the peripheral corneal thickness and the excess scleral tissue was removed using scissors.25 The resulting ring of limbal tissue was cut into six pieces with scissors, each explant being approximately 5 mm in length. The superficial limbal explants were sutured on a plastic lamella (320 mm2; one explant per lamella; ThermanoxR, Nunc, Illkirch, France) epithelial side up using a single 5/0 polyester suture. They were cultured using 6-well plates (907 mm2; Becton Dickinson, Rungis, France) with 2 ml of medium for 3 weeks at 37°C (5% CO2). The medium was changed twice a week.

Culture Medium

The six explants obtained from 1 donor cornea were cultured in the following six culture conditions (one explant per culture condition): basal medium, cholera toxin-supplemented medium, and isoproterenol-sup-plemented medium (four different concentrations).

The basal culture medium was composed of a 3:1 mixture of calcium-free Dulbecco’s Modified Eagle’s Medium (Dutscher, Brumath, France) and Ham F12 medium (Invitrogen, CergyPontoise, France) with 10% fetal bovine serum (Invitrogen), 1 mM/ml HEPES buf-fer (Invitrogen), 5 µg/ml human recombinant insulin (Actrapid®; Novo Nordisk, Paris, France), 0.4 µg/ml hydrocortisone (Pharmacia, Pfizer, Paris, France), 4 µM/ml l-glutamine (Invitrogen), 2 pM/ml tri-iodothyronine (Sigma, Saint Quentin en Yvelines, France), 200 nM/ml adenine (Sigma), 100 IU/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen), 0.25 µg/ml amphot-ericin B (Invitrogen), and 10 ng/ml human recombinant Epithelial Growth Factor (Sigma).

Cholera toxin (Sigma) was added to the basal culture medium at 10−7 M (50 µl/50 ml). Isoproterenol (Hospira, Asnières, France; 0.20 mg/1 ml) was added to the basal culture medium at four different concentrations: 0.5 µg/ml, 1 µg/ml, 2 µg/ml, and 4 µg/ml (one concentration per dish).

Assessment of Explant Outgrowth Area, Cell Proliferation, and Cell Morphometry

Cell cultures were observed once a week for 3 weeks with a phase contrast microscope (Olympus IX 70, Tokyo, Japan). The explant outgrowth area was assessed at 7, 14, and 21 days using a grid placed under the culture plate well. Photographs of the cultured cells were taken near the explant, at the edge of the explant outgrowth, and in between. Cell morphology was analysed at day 14 and 21 using the Image J 1.42q, National Institute of Health, USA-software. Photographs of mono-layered cell sheets taken between and at the edge of the explant outgrowth were used for morphometric analysis. After 3 weeks of culture, cells were dissociated enzymatically and counted.

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Ex vivo Confocal Microscopy

Laser scanning ex vivo confocal microscopy was performed on confluent cultures at day 21 using the Heidelberg Retina Tomograph III Rostock Corneal Module (Heidelberg Engineering GmbH, Heidelberg, Germany). For each lamella, images were taken from each level and tissue thickness measurements were performed.

Immunocytochemistry

Immunocytochemical staining was performed after 3 weeks of culture to evaluate the expression of differ-ent markers of limbal epithelial cells and progenitors as previously described.25 Briefly, cultured cells were enzymatically dissociated and concentrated by cytospin (450T/min, 20 min). After washing in PBS, the tissue sections and cultivated cells were fixed for 10 min with 4% paraformaldehyde and incubated for 30 min in PBS containing 1% bovine serum albumin (BSA) and 0.3% Triton X 100 to permeabilize the cells and to block non-specific staining. The endogenous peroxidases were quenched with 0.3% H2O2 during 10 min. Cells were incubated for 30 min at room temperature with primary antibodies against cytokeratin 3 1:200 (Clone AE-5; DakoTrappes, France), cytokeratins 4, 5, 6, 8, 10, 13, and 18 1:100 (MNF116; Dako), cytokeratin 19 1:50 (clone BA17, Dako), vimentin 1:200 (clone V9, Dako), and Delta N p63α 1:50 (clone 4A4, Dako) followed by incubation with the biotinylated secondary antibody using a LSAB2 system-HRP Kit (Dako) according to the manufacturer’s instructions. 3,3’ Diaminobenzidine (DAB) was used as peroxidase substrate and specimens were counterstained with haematoxylin.

