Human Limbal Epithelial Cells Research Articles

Ex vivo expanded autologous limbal epithelial cells on amniotic membrane using a culture medium with human serum as single supplement

In patients with limbal stem cell deficiency (LSCD), transplantation of ex vivo expanded human limbal epithelial cells (HLECs) can restore the structural and functional integrity of the corneal surface. However, the protocol for cultivation and transplantation of HLECs differ significantly, and in most protocols growth additives such as cholera toxins, exogenous growth factors, hormones and fetal calf serum are used. In the present article, we compare for the first time human limbal epithelial cells (HLECs) cultivated on human amniotic membrane (HAM) in a complex medium (COM) including fetal bovine serum to a medium with human serum as single growth supplement (HSM), and report on our first examinations of HLECs expanded in autologous HSM and used for transplant procedures in patients with LSCD. Expanded HLECs were examined by genome-wide microarray, RT-PCR, Western blotting, and for cell viability, morphology, expression of immunohistochemical markers and colony forming efficiency. Cultivation of HLECs in HSM produced a multilayered epithelium where cells with markers associated with LESCs were detected in the basal layers. There were few transcriptional differences and comparable cell viability between cells cultivated in HSM and COM. The p63 gene associated with LESCs were expressed 3.5 fold more in HSM compared to COM, and Western blotting confirmed a stronger p63α band in HSM cultures. The cornea-specific keratin CK12 was equally found in both culture conditions, while there were significantly more CK3 positive cells in HSM. Cells in epithelial sheets on HAM remaining after transplant surgery of patients with LSCD expressed central epithelial characteristics, and dissociated cells cultured at low density on growth-arrested fibroblasts produced clones containing 21 ± 12% cells positive for p63α (n = 3). In conclusion, a culture medium without growth additives derived from animals or from animal cell cultures and with human serum as single growth supplement may serve as an equivalent replacement for the commonly used complex medium for ex vivo expansion of HLECs on HAM.

Molecular profile of organ culture-stored corneal epithelium: Lgr5 is a potential new phenotypic marker of residual human corneal limbal epithelial stem cells

Long-term preservation of corneal limbal epithelium may decrease its quality and change the molecular signature of the limbal epithelial stem cells. In this study we have investigated the molecular profile of isolated corneal epithelial cells that have been in storage for an extended time. Isolated cells were characterised by the expression profile of different cytokeratins and markers of squamous metaplasia (vimentin and α‑actin). Furthermore, we examined global markers of adult stem cells including p63α and ABCG2 but also LGR5 as a novel stem cell marker. Immunocytochemical staining and PCR analysis of p63α, ABCG2 and LGR5 revealed the existence of side-population cells with a stem-cell phenotype and maintenance of corneal limbal stem cell properties. LGR5 expression can be related to cellular stemness and can be considered as a new phenotypic marker of residual human corneal limbal stem cells. However, the existence of CK10 together with co-expressed α-actin and vimentin suggests that the corneas investigated were under oxidative stress and showed evidence of squamous metaplasia.

Inhibition of Sevoflurane Postconditioning Against Cerebral Ischemia Reperfusion-Induced Oxidative Injury in Rats

The volatile anesthetic sevoflurane is capable of inducing preconditioning and postconditioning effects in the brain. In this study, we investigated the effects of sevoflurane postconditioning on antioxidant and immunity indexes in cerebral ischemia reperfusion (CIR) rats. Rats were randomly assigned to five separate experimental groups I–V. In the sham group (I), rats were subjected to the same surgery procedures except for occlusion of the middle cerebral artery and exposed to 1.0 MAC sevoflurane 90 min after surgery for 30 min. IR control rats (group II) were subjected to middle cerebral artery occlusion (MCAO) for 90 min and exposed to O2 for 30 min at the beginning of reperfusion. Sevoflurane 0.5, 1.0 and 1.5 groups (III, IV, V) were all subjected to MCAO for 90 min, but at the beginning of reperfusion exposed to 0.5 MAC, 1.0 MAC or 1.5 MAC sevoflurane for 30 min, respectively. Results showed that sevoflurane postconditioning can decrease serum tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), nitric oxide (NO), nitric oxide synthase (NOS) and increase serum interleukin-10 (IL-10) levels in cerebral ischemia reperfusion rats. In addition, sevoflurane postconditioning can still decrease blood lipid, malondialdehyde (MDA) levels, infarct volume and increase antioxidant enzymes activities, normal pyramidal neurons density in cerebral ischemia reperfusion rats. It can be concluded that sevoflurane postconditioning may decrease blood and brain oxidative injury and enhance immunity indexes in cerebral ischemia reperfusion rats.

Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23 °C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23 °C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell–cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03 ± 0.38 microvilli/μm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69 ± 0.54 microvilli/μm; P = 0.98 and 0.89 ± 1.0 microvilli/μm; P = 0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07 ± 1.0 microvilli/μm; P = 0.47 and 0.07 ± 0.07 microvilli/μm; P = 0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4 ± 0.3 cell layers, as opposed to 4.0 ± 0.9 cell layers; P = 0.89 after 4 days of HEPES-MEM storage and 2.8 ± 0.6 cell layers; P = 0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7 ± 0.2 cell layers; P = 0.46 and 3.4 ± 0.4 cell layers; P = 0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4% ± 3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1% ± 4.5%; P = 0.99 and 85.1% ± 13.7%; P = 0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7% ± 15.2%; P = 0.97 and 79.8% ± 15.7%; P = 0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23 °C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

A Novel Mechanism of Increased Infections in Contact Lens Wearers

It is well documented that contact lens wearers have much higher incidences of corneal infections compared with those of non-contact lens wearers, although the exact cause(s) of this increased susceptibility has not been identified. A distinct subset of mucins (MUCs) is present on the ocular surface, acting to protect the integrity of the corneal epithelium. This study was performed to determine whether multipurpose contact lens solutions (MPCLSs) can cause increased infections in the cornea by destroying the protective cell-bound mucin layer. An immortalized human corneal limbal epithelial cell line was treated in the presence of four commonly used MPCLSs or PBS and the expression and release of MUC-16 was assessed. Cells were also cultured with Pseudomonas aeruginosa after MPCLS treatment and internalization of bacteria was assessed by quantitative genomic PCR. Loss of MUC-16 was then correlated with infection rates. Each of the four commonly used MPCLSs examined in this study differentially affected mucin release. The relative effect was correlated with an increase in infection of corneal epithelial cells by P. aeruginosa. The results of this study are consistent with the hypothesis that MPCLSs cause increased infections in the cornea by destroying the protective cell-bound mucin layer.

Effect of Retinoic Acid on Epithelial Differentiation and Mucin Expression in Primary Human Corneal Limbal Epithelial Cells

Purpose: Retinoic acid (RA) is essential for epithelial differentiation and maintenance of the mucous phenotype. This study investigated the effect of RA on corneal epithelial differentiation and mucin expression in a primary human corneal limbal epithelial cell (HCLEC) culture model.Methods: HCLECs were grown in RA-supplemented media at various concentrations (0, 10−9 to 10−6 M). Stratified HCLECs were examined using immunohistochemical or immunofluorescent staining for p63, ABCG2, CK3, CK19, and Western blotting for ABCG2 and CK12 to assess differentiation. Ultrastructural morphology was investigated using scanning and transmission electron microscopy. They were incubated with rose bengal dye to examine barrier function. The effects of RA on the expression of MUC1, -4, and -16 were analyzed by immunohistochemistry, quantitative real-time PCR and Western blot analysis.Results: HCLEC grown without RA showed hyperkeratosis, whereas those grown with 10−8 to 10−7 M RA induced non-keratinized stratified epithelium with a normal appearance. Under these conditions, p63, ABCG2, CK3, CK19, MUC1, -4, and -16 staining patterns were similar to in vivo limbal epithelium. A higher concentration (10−6 M) of RA resulted in abnormal differentiation. HCLECs grown with RA were tightly apposed and maintained intact barrier function against dye penetration. In addition, MUC1, -4, and -16 expressions were highly associated with RA concentrations.Conclusions: This study showed that cultured HCLEC could mimic physiologic and functional phenotypes by controlling RA concentrations in medium. Also, our results suggested modulating effect of RA on differentiation and mucin expression in corneal epithelium.

