Human Limbal Epithelial Cells Research Articles

Sterility control and long-term eye-bank storage of cultured human limbal epithelial cells for transplantation

Background/aims:To assess sterility of cultured human limbal epithelial cells (HLEC) and to investigate the viability, morphology and phenotype of cultured HLEC following 2 and 3 weeks of organ culture storage.Methods:HLEC...

Utilization of human limbal mesenchymal cells as feeder layers for human limbal stem cells cultured on amniotic membrane

Various cell culture techniques for limbal epithelial cells are currently being used for the transplantation of cultured limbal stem cells. In this study, we explored the possibility of using human limbal mesenchymal cells (HLMCs) as feeder layer for the human limbal epithelial cells (HLECs). Single cell suspension of HLECs was seeded onto denuded amniotic membranes with inactivated 3T3 fibroblasts or HLMCs as feeder layer. Expressions of Cytokeratin 3, Np63 and connexin 43 (Cx43) of the cultured epithelial cells were determined at 28 days and the ultrastructure of the epithelium was examined by transmission electron microscope after 14 days and 28 days of cultivation. In both groups, cells were differentiated into multilayer epithelium at 28 days. Basal cells of the cultured epithelium showed a strong nuclear labeling of Np63, but lacked CK3 and Cx43 expression. Transmission electron microscopy examination showed that there were abundant desmosomal contacts between the cells. The key feature the cultured epithelium was occurrence of a typical basement membrane. These results suggested that HLMCs can be used as an alternative feeder layer for HLECs, which makes the bioengineering product biologically safer for the clinical applications.

F.125. Effects of Dialyzable Leukocyte Extracts on VEGF and IL-17 Secretion by Human-limbal Epithelial Cells

F.125. Effects of Dialyzable Leukocyte Extracts on VEGF and IL-17 Secretion by Human-limbal Epithelial Cells

S.91. Changes in the Proinflammatory Cytokine Profile in Human Limbal Epithelial Cells Treated with Dialyzable Leukocyte Extracts

S.91. Changes in the Proinflammatory Cytokine Profile in Human Limbal Epithelial Cells Treated with Dialyzable Leukocyte Extracts

Expression of Membrane-Associated Mucins in Limbal Stem Cell Deficiency and after Transplantation of Cultivated Limbal Epithelium

Purpose: Membrane-associated mucins are important components of the ocular tear film. Our objective was to characterize the expression of membrane-associated mucins MUC1, MUC4, and MUC16 in healthy cornea, healthy conjunctiva, fibrovascular tissue (pannus) covering the corneal surface in limbal stem cell deficiency (LSCD), human limbal epithelial cells cultivated on intact amniotic membrane (HLEC-AM), and corneal epithelium after transplantation of cultivated limbal epithelium (TCLE). Methods: Total RNA was isolated from six samples of healthy human cornea, healthy conjunctiva, pannus, limbal epithelial cell cultures, and four corneal samples after TCLE. The expression of MUC1, MUC4, and MUC16 was evaluated by real-time polymerase chain reaction (PCR), Western blotting, and immunofluorescence. Results: Conjunctiva showed a significantly higher expression of MUC1 and MUC4 than cornea, but the expression of MUC16 was similar in both tissues. The expression of MUC1 in HLEC-AM increased, MUC16 decreased, and MUC4 showed no significant difference when compared to cornea. The expression of all studied mucins in conjunctiva was significantly higher than in HLEC-AM. Pannus revealed a similar expression pattern of membrane-associated mucins to conjunctiva. The expression of all studied mucins in pannus was significantly higher than in cornea or HLEC-AM. A corneal expression pattern of membrane-associated mucins was found in all four samples of cornea after TCLE. Conclusion: A tissue-specific expression of MUC1 and MUC4 but not MUC16 was found in human cornea and conjunctiva. LSCD led to a conjunctival expression pattern of mucins on the corneal surface. A corneal phenotype could be restored after TCLE, confirming the potential of this method to reconstruct the corneal surface.