Flow Cytometry Analysis

The flow cytometry analysis was performed on a BD FACSCalibur system (Becton Dickinson, Heidelberg, Germany) as previously described.25 Briefly, Cell sus-pensions (105 cells/ml) in PBS/BSA buffer were fixed with 4% paraformaldehyde (Fix Buffer I, BD) for 10 min at 37°C. After washing with PBS/BSA, cells were permeabilized with Perm Buffer III (BD Bioscience) and incubated for 30 min at 4°C. Aliquots were distributed into different test tubes for primary antibody binding (CK-3, vimentin, CK 4, 5, 6, 8, 10, 13, and 18, CK-19, Delta N p63α,) and incubated at room temperature for 30 min. An anti-mouse IgG FITC secondary antibody was added, and cells were incubated for 30 min at room temperature. Relative size, granularity or inter-nal complexity, and relative fluorescence intensity were registered with the Cell Quest Pro software (BD Bioscience).

Colony Formation Assay

The clonal growth ability of cultured limbal epithelial cells was evaluated by determining colony-forming effi-ciency (CFE). Swiss albino 3T3 fibroblasts were treated with 4 µg/ml mitomycin C for 2 h and then trypsinized and plated at a density of 105 onto six-well culture dishes (Two different concentrations of 3T3 (i.e. 2.4 × 105 and 105 cells per well) were tested. As no differences in the number of clones were observed, the lowest concentra-tion was used). The cells cultured for 14 or 21 days in basal medium, basal medium supplemented by 2 µg/ml isoproterenol, and basal medium supplemented with cholera toxin were seeded at a density of 1000 cells/well in six-well culture dishes on 3T3 fibroblasts feeder layers.12 Cultures were incubated at 37°C under 5% CO2. A colony was defined as a group of eight or more con-tiguous cells as described elsewhere.26–28 The colonies were fixed on day 12 and stained with crystal violet, and photographed.29 The CFE was defined as follows: CFE (%) = (number of colonies counted/ number of cells seeded) × 100.

Assessment of Specific Messengers RNAs in Cell Culture by Reverse Transcription And Polymerase Chain Reaction

Total RNA was isolated using the MagNA Pure Compact RNA isolation Kit (Roche Diagnostics, Mannheim, Germany) after 21 days of culture according to the manufacturer’s instructions. The RNAs extracted from cultures were quantified by 260 nm absorption. Reverse transcription and polymerase chain reaction (RT-PCR) was performed using the Qiagen One Step RT-PCR Kit (Qiagen, Courtaboeuf, France) which provides enzymes for both the reverse transcription and the nucleic acid amplification. The first-strand cDNA was synthesized after incubation at 50°C for 30 min. PCR conditions were 95°C for 15 min followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and finally at 72°C for 10 min. The primers for human CK-3, CK-19, vimentin, Delta N p63α, and β-actin (ACTB), a housekeeping gene used as an internal control, have been previously report-ed.25 The amplified products were separated using 2% agarose gel electrophoresis stained with ethidium bro-mide for molecular mass assessment.

Statistical Analysis

One-way way ANOVA was used to analyse the influ-ence of culture conditions on the various quantitative variables (i.e. cell growth, cell morphology, and cell phenotype) and the Brown–Forsythe test was used to examine homogeneity of variance between culture con-ditions. Where a significant difference was detected by

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ANOVA, a post-hoc analysis was performed.30 A p value <0.05 was considered as statistically significant.