Stratified corneal limbal epithelial cells are protected from UVB-induced apoptosis by elevated extracellular K+

The goal of this study was to determine whether elevated [K+] protects stratified corneal epithelial cells from entering apoptosis following exposure to ambient levels of UVB radiation. Human corneal limbal epithelial (HCLE) cells were stratified to form multilayered constructs in culture. The cells were exposed to UVB doses of 100–250 mJ/cm2 followed by incubation in medium with 5.5–100 mM K+. The protective effect of K+ was determined by measuring the caspase-3 and -8 activity and TUNEL staining of the stratified HCLE constructs. In response to UVB exposure, activation of apoptotic pathways peaked at 24 h. Caspase-8 in stratified cells was activated by exposure to UVB at 100–250 mJ/cm2, and activity was significantly reduced in response to 50 or 100 mM K+. Caspase-3 was activated in the stratified cells in response to 100–250 mJ/cm2 UVB and showed a significant reduction in activity in response to 25, 50 or 100 mM K+. DNA fragmentation, as indicated by TUNEL staining, was elevated after exposure to 200 mJ/cm2 UVB, and decreased following incubation with 25–100 mM K+. These results show that in a culture system that models the intact corneal epithelium, elevated extracellular K+ can reduce UVB-induced apoptosis which is believed to be initiated by loss of K+ from cells. This is the basis of damage to the corneal epithelium caused by UVB exposure. Based on these observations it is suggested that the relatively high K+ concentration in tears (20–25 mM) may play a role in protecting the corneal epithelium from ambient UVB radiation.

Laser scanning confocal microscopy-assisted obtain of limbal tissue for the ex vivo culture of human limbal epithelial stem cells and identification

Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane Key words: Cornea/limbus ; stem cells ; Laser scanning confocal microscopy; Cell culture; Markers

Effect of Sub-Atmospheric Oxygen on the Culture of Rabbit Limbal Epithelial Cells

Purpose: To investigate the effect of sub-atmospheric oxygen tensions on the maintenance and expansion of limbal epithelial cells in vitro.Materials and Methods: Limbal epithelial cells were isolated from rabbit corneas and cultured in 21%, 14%, 8%, and 2% oxygen in a co-culture system with Mitomycin C growth arrested 3T3 fibroblasts. The colony forming efficiency, proliferative ability, and cell cycle distribution of cells cultured in these different oxygen tensions were determined, and semi-quantitative RT-PCR was used to detect expression of putative limbal epithelial stem cell markers, such as ABCG2 and p63α.Results: Of the four different oxygen tensions studied, 14% and 2% supported the highest and lowest colony forming efficiency values, respectively. A greater proportion of cells were found in S and G2/M phases of the cell cycle in primary 14% and 8% oxygen cultures compared to atmospheric controls. Differentiation was promoted at oxygen tensions of 8% and below. Cells with a differentiated phenotype and limited proliferative capacity were observed in 2% oxygen. Semi-quantitative RT-PCR analysis showed that the putative limbal epithelial stem cell markers ABCG2 and p63α were expressed in 21%, 14%, and 8% oxygen, with a trend of lower expression in 8% oxygen being observed.Conclusions: Limbal epithelial cells are sensitive to in vitro changes in oxygen tension. A sub-atmospheric oxygen tension of 14% promoted the maintenance and expansion of cells with limbal epithelial stem cell characteristics in a feeder co-culture system and is, therefore, recommended for the culture of rabbit limbal epithelial cells. This may also have relevance for the culture of human limbal epithelial stem cells for therapeutic application.

Epidermal Differentiation and Loss of Clonal Growth Potential of Human Limbal Basal Epithelial Progenitor Cells during Intrastromal Invasion

Intrastromal invasion by limbal basal epithelial progenitor cells in explant cultures is associated with epithelial-mesenchymal transition. It remains unclear whether intrastromal invasion is contingent on culturing conditions and whether invaded cells retain their progenitor status and original lineage. Human limbal explants were cultured on various culture substrates, with or without air-lifting (AL), and subjected to hematoxylin and eosin staining and immunostaining to pan-cytokeratins, p63α, ΔNp63, Pax6, CK10, and CK12. Single cells obtained by trypsin/EDTA from dispase-isolated epithelial sheets from both the outgrowth and the surface epithelium, or by collagenase from the remaining stroma, were seeded on 3T3 feeder layers. Intrastromal invasion was verified in all seven explant cultures by positive pan-cytokeratin staining. Immunofluorescence staining revealed that invaded epithelial cells were positive for p63α and ΔNp63, with or without nuclear staining of Pax6. Double immunostaining to CK10 and CK12 revealed that squamous metaplasia induced by AL was noted on the surface epithelium but not in intrastromally invaded epithelial cells. On 3T3 feeder layers, both the outgrowth and the surface epithelium yielded significant numbers of holoclones and meroclones positive to ΔNp63 but negative to CK10 and CK12. In contrast, intrastromally invaded epithelial cells generated only paraclones negative to ΔNp63 and CK12 but positive to CK10 regardless of culturing conditions. Intrastromal invasion by limbal basal epithelial progenitor cells is universal in all explant culture conditions, explaining why there is a gradual decline of outgrowth potential. Alteration of the limbal stromal niche leads invaded epithelial cells to adopt an epidermal fate.