Correlation of Cell Cycle Kinetics with p63 Expression in Human Limbal Epithelial Cells Expanded on Intact Human Amniotic Membrane

Aim: To analyse the correlation of p63 expression and cell cycle kinetics of human limbal epithelial cells (HLECs) expanded on amniotic membrane (AM) or plastic. Methods: Primary HLECs were cultured either on cryopreserved intact AM or plastic dishes for 2 weeks. Cells were labelled with 5 μM 5-bromo-2′-deoxyuridine (BrdU) for 3 days, followed by an interval of either 7 or 14 days in BrdU-free medium. The expression of p63 and BrdU labelling was detected by immunocytochemistry. Results: More cells on AM (56%) than on plastic (24%) retained their BrdU label after the 7-day interval (p < 0.001). The difference was even more pronounced after 14 days (20 and 2%, p < 0.001). All BrdU-labelled cells were also p63 positive. 2.5-fold more cells on AM (54%) than on plastic (21%) were BrdU positive/p63 positive after 7 days (p < 0.001). It increased to a 9-fold difference after 14 days (p < 0.001). The BrdU label was lost more quickly than the p63 expression during the observation period, indicating that p63 expression was not confined to stem cells but existed also in transient amplifying cells. Conclusions: The combination of p63 expression and BrdU label retention is a better criterion to characterize stemness than either marker alone. AM as a culture substrate preserves stemness better than plastic.

Bovine Lactoferrin Stimulates Human Corneal Epithelial Alkali Wound Healing In Vitro

The purpose of this study was to investigate the effect of bovine lactoferrin (BLF) on human corneal epithelial wound healing using an in vitro alkali-induced wound model and to understand its role in promoting wound healing. Confluent human corneal limbal epithelial (HCLE) cells wounded using 0.5 microL of 0.1 M sodium hydroxide were treated with BLF (0, 0.1, 1, 2.5, and 5 mg/mL) or anti-human interleukin-6 (IL-6) receptor neutralizing antibody (anti-IL-6 antibody; 1, 10, and 50 microg/mL) or tyrphostin AG1295 (an inhibitor of platelet-derived growth factor [PDGF] receptor kinase; 1 and 10 microM), IL-6, or PDGF-BB. The conditioned medium collected for BLF treatment (0 and 5 mg/mL) was analyzed using a protein array for a number of cytokines/growth factors involved in corneal wound healing. A preliminary animal study using mice was carried out to determine the effect of BLF on alkali wounds. BLF at 2.5 and 5 mg/mL promoted wound healing (P<0.01). During wound closure, BLF upregulated PDGF-BB 180-fold and IL-6 10-fold compared with control. Treatment with tyrphostin AG1295 (10 microM; P<0.01) or anti-IL-6 antibody (50 microg/mL; P<0.01) in the presence of BLF inhibited wound closure, whereas the addition of exogenous IL-6 and PDGF-BB promoted wound closure. Preliminary animal studies have shown that BLF (5 mg/mL) promotes alkali wound healing in vivo. These results suggest that BLF at >or=2.5 mg/mL stimulates HCLE wound healing, and this stimulation is mediated through the upregulation of PDGF or IL-6.

Localization of the low‐affinity nerve growth factor receptor p75 in human limbal epithelial cells

Biological effects of nerve growth factor (NGF) are mediated through receptors known as nerve growth factor receptors (NGFR), which include p75 and TrkA. This study was initiated after identifying NGFR as an up-regulated gene in the limbus by cDNA microarray analysis and we postulate that its expression may be indicative of a stem/progenitor cell phenotype. Immunohistochemistry was performed on normal human adult (n= 5) and foetal (n= 3) corneal tissue using antibodies directed against p75, TrkA, NGF, p63, ABCG2 and CK3/12. Limbal, conjunctival and pterygium tissue was obtained from patients (n= 10) undergoing pterygium resection and used for immunohistochemical assessment. Paraffin-embedded archival human skin specimens (n= 4) were also evaluated. In vitro expression of NGFR was determined in limbal, conjunctival and pterygium-derived epithelial cells. p75 was selectively expressed by basal epithelial cells in pterygia, conjunctiva and limbus, but was absent in the central cornea. These results were confirmed with two additional p75 specific antibodies. In contrast, TrkA was found in full-thickness pterygium, conjunctival, limbal and corneal epithelium in both adult and foetal eyes. p75 expression was identified in a small percentage, while TrkA was found on the entire population of cultured conjunctival, limbal and pterygium-derived epithelial cells. This receptor was also observed in selective regions of the human epidermis and hair follicle bulge. Our results illustrate the selective expression of p75 in basal pterygium, conjunctival and limbal epithelium, while staining was absent in adult and foetal central cornea. p75 may represent an additional ocular surface epithelial stem/progenitor cell signature gene.