RESULTS

Donor Tissue

Sixteen corneas (mean donor age: 65 years, mean death-to-preservation time: 24 h; mean preservation time of 18 days) were used

Assessment of Explant Outgrowth Area, Cell Proliferation, and Cell Morphometry

After 3 weeks of culture, the number of viable cells per well was significantly enhanced with 0.5 µg/ml, 1 µg/ml,

and 2 µg/ml isoproterenol (Figure 1A). Figure 1B shows mean explant outgrowth area according to culture time in each group. No significant differ-ences in outgrowth area were found between groups (p > 0.05).Three different cell layers could be distin-guished by confocal microscopy after 3 weeks of cul-ture (Figure 2): a cubical basal cell layer, a polygonal intermediate cell layer, and a flat superficial cell layer with well-defined cell borders and distinct nuclei. The cell sheet thickness was about 50 µm. No major differ-ences in cell morphology between culture conditions were observed. However, phase-contrast microscopy showed differences in cell morphology after 2 weeks of culture according to culture conditions (Figure 3). In isoproterenol-supplemented basal medium, cells were smaller, polygonal, and regular, with a high nuclear/cytoplasmic ratio. In cholera toxin-supplemented basal medium, cells were larger and featured lower

FIGURE 1 Cell proliferation and explant outgrowth area according to culture medium. A: number of living cell per well after 3 weeks of culture. B: Mean area of explant outgrowth after 7, 14, and 21 days of culture. Isoproterenol at 0.5, 1, and 2 g/ml significantly increased cell proliferation when added to the basal culture medium whereas, cholera toxin and 4 g/ml isoproterenol did not significantly increase cell proliferation. Shown is mean + SEM. $ = comparison with basal medium (A). The most important outgrowth area is observed with isoproterenol at 1 and 2 g/ml (B). *Comparison with cholera toxin-supplemented basal medium. NS = not significant (p > 0.05).

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nuclear/cytoplasmic ratio. After 3 weeks of culture, no major differences in cell morphology between culture conditions were observed by phase-contrast microscopy.

The Image J software (Figure 4) for cell morphology allowed assessing five parameters: the mean cell area (µm2) and its coefficient of variation, mean perimeter, mean circularity, and mean Feret diameter. Data were

FIGURE 2 Laser scanning confocal microscopy. Confluent cell sheets cultured for 21 days in basal medium, cholera toxin-supplemented basal medium and 2 g/ml isoproterenol-supplemented basal medium. No major differences in cell morphology between culture condi-tions were observed. A: basal cells; B: intermediate cells; C: superficial cells.

FIGURE 3 Limbal epithelial cells cultured from human explants. Cell cultured for 14 days in basal medium, cholera toxin-supplemented basal medium, and isoproterenol-supplemented basal medium assessed by phase-contrast microscopy (×10). Cells were smaller with isoproterenol-supplemented basal media.

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studied after 14 and 21 days of culture. Results obtained for the mean cell area were consistent with those obtained for the mean Feret diameter and the mean perimeter. Cholera toxin had no significant influences on cell mor-phometry whereas, isoproterenol treatment resulted in significantly smaller cells at day 14 but not at day 21.

No differences were found for cell circularity and coefficient of variation of cell area between culture con-ditions (data not shown).

According to cell proliferation and cell morphometric analysis, isoproterenol at a concentration of 2 µg/ml was used in further studies.

Immunocytochemistry

Figure 5 shows the percentage of stained cells after 21 days of culture in the three different media. Ninety-one percent and 83% of cells cultured in 2 µg/ml isoproterenol-supplemented basal medium were posi-tive, respectively, for CKs-4,-5,-6,-8,-10,-13,-18 and for CK-3. Thirty-seven percent of cells cultured in 2 µg/ml isoproterenol-supplemented basal medium were positive for Delta N p63α. Sixty percent and 43% of cells cultured in 2 µg/ml isoproterenol-supplemented basal medium were positive, respectively, for vimentin and CK-19. No significant differences for the various culture conditions were found for the five antigens studied here. The progenitor markers (i.e. Delta N p63α, vimentin, and CK-19) were mainly expressed by small cells.

