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1.  Structure of corneal layers, collagen fibrils, and proteoglycans of tree shrew cornea 
Molecular Vision  2011;17:2283-2291.
The stroma is the major part of the cornea, in which collagen fibrils and proteoglycans are distributed uniformly. We describe the ultrastructure of corneal layers, collagen fibrils (CF), and proteoglycans (PGs) in the tree shrew cornea.
Tree shrew corneas (5, 6, and 10 week old animals) and normal human corneas (24, 25, and 54 years old) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in a sodium acetate buffer. The tissue was processed for electron microscopy. The ‘iTEM Olympus Soft Imaging Solutions GmbH’ program was used to measure the corneal layers, collagen fibril diameters and proteoglycan areas.
The tree shrew cornea consists of 5 layers: the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium. The epithelium was composed of squamous cells, wing cells and basal cells. The Bowman’s layer was 5.5±1.0 µm thick and very similar to a normal human Bowman’s layer. The stroma was 258±7.00 µm thick and consisted of collagen fibril lamellae. The lamellae were interlaced with one another in the anterior stroma, but ran parallel to one another in the middle and posterior stroma. Collagen fibrils were decorated with proteoglycan filaments with an area size of 390 ±438 nm2. The collagen fibril had a minimum diameter of 39±4.25 nm. The interfibrillar spacing was 52.91±6.07 nm. Within the collagen fibrils, very small electron-dense particles were present.
The structure of the tree shrew cornea is very similar to that of the normal human cornea. As is the case with the human cornea, the tree shrew cornea had a Bowman's layer, lamellar interlacing in the anterior stroma and electron-dense particles within the collagen fibrils. The similarities of the tree shrew cornea with the human cornea suggest that it could be a good structural model to use when studying changes in collagen fibrils and proteoglycans in non-genetic corneal diseases, such as ectasia caused after LASIK (laser-assisted in situ keratomileusis).
PMCID: PMC3171502  PMID: 21921979
2.  Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture 
Experimental eye research  2003;77(2):211-217.
The authors have previously documented decreased epithelial basement membrane (BM) components and α3β1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar–collagen gel at the air–liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 α5 chain, nidogen-1/entactin, integrin α3β1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of α3β1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized α3β1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.
PMCID: PMC2909880  PMID: 12873452
diabetic retinopathy; cornea; organ culture; basement membrane; integrin; laminin; nidogen; stromelysin; matrix metalloproteinase; MMP-10; tenascin-C; fibrillin-1; α-enolase; keratin 3
3.  Basement Membrane Dissolution and Reassembly by Limbal Corneal Epithelial Cells Expanded on Amniotic Membrane 
To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM.
Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin α6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography.
All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM.
After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.
PMCID: PMC1569675  PMID: 16723447
4.  Keratoconus in a Cynomolgus Monkey 
In a seven-year-old male cynomolgus monkey, erythema of the upper eyelid and forehead and corneal opacity, edema and conical protrusion in the eye were observed. At necropsy, ophthalmological and serological examinations revealed binocular corneal opacity and conical protrusion and a high IgE level, respectively. Thinning of the epithelium and stroma of the cornea were noted histopathologically. At the center of the corneal epithelium, the number of epithelial cells was reduced, their cytoplasm was poorer and the basal cells were flatter than at the periphery. Bowman’s membrane was folded with partial loss or breakage. Collagen fibers were compacted or disarranged, and the keratocytes were increased in the stroma, with focal pyknosis or loss of the endothelium and folding of Descemet’s membrane. Electron microscopical examination revealed atrophy of the corneal epithelial basal cells. This is the first report of a case of keratoconus in a cynomolgus monkey.
PMCID: PMC3234653  PMID: 22272012
keratoconus; high serum IgE; atopy-like mechanism; cynomolgus monkey
5.  Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types 
Molecular Vision  2010;16:2192-2201.
The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach.
Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed.
Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1.
This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.
PMCID: PMC2994343  PMID: 21139684
6.  The Fate of Limbal Epithelial Progenitor Cells during Explant Culture on Intact Amniotic Membrane 
The clinical success of treating corneas with total limbal stem cell deficiency using limbal biopsy explants cultured on intact amniotic membrane (iAM) relies on ex vivo expansion of limbal epithelial progenitor cells. However, the ultimate fate of limbal epithelial progenitor cells in the explant remains unclear.
Human limbal explants were cultured on iAM for 2 weeks and then removed and transferred to a new iAM until passage 3. The outgrowth surface area of each passage was measured and compared. For each passage, clonogenicity on 3T3 fibroblasts feeder layers was compared among progenitor cells removed from the outgrowth, the explant surface, and the remaining stroma. Cryosections of the explant and the outgrowth were detected with p63, vimentin, pancytokeratin, and the basement membrane components type VII and IV collagen and laminin 5 antibodies.