A Novel Cell-Associated Protection Assay Demonstrates the Ability of Certain Antibiotics To Protect Ocular Surface Cell Lines from Subsequent Clinical Staphylococcus aureus Challenge

In vivo effectiveness of topical antibiotics may depend on their ability to associate with epithelial cells to provide continued protection, but this contribution is not measured by standard antibiotic susceptibility tests. We report a new in vitro method that measures the ability of test antibiotics azithromycin (AZM), erythromycin (ERY), tetracycline (TET), and bacitracin (BAC) to associate with mammalian cells and to protect these cells from destruction by bacteria. Mammalian cell lines were grown to confluence using antibiotic-free medium and then incubated in medium containing a single antibiotic (0 to 512 μg/ml). After incubation, the cells were challenged with Staphylococcus aureus ocular isolates, without antibiotics added to the culture medium. Epithelial cell layer integrity was assessed by gentian violet staining, and the minimum cell layer protective concentration (MCPC) of an antibiotic sufficient to protect the mammalian cells from S. aureus was determined. Staining was also quantified and analyzed. Bacterial viability was determined by culture turbidity and growth on agar plates. Preincubation of Chang and human corneal limbal epithelial cells with AZM, ERY, and TET at ≥64 μg/ml provided protection against AZM-susceptible S. aureus strains, with increasing protection at higher concentrations. TET toxicity was demonstrated at >64 μg/ml, whereas AZM displayed toxicity to one cell line at 512 μg/ml. BAC failed to show consistent protection at any dose, despite bacterial susceptibility to BAC as determined by traditional antibiotic susceptibility testing. A range of antibiotic effectiveness was displayed in this cell association assay, providing data that may be considered in addition to traditional testing when determining therapeutic dosing regimens.

Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes

Membranes prepared from a protein, fibroin, isolated from domesticated silkworm ( Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and distribution of ΔNp63 + progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.

Comparação entre membrana amniótica com e sem epitélio como substrato para cultura de células epiteliais do limbo ex vivo

To evaluate the efficacy and ultrastructural aspects of human limbal epithelial cells cultured on amniotic membrane (AM) with and without epithelium. Limbal epithelial cell cultures were established from cadaveric cor neo-scleral rim explants derived from 6 different donors. The explants from each donor were placed under 3 different groups: on human preserved AM with epithelium (Group 1), AM deepithelialized with trypsin (Group 2) and control (Group 3). The epithelial cell migration was evaluated under phase contrast microscopy. After 15 days, the amniotic membrane with cells cultures were removed and submitted to scanning and transmission electron microscopy to check for epithelial migration and adhesion. All epithelial cell cultures from the controls grew over the botton of the culture plate wells until reaching confluence. Epithelial cultures grew over all but one denuded amniotic membrane. In the group amniotic membrane with epithelium, epithelial cell growing was observed only in 1 well. Using this model, denuded amniotic membrane appeared to be the best substrate for epithelial cell migration and adhesion comparing to amniotic membrane with epithelium. Removal of amniotic membrane epithelial seems to be an important step for establishing limbal epithelial cell culture on amniotic membrane.

Potassium ion fluxes in corneal epithelial cells exposed to UVB

The goal of this study was to investigate the efflux of K+ from human corneal limbal epithelial cells (HCLE) exposed to ambient levels of UVB, which is known to cause apoptosis, and to examine the effect of K+ channel blockers on loss of potassium induced by UVB. HCLE cells were exposed to 100–200 mJ/cm2 UVB, followed by incubation in culture media with 5.5–100 mM K+, BDS-1, Ba2+ or ouabain. To measure intracellular cations, cells were washed in 280 mM sucrose and lysed in DI water. K+ and Na+ levels in lysates were measured by ion chromatography. HCLE cells showed maximal loss of K+i 10 min after exposure to UVB and 5.5 mM K+ media, with recovery of normal K+ levels after 90 min. Treatment with 1 μM BDS-1 following UVB exposure reduced the loss of K+ by HCLE cells. Exposure to 0.1–5 mM Ba2+ inhibited UVB-induced K+ loss in a time and dose-dependent manner. These results confirm that blocking K+ channels in HCLE cells exposed to UVB prevents efflux of K+, confirming that UVB activates K+ channels in these cells. Electrophysiology data show that K+ channels remain highly active at least 90 min after UVB exposure. HCLE cells exposed to UVB and incubated in 0.01–1 μM ouabain did not recover from UVB-induced K+ loss. These data suggest that the Na/K pump may act to restore [K+]i to control levels in HCLE cells following UVB exposure and that the pump is not damaged by exposure to UVB. Incubation of HCLE cells exposed to UVB in medium with 25–100 mM K+ media prevented K+ efflux at extracellular concentrations as low as 25 mM (the concentration in tear fluid), maintaining control levels of K+i. In all experiments inward fluxes and intracellular Na+ levels mirrored K+ changes, albeit at the expected lower concentrations. The prevention of UVB-induced Ki+ loss by 25 mM Ko+ is consistent with the possible contribution of the relatively high K+ concentration in tears to protection of the corneal epithelium from ambient UVB.