Flow Cytometry

The analysis of confluent cultures after 3 weeks showed similar Side Scatter (SSC) / Forward Scatter (FSC) scat-ter plots whatever the medium used (Figure 6A). The study cell population featured an FSC value between 250 and 1000 in flow cytometry. For determining the percentage of small cells, a 400-FSC cut-off value was chosen. However, differences were shown when the percentage of small cells was analysed. This figure was, respectively, 77%, 61%, and 58%, with isoproterenol-supplemented basal medium, basal medium, and chol-era toxin-supplemented basal medium.

Figure 6B shows the level of expression of the vari-ous markers assessed by flow cytometry. No significant

FIGURE 4 Mean cell area after 14 and 21 days of culture. The mean cell area at day 14 was significantly lower in isoproterenol-supple-mented medium than in basal culture medium (or cholera toxin-supplemented basal medium). At day 21, no significant differences in cell area could be found between the various culture media. Shown is mean + SEM. *Comparison with basal medium. $Comparison with cholera toxin-supplemented basal medium. NS = not significant (p > 0.05).

FIGURE 5 Expression of CKs-4,-5,-6,-8,-10,-13,-18 (MNF116), CK-3 (AE5), CK-19, vimentin, and Delta N p63α in primary cultured human limbal epithelial cells assessed by immunocytochemis-try. No significant differences in the percentage of stained cells between the three culture media were found for any of the five antibodies (p > 0.1). Shown is mean + SEM.

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differences were found between the three culture media.

Clonal Growth Assay

After 14 days of culture, the average CFE was, respec-tively, 1.09%, 1.05%, and 1.15% for basal medium, cholera toxin-supplemented basal medium, and isoproterenol-supplemented basal medium (p = 0.98) (Figure 7A). After 21 days of culture, no clones were obtained whatever culture medium used (Figure 7B).

RT-PCR

The analysis of the expression profiles for various RNAs (CK-3, CK-19, vimentin, and Delta N p63α) after 21 days of culture showed high levels of expression of the mRNAs encoding CK-3 and vimentin. The expression of mRNA of CK-19 and Delta N p63α was weaker (Figure 8).

FIGURE 6 Flow cytometry of limbal epithelial cells cultured in basal medium, cholera toxin-supplemented basal medium, and isopro-terenol-supplemented basal medium. Similar SSC/FSC scatter plots were found whatever the medium used (A). Expression of CKs-4,-5,-6,-8,-10,-13,-18 (MNF116), CK-3 (AE5), CK-19, vimentin, and Delta N p63α in primary cultured human limbal epithelial cells assessed by flow cytometry (B). No significant differences in the percentage of stained cells between the three culture media were found for any of the five antibodies (p > 0.1). Shown is mean + SEM.

FIGURE 7 Colony-forming assay. Cells cultured for 2 (A) and 3 (B) weeks. Phase contrast image showing one representative colonie (C).

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DISCUSSION

In the explant culture system, the limbal stem cell niche is maintained and limbal fibroblaste may act as feeders.25,31–35

In the present study, immunocytochemistry showed that 20% of cultured cells were negative for pan-cytok-eratins. These cells may correspond to limbal epithelial stem cells and to limbal fibroblasts that may act as feeders.32 The retained stroma appears to preserve some stem-cell niche qualities.