A Subset of Human Limbal Epithelial Cells With Greater Nucleus-to-Cytoplasm Ratio Expressing High Levels of p63 Possesses Slow-Cycling Property

The purpose of this study was to evaluate the subset of limbal epithelial cells with greater nucleus-to-cytoplasm (N/C) ratio expressing high levels of p63 for their slow-cycling property, a characteristic feature of stem cells (SCs). Limbal and peripheral corneal explant cultures were pulse labeled with 5-5-bromo-2'-deoxyuridine (BrdU) for 5 days, followed by a period of 3-week chase. Cultured explants were cryosectioned and stained for BrdU. The epithelial cells in the outgrowth and those remaining on the explant were isolated and subjected to cytospin and double immunostaining for BrdU and p63, followed by identification of label-retaining cells (LRCs) and quantification of p63 expression using confocal microscopy. A distinct population of small cells with large N/C ratio expressing high levels of p63 retained the BrdU label after 21-day chase. Further, this population of LRCs, negative for the differentiation marker K3, was observed in the epithelial outgrowth of limbal but not in that of peripheral cornea. LRCs were seen to migrate along the cut edge of limbal explants in culture and were also observed as clusters of small cells in the outgrowth, which contained cells with the ability to form holoclone colonies. These results demonstrate that the small cells with large N/C ratio and high levels of p63 have BrdU label retaining slow-cycling property, thus confirming that these 2 parameters in combination may serve as a precise marker for identification and quantification of ex vivo-expanded limbal SCs. This method would be useful to standardize the optimal culture conditions that can maintain and expand SCs for therapeutic applications.

The effect of amniotic membrane preparation method on its ability to serve as a substrate for the ex-vivo expansion of limbal epithelial cells

Human amniotic membrane (HAM) is employed as a substrate for the ex-vivo expansion of limbal epithelial cells (LECs) used to treat corneal epithelial stem cell deficiency in humans. The optimal method of HAM preparation for this purpose is unknown. This study evaluated the ability of different preparations of stored HAM to serve as substrates for LEC expansion ex-vivo. The effect of removing the amniotic epithelial cells (decellularisation) from HAM prior to seeding of LECs, the effect of glycerol cryopreservation and the effect of peracetic acid (PAA) sterilization and antibiotic disinfection were evaluated using different HAM test groups. Human LECs were cultured on each preparation and the following outcomes were assessed: confluence of growth, cell density, cell morphology and expression of the putative LESC markers deltaN-p63alpha and ABCG2. Removing amniotic epithelial cells prior to seeding of LECs resulted in a higher percentage of confluence but a lower cell density than intact HAM suggesting that decellularisation does not increase proliferation, but rather that it facilitates migration of LECs resulting in larger cells. Decellularisation did not affect the percentage of cells expressing the putative LESC markers deltaN-p63alpha (≤4% in both intact and acellular groups) and ABCG2 (≤3% in both intact and acellular groups). Glycerol cryopreservation of HAM resulted in poor morphology and a low proportion of cells expressing deltaN-p63alpha (≤6%) and ABCG2 (≤8%). HAM frozen at −80 °C in Hank's Balanced Salt Solution (HBSS) was superior, demonstrating excellent morphology of cultured LECs and high levels of deltaN-p63alpha (≤68%) and ABCG2 (≤62%) expression ( p < 0.001). The use of PAA or antibiotics to decontaminate HAM does not appear to affect this function. The variables affecting the ability of HAM to serve as a substrate for LEC expansion ex-vivo are poorly understood. The use of glycerol as a cryoprotectant impairs this ability whereas simple frozen HAM appears to work extremely well for this purpose.