Cell Delivery with Fixed Amniotic Membrane Reconstructs Corneal Epithelium in Rabbits with Limbal Stem Cell Deficiency

To explore the feasibility and efficacy of a cell delivery system using amniotic membrane (AM) fixed by a novel biomembrane-fixing device (BMFD) for corneal epithelium reconstruction in rabbits with limbal stem cell deficiency (LSCD). Sixty female rabbits with LSCD were created and randomly assigned to three groups of 20 each: LSCD rabbits without treatment (the control), LSCD rabbits treated with BMFD-fixed AM (BMFD-AM), and rabbits treated with male human limbal epithelial cells delivered with BMFD-fixed AM (BMFD-AM+cells). They were followed up with slit lamp observation and corneal fluorescein staining for 14 days. Cytokeratin K3 and K4 and mucin 5AC were used to evaluate corneal conjunctivalization. Sry gene detection was used to trace the delivered cells. The corneal re-epithelialization time was 5.60 ± 0.46 days in the BMFD-AM+cell group, significantly shorter (P < 0.05) than in the LSCD (12.45 ± 0.65 days) and the BMFD-AM (9.25 ± 0.51 days) groups. Conjunctivalization and neovascularization were observed to be severe in the LSCD group and moderate in the BMFD-AM group. The prevention of conjunctivalization in the BMFD-AM+cell group was evidenced by positive K3/K12 and negative MUC5AC and K4 observed on re-epithelialized corneal epithelium. The histologic sections at different time points and positive Sry gene expression indicated that the delivered cells adhered to the wounded corneal surface and proliferated well. These findings demonstrate that the BMFD with fixed AM served well as a cell delivery system for the ocular surface. The delivered limbal epithelial cells promoted corneal re-epithelialization and prevented corneas from conjunctivalization and neovascularization in rabbits with experimental LSCD.

The Porcine Limbal Epithelial Stem Cell Niche as a New Model for the Study of Transplanted Tissue-Engineered Human Limbal Epithelial Cells

Transparency of the human cornea is dependent upon the integrity of its epithelium and hence a population of limbal epithelial stem cells (LESCs). We have previously shown that LESCs reside in limbal epithelial crypts at the periphery of the human cornea. In this study the anatomy and functionality of the porcine limbus was evaluated for the first time as a novel model of the human limbus. Scanning electron microscopy, confocal microscopy, and histology revealed common structures in the porcine and human limbus in terms of the location and topography of palisades of Vogt and limbal epithelial crypts. Epithelial cells harvested from crypt regions achieved higher colony forming efficiency than cultures established from the noncrypt regions and central cornea. Also, expression of the putative SC markers p63α and integrin β1 brightness was higher in the basal layer of the crypt regions, as shown by immunocytochemistry. De-epithelialized porcine corneas were used as an in vitro organ culture model to study the fate of transplanted human epithelium cultured from the limbus. Multilayered epithelium was observed after ∼1 week. Subsequently, wounds were inflicted on the central corneal epithelium. The wounded tissue healed within 5-7 days, and multilayering of the central corneal epithelium was re-established. The transplanted epithelia were repeatedly wounded at least four times and the wounds healed by 1 week. Putative SC marker expression of the transplanted epithelia was confirmed using immunohistochemistry. These results demonstrate that the porcine limbus shares features with the human limbus and as such provides a suitable model for the study of cultured limbal epithelial cell transplantation. These data have significant clinical value as this model can provide information on LESC fate post-transplantation and their ability to respond to injury, which is not possible to study in patients.