Isoproterenol at 0.5, 1, and 2 µg/ml increased explant outgrowth with a maximum effect obtained at 1 and 2 µg/ml. This effect is consistent with the results of the study published by King et al.23 It was demonstrated by counting cells after 3 weeks of culture. Conversely, cholera toxin did not significantly increase explant out-growth. However, the various culture media assessed in the present study included 10% fetal bovine serum which may mask the mitogenic effect of cholera toxin. Using a serum-free clonal growth assay, Kruse and Tseng previously showed that cholera toxin was mito-genic for corneal and stem cell-containing limbal epi-thelium.17 Measurement of the cell sheet area was not significantly influenced by addition of cholera toxin or isoproterenol to the basal culture medium. However, culture conditions associated with the highest numbers of cells after 3 weeks of culture featured the smallest cell size. This may result in absence of significant dif-ferences in cell sheet area among culture conditions.

Cells cultured with isoproterenol featured lower size. This effect was found after 2 weeks of culture and it appeared to be dose-dependent. After 3 weeks, differ-ences in cell size between culture media did not reach statistical significance. Precise relationship between cell size, cell growth, and cell sheet area could not be obtained in the present study. This would require in situ assessment of the three parameters on the whole cell sheet by the same technique. The confocal microscopy technique used in this study does not permit such an analysis.

Currently, two systems are usually available to promote the growth of limbal epithelial cells: one with the support of human amniotic membrane36,37 and the other with 3T3 fibroblasts co-culture.38,39 Several papers reported the advantages of using human amniotic membrane for the growth of limbal cells; however, besides the limitations to obtain human tissues for cell expansion, the limbal cells growing on the membrane cannot be directly observed. In the present study, cells were grown on plastic which permits assessment of cell morphology.

We used five markers to characterize cell phenotype in culture. Delta N p63α, vimentin, and cytokeratin 19 were used to label progenitors. Epithelial cells were identified by expression of pan-cytokeratin and dif-ferentiated corneal epithelial cells were identified by expression of cytokeratin 3. Cholera toxin and iso-proterenol did not significantly alter cell phenotypes. In addition, the CFE was not influenced by culture medium. Nevertheless, in this work, the phenotypes were assessed after 3 weeks of culture and this could have been in favor of cell differentiation (cytokeratin 3 was expressed under all conditions and assessed by immunocytochemistry, flow cytometry, and RT-PCR). In addition, different regions of the limbus have differ-ent stem cell concentrations. Explants retrieved from the same cornea may feature different numbers of lim-bal stem cells. However, stem cell concentration may also be different according to donors. Unless the exact number of stem cells in the explant at the beginning of culture can be precisely assessed non-invasively, it is not possible, with the explant culture system, to make sure that all culture dishes feature the same number of stem cells at that time.

Vimentin is an intermediate filament that is found in mesenchymal cells other than muscle.40 Lauweryns et al. identified a subpopulation of ‘‘transitional cells’’ in normal limbal tissue, that co-expressed CK-19 and vimentin, and speculated that these might be stem cells.41 Vimentin has been reported to be frequently upregulated in cultured cells.42 We found upregula-tion of vimentin in the epithelial cells of the cultured explants from 3 weeks.

According to the results of the CFE assay, progenitors appear to be better preserved after 2 weeks of culture. Extending the culture time beyond 2 weeks resulted in loss of CFE.

FIGURE 8 Electrophoresis of amplicons obtained after reverse transcription and amplification of mRNAs extracted from cultured cells. Column A, basal medium; column B, cholera toxin-supple-mented basal medium; column C, isoproterenol-supplemented basal medium (2 µg/ml).

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In conclusion, in feeder-free explant culture system, isoproterenol enhances growth of human limbal epithelial cells, it decreases cell size in 2-week cultures, and it allows growth of progenitors (Delta N p63α+/vimentin+/CK19+ small cells). It appears to be more efficient than cholera toxin for enhancing cell growth and decreasing cell size in two-week cultures. Conversely, the percentage of progeni-tors after 3 weeks was not modified by addition of iso-proterenol or cholera toxin to the basal culture medium. Isoproterenol-supplemented medium (2 µg/ml) appears to be useful and safe for growing human limbal epithelial progenitors from limbal explants with no feeders before transplantation to patients with limbal deficiency. Further studies are needed to determine the best culture time.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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