Ophthalmic formulations of the intraocular hypotensive melatonin agent 5-MCA-NAT

Melatonin is a hormone responsible for the regulation of circadian and seasonal rhythms. This hormone is synthesised in many tissues in the body including the eye, where it regulates important processes. During the recent years, the role of melatonin in the control of IOP has been investigated and it has been demonstrated that melatonin receptors are present and involved in the dynamics of the aqueous humour. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a selective MT3 melatonin receptor agonist. Topical application of this product produces a clear reduction in intraocular pressure (IOP) in New Zealand white rabbits and in glaucomatous monkeys. In this work, the potent ocular hypotensive 5-MCA-NAT has been dissolved in excipients used in currently marketed drug formulations. Until now, this melatonin analogue had been dissolved in either DMSO or ethanol neither of which is suitable for ocular topical application in humans. Solubility assays in the different solvents were performed by the observation of the presence of drug crystals under optical microscopy. 5-MCA-NAT was completely dissolved in propylene glycol (PG) and polyethylene glycol 300 (PEG 300) within 24h. Ophthalmic formulations were prepared from different ratios of PG:PBS and the commercialized Systane® product. Quantification of 5-MCA-NAT in the vehicles was assessed by HPLC. In vitro cytotoxicity of the formulations was evaluated by the MTT method and in vivo tolerance of 5-MCA-NAT in the solvents was analyzed by biomicroscopy and specular microscopy. Systane® and proportions of PG:PBS up to 10% of PG did not show cytotoxicity in human corneal limbal epithelial cells (HCLE). In vivo experiments showed that the higher the ocular tolerance, the less amount of PG present. The ocular hypotensive effect of 5-MCA-NAT dissolved in the new formulations was checked measuring IOP for 8h after instillation of the substance. The best effect lowering IOP was obtained with 5-MCA-NAT dissolved in PG and diluted with PBS (PG 1.43%) in which 5-MCA-NAT produced a reduction of 28.11±2.0% and the effect lasted about 7h. In conclusion, new formulations accepted for ocular topical treatments different from DMSO or ethanol were capable of dissolving the melatonin analogue 5-MCA-NAT, preserving its ocular hypotensive ability. Therefore, the use of 5-MCA-NAT may be possible in the treatment of ocular hypertension and glaucoma.

Recent Advances in Cultivated Epithelial Transplantation

Currently, cultivated epithelial transplantation usually uses ex vivo-expanded epithelial cells, with or without biological carriers. Our group has carried out approximately 100 such procedures and has conducted research in this area. The results of a retrospective study indicate that cultivated limbal epithelial transplantation with amniotic membrane (AM) and airlifting may be beneficial at avoiding sight-threatening complications in patients with severe chronic cicatricial keratoconjunctivitis. Our experience in cultivated oral mucosal epithelial transplantation indicates that this technique is useful in achieving a stable ocular surface. However, the treatment for severe ocular surface diseases such as Stevens-Johnson syndrome remains unsatisfactory. In addition to using epithelial sheets with AM, we have developed a technique for generating carrier-free sheets using fibrin sealants. These sheets seem to contain more differentiated epithelium than those obtained with AM while retaining similar levels of colony-forming progenitor cells. A clinical trial of this technique is currently underway. We have also generated epithelial sheets using as biological carrier silk fibroin film, which offers a more transparent medium than conventional sheets. In terms of isolation and cultivation of corneal epithelial stem/progenitor cells, we found that single murine limbal cells exhibited clonal growth and generated stratified epithelial sheets. We also reported that hypoxic culture (2%) enhanced proliferation in human limbal epithelial cells while inhibiting differentiation. This technique may help maintain progenitor cells during ex vivo expansion of epithelial cells. Although these advances are expected to improve clinical outcomes in patients with ocular surface disorders, further improvements, such as the development of cultivation methods that do not require 3T3 feeder cells or real-time assessment of cultivated sheets, are required in the near future.

PHEMA Hydrogels Modified through the Grafting of Phosphate Groups by ATRP Support the Attachment and Growth of Human Corneal Epithelial Cells

Converting the surface of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel into a cell-adhesive surface has been successfully achieved through a method based on atom transfer radical polymerization (ATRP) grafting. Following activation of the surface hydroxyl groups of PHEMA by bromination, surface-initiated ATRP of mono(2-methacryloyloxyethyl) phosphate (MMEP) was conducted in a methanol-water system with Cu(I)Br as catalyst at room temperature. The conversion of PHEMA hydroxyl groups to brominated isobutyryl groups and the occurrence of grafting of PMMEP were confirmed by infrared and X-ray photoelectron spectroscopies. Cell attachment experiments were conducted by culturing human corneal limbal epithelial cells on the PMMEP-grafted PHEMA, and on unmodified PHEMA and tissue culture plastic for comparison. The results showed that the grafted PMMEP was homogeneously distributed, and the phosphate groups appeared to significantly promote the attachment, spreading and growth of cells, at a level comparable to the tissue culture plastic.