The outgrowth surface area significantly decreased from passage (P)1 to P3. The total number of epithelial cells that were isolated from the explant surface also decreased from before culture (P0) to P1, became stable from P1 to P2, but was uncountable at P3. Clonogenicity significantly declined from P1 to P3 for the epithelium derived from the explant surface and the outgrowth epithelium; the extent was less in the former than in the latter at P2 and P3. In addition, groups of epithelial cells invaded the limbal stroma of the explants from P1 to P3; p63(+)/pancytokeratin(−) and p63(+)/vimentin(+) cells also presented in the limbal stroma. Increasing fibroblast, but not epithelial, colonies were observed from cells isolated from the remaining limbal stroma when seeded on 3T3 fibroblast feeder layers from P1 to P3.
During ex vivo expansion on iAM, some limbal epithelial progenitor cells indeed migrate onto iAM from the explant surface, whereas some also invade the limbal stroma, very likely undergoing epithelial-mesenchymal transition. This new information should be taken into account in formulating new strategies to improve the expansion protocol.
PMCID: PMC3197022  PMID: 17251456
7.  Decellularized Human Cornea for Reconstructing the Corneal Epithelium and Anterior Stroma 
In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.
PMCID: PMC3338110  PMID: 22082039
A rare, familial early-onset form of Fuchs corneal dystrophy (FCD) is caused by mutation in the COL8A2 gene. This study describes the aberrant pattern of distribution of collagen type VIII and basement membrane components in Descemet’s membrane (DM) and endothelium of three individuals with the same L450W mutation that represent different stages of early-onset FCD.
Immunohistochemical studies with bright field, fluorescence, and confocal microscopy characterized the pathology of sectioned corneal buttons with antibodies against COL8A1, COL8A2, COL4, laminin, and fibronectin. A portion of each was processed for electron microscopy.
Histologic examination of pathologic changes in case 1 demonstrated relative preservation of the endothelium, whereas in case 2 much of this layer was atrophic and in case 3 there was complete loss of the endothelium. DM also increased in thickness to 25 μm for case 1, to 31 μm for case 2, and to 38 μm for case 3. Case 1 was the only specimen to reveal shallow warts along the posterior surface of DM, whereas the most advanced specimen, case 3, showed evidence of earlier nodularity that had been buried by the accretion of further extracellular matrix material. The posterior aspect of DM in this specimen had the unusual property of lighter staining relative to the anterior region of DM, laid down earlier in life. Immunocytochemistry revealed increased expression and complex, sharply defined patterns of deposition of collagen VIII, collagen IV, laminin, and fibronectin. Ultrastructurally, the posterior nonbanded layer of DM was intermixed with banded collagen, and the posterior region of DM showed a high density of foci of spindle-shaped structures with intense-staining bands, spaced at ~ 120 nm. Finally, ultrastructural studies of the endothelium in case 1 revealed unusual accumulation of swelling mitochondria. The endothelial cells also had large amounts of abnormal prominent rough endoplasmic reticulum. Type VIII collagen alpha 2 immunogold signal was associated with the highly granular ribosomes of the rough endoplasmic reticulum of these patients.
Microscopic and electron microscopic examination revealed pathological changes in DM of L450W COL8A2 mutants that were consistent with several-fold increased growth of the extracellular matrix and progressive deposition and synthesis of extracellular material by endothelial cells. As with late-onset FCD, this is accompanied by attenuation and eventual loss of the endothelium itself. Whether the abnormal deposition of collagen, laminin, and fibronectin contributes to the dysfunction and death of the endothelium remains to be determined.
PMCID: PMC1809915  PMID: 17471329
9.  Compliance profile of the human cornea as measured by atomic force microscopy 
Micron (Oxford, England : 1993)  2012;43(12):1293-1298.
The ability to accurately determine the elastic modulus of each layer of the human cornea is a crucial step in the design of better corneal prosthetics. In addition, knowledge of the elastic modulus will allow design of substrates with relevant mechanical properties for in vitro investigations of cellular behavior. Previously, we have reported elastic modulus values for the anterior basement membrane and Descemet’s membrane of the human cornea, the surfaces in contact with the epithelial and endothelial cells, respectively. We have completed the compliance profile of the stromal elements of the human cornea by obtaining elastic modulus values for Bowman’s layer and the anterior stroma. Atomic force microscopy (AFM) was used to determine the elastic modulus, which is a measure of the tissue stiffness and is inversely proportional to the compliance. The elastic response of the tissue allows analysis with the Hertz equation, a model that provides a relationship between the indentation force and depth and is a function of the tip radius and the modulus of the substrate. The elastic modulus values for each layer of the cornea are: 7.5 ± 4.2 kPa (anterior basement membrane), 109.8 ± 13.2 kPa (Bowman’s layer), 33.1 ± 6.1 kPa (anterior stroma), and 50 ± 17.8 kPa (Descemet’s membrane). These results indicate that the biophysical properties, including elastic modulus, of each layer of the human cornea are unique and may play a role in the maintenance of homeostasis as well as in the response to therapeutic agents and disease states. The data will also inform the design and fabrication of improved corneal prosthetics.
PMCID: PMC3622051  PMID: 22421334
10.  Limbal fibroblast conditioned media: A non-invasive treatment for limbal stem cell deficiency 
Molecular Vision  2011;17:658-666.
Limbal fibroblasts are known to regulate the maintenance and differentiation of the corneal epithelium including the limbal epithelial stem cells. This