An Immunoprotective Privilege of Corneal Epithelial Stem Cells Against Th17 Inflammatory Stress by Producing Glial Cell-Derived Neurotrophic Factor

Adult stem cells are well known for their self-renewal and regenerative capacity. The mechanisms protecting these cells from inflammatory damage have not been well elucidated. This study investigated the immunoprotective properties of corneal epithelial stem cells from inflammation by producing glial cell-derived neurotrophic factor (GDNF). Primary human limbal epithelial cells (HLECs) cultured from limbal explants were treated with interleukin (IL)-17A, tumor necrosis factor (TNF)-α, or hyperosmotic media, with or without GDNF or nuclear factor kappa B (NF-κB) inhibitor (NF-κB-I) for 4-48 hours. Inflammatory mediators and Th17-inducing cytokines were determined by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunobead assays. NF-κB activation was detected by p65 phosphorylation, immunostaining and Western blotting. GDNF and its receptor, GDNF family receptor α-1, were exclusively immunolocalized in the basal layer of limbal epithelium, whereas IL-17 receptor was negative in these cells. Exogenous IL-17A stimulated the expression and production of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and chemokine IL-8 by HLECs. Th17-inducing cytokines, transforming growth factor (TGF)-β1, IL-6, IL-23, and IL-1β, were significantly increased at mRNA and protein levels by HLECs exposed to TNF-α or hyperosmotic media. IL-17 activated NF-κB by p65 phosphorylation at serine 536 and nuclear translocation. GDNF or NF-κB-I blocked IL-17-induced NF-κB p65 activation and production of inflammatory mediators. Furthermore, GDNF suppressed the production of Th17-inducing cytokines through inhibiting NF-κB activation. These findings demonstrate that limbal progenitor cell-produced neurotrophic factor GDNF suppresses IL-17-mediated inflammation via NF-κB signaling pathway. This may represent a unique immunoprotective property of limbal stem cells against inflammatory challenges on the ocular surface.

Critical Appraisal of Ex Vivo Expansion of Human Limbal Epithelial Stem Cells

The stem cells (SCs) of the corneal epithelium located in the limbal basal layer are the ultimate source to maintain corneal epithelial homeostasis. Like other adult tissue-specific SCs, self renewal and fate decision of limbal SCs are regulated by a specialized in vivo microenvironment, termed "niche". Loss of limbal SCs or dysfunction of the limbal niche renders corneas with a unique clinical disease labeled limbal stem cell deficiency (LSCD). Besides transplantation of autologous or allogeneic limbal SCs or amniotic membrane, a new strategy of treating LSCD is to transplant a bio-engineered graft by expanding limbal SCs ex vivo. Herein, we conduct a critical appraisal of six protocols that have successfully been practiced in treating human patients with LSCD, and identify issues whether niche regulation has been disrupted or maintained during isolation and expansion. Consequently, we propose a future direction that may circumvent the potential pitfalls existing in these conventional protocols by preserving the interaction between limbal SCs and their native niche cells during isolation and expansion. Such an approach may one day help realize considerable promise held by adult SCs in treating a number of diseases.

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

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

The Antigenicity of Ex Vivo Cultivated Human Corneal Limbal Epithelial and Stromal Cells: Temporal Changes In Vitro

To investigate the effect of ex vivo culture on the expression of human leukocyte antigen (HLA) in human corneal limbal epithelial cells (LECs), and to evaluate the expression of HLA and costimulatory molecules on human corneal limbal stromal cells (LSCs). The expression of HLA-ABC and HLA-DR were evaluated using flow cytometry on cultured human LECs and LSCs after serial passage and compared with that on freshly isolated cells. Additionally, the expression of costimulatory molecules CD80, CD86, CD40, and immunoregulatory molecule HLA-G were investigated on LSCs with or without an addition of interferon (IFN) γ (250 U/mL, 4 days). HLA-ABC and HLA-DR were expressed in 99.12 ± 1.02% and 25.86 ± 4.34% of freshly isolated LECs, respectively. After culture, the HLA-DR-expressing cells significantly decreased with serial passage, whereas the percentage of HLA-ABC-expressing cells did not change. More than 90% of LSCs expressed HLA-ABC and HLA-G, whereas the expression of HLA-DR, CD80, CD86, and CD40 was negligible. This expression profile did not change after serial passage. However, the treatment with IFN-γ induced a significant increase in HLA-DR (0.17% vs. 17.68%) and CD40 (0.19% vs. 13.19%) expression on LSCs. The immunogenicity of LECs can be lowered after ex vivo cultivation, whereas LSCs can be immunogenic in the presence of IFN-γ.