NFAT5 Induction and Its Role in Hyperosmolar Stressed Human Limbal Epithelial Cells

To introduce a tonicity response gene regulator, NFAT (nuclear factor of activated T-cell)-5 and determine its expression mechanism and specific roles in human limbal epithelial cell (HLECs) subjected to hyperosmolar stress. NFAT5 expression was determined in various hyperosmolar conditions in HLECs by RT-PCR and Western immunoblot analyses. NFAT5 translocation during hyperosmolar stress was observed by immunocytochemistry. NFAT5-related signal transduction activity was measured on the basis of inhibition of NF-kappaB (nuclear factor-kappaB), and MAPK activity. TNF-alpha and IL-1beta, -6, and -8 levels were determined after inhibition of NFAT5 and/or NF-kappaB. Hyperosmotic apoptotic cell death, with or without inhibition of NFAT5, was measured by flow cytometry. NFAT5 was induced and translocated to the nucleus under conditions of hyperosmolar stress. It was inhibited by SB239063, a p38 MAPK inhibitor. Among the inflammatory cytokines induced in hyperosmolar stress conditions, IL-1beta and TNF-alpha levels were significantly reduced after inhibition of NFAT5. Of interest, even after 48 hours of hyperosmolar stress, 45% of HLECs survived. HLEC apoptosis increased markedly as a result of NFAT5 suppression. Moreover, most of the HLECs underwent cell death on dual inhibition of NF-kappaB and NFAT5. NFAT5 is induced and translocates to the nucleus in HLECs undergoing hyperosmolar stress through activation of p38. IL-1 beta and TNF-alpha are induced via NFAT5 activation. Our data collectively indicate that NFAT5 may be an important gene regulator and survival factor in hyperosmolar stressed HLECs.

Bombyx moriSilk Fibroin Membranes as Potential Substrata for Epithelial Constructs Used in the Management of Ocular Surface Disorders

Membranes were prepared from fibroin, a protein isolated from the domesticated silkworm (Bombyx mori) silk, and evaluated as a potential substratum for corneal limbal epithelial cells. These membranes (i.e., B. mori silk fibroin [BMSF] membranes) were cast from dialyzed solutions of fibroin protein (4% w/v) dispensed into 35-mm-diameter culture dishes and dried at room temperature (23-24 degrees C). The resulting material was transparent, easy to handle, and supported levels of human limbal epithelial (HLE) cell growth comparable to that observed on tissue culture plastic. Remarkably, these results were obtained utilizing a commercial serum-free medium (CnT-20) designed for the ex vivo expansion of corneal epithelial progenitor cells. The potential benefits of serum proteins on this culture system were examined through addition of fetal bovine serum (FBS) either to fibroin stocks prior to membrane casting or by supplementation of the CnT-20 medium. Membranes cast in the presence of FBS displayed increasing opacity and induced little change in HLE growth. Supplementation of CnT-20 medium with FBS deterred cell growth on all substrata, including tissue culture plastic control substrata. The remarkable properties of BMSF membranes demonstrated under serum-free conditions warrant investigation of this material as a substratum in the creation of tissue-engineered constructs for the restoration of diseased or damaged ocular surface.

A Putative Role for RHAMM/HMMR as a Negative Marker of Stem Cell-Containing Population of Human Limbal Epithelial Cells

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is, therefore, crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript, we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH(dim) cells expressed significantly higher levels of DeltaNp63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH(bright) cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker, RHAMM/HMMR, which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size, express negligible CK3, have higher levels of DeltaNp63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization. Disclosure of potential conflicts of interest is found at the end of this article.

Efficient Cultivation Conditions for Human Limbal Epithelial Cells

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding casette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Effects of Organ Culture and Optisol-GS Storage on Structural Integrity, Phenotypes, and Apoptosis in Cultured Corneal Epithelium

A previous report has described the use of eye bank storage of cultured human limbal epithelial cells (HLECs) to provide a reliable source of tissue for treating limbal stem cell deficiency. In the present study, conventional organ culture (OC) storage and Optisol-GS (Bausch & Lomb, Irvine, CA) storage of cultured HLECs were compared. Three-week HLEC cultures were either organ cultured at 31 degrees C or 23 degrees C or stored in Optisol-GS at 5 degrees C in a closed container for 1 week. Morphology was studied by light microscopy and transmission electron microscopy, and phenotypic characterization was assessed by immunohistochemistry. Apoptosis was evaluated by real-time RT-PCR microarray analysis, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). The ultrastructure was preserved at 23 degrees C, while storage at 31 degrees C and 5 degrees C was associated with enlarged intercellular spaces, separation of desmosomes, and detachment of epithelial cells. Cultured HLECs remained undifferentiated in all storage conditions. The expression of the antiapoptotic gene BCL2 was prominently upregulated in storage at 23 degrees C and 5 degrees C. Downregulation of BCL2A1, BIRC1, and TNF and upregulation of CARD6 in 23 degrees C and 5 degrees C storage conditions suggests a reduction in nuclear factor-kappaB activity. No significant increase in cleaved caspase-3 and TUNEL staining was observed in response to eye bank storage, and the labeling indices of cleaved caspase-3 (range, 0.0%-4.7%) and TUNEL (range, 0.0%-7.8%) were low. These data indicate that OC storage of cultured HLECs at ambient temperature is superior to OC storage at 31 degrees C and Optisol-GS storage at 5 degrees C and that apoptosis is minimal after eye bank storage of cultured HLECs.

A Xenobiotic-Free Culture System for Human Limbal Epithelial Stem Cells

Murine 3T3 feeder cells are commonly used for stem cell expansion. Although 'feeder-free' systems are being developed for a variety of stem cells including embryonic, the use of feeder cells currently remains optimal for the expansion of epithelial stem cells. In this study, MRC-5, a human embryonic fibroblast cell line, has been investigated for its potential use as a feeder layer in human limbal epithelial (HLE) cell expansion under serum-free conditions, with the aim of developing a xenobiotic-free culture system for therapeutic corneal regeneration applications. MRC-5 feeder cells were compared with J2 3T3 mouse fibroblasts, in both serum-supplemented and serum-free conditions, in terms of their relative ability to support HLE cell metabolic activity, expression of the putative stem cell markers ABCG2 and P63 alpha, cell differentiation using the cornea-specific cytokeratin 3 antibody and colony-forming efficiency. The proportion of HLE stem cells maintained was determined by functional colony-forming efficiency assays. The metabolic activity results showed that HLE cells cultured on MRC-5 fibroblasts under serum-free conditions proliferated as well as cells cultured on J2 cells under serum-free conditions. Moreover, the HLE cultured on MRC-5 fibroblasts under serum-free conditions expressed high levels of putative stem cell markers ABCG2 and P63 alpha and low levels of the differentiation marker CK3, indicating that they retained poorly differentiated 'stem cell-like' characteristics under those culture conditions. Clonal analysis of HLE cells cultured on growth-arrested feeder layers of J2 and MRC-5 fibroblasts showed that cells expanded on MRC-5 and J2 fibroblasts in serum-free conditions had a colony-forming efficiency of approximately 1.5%, indicating the maintenance of stem cells. These results demonstrate feasibility of expanding HLE cells for clinical purposes by using a human fibroblast cell line as a feeder layer, avoiding the use of bovine serum, while preserving the proliferative potential and stem cell characteristics of HLE cells.

Silk as Substratum for Cell Attachment and Proliferation

Silk fibroin (SF), isolated from silkworm (Bombyx mori) cocoons, is a natural biodegradable polymer. Over the past decade, there was some interest in using SF as a biomedical material. As part of a project to develop tissue-engineered constructs for the surgical restoration of the ocular surface (cornea, conjunctiva), we have investigated the capacity of SF to function as a substratum for the attachment and growth of corneal stem/progenitor cells harvested from the corneoscleral limbus of donor human corneal tissue. SF membranes were produced from cocoons following a protocol involving successive dissolution steps, filtration, dialysis, evaporation, and methanol treatment. Human limbal epithelial cells were harvested from donor tissue and seeded onto SF membranes. After 5 days, the culture was fixed and stained with specific agents to visualize the cells. The study indicated profuse cellular attachment and growth. SF membranes appear to be suitable as a substratum for the repair of damaged ocular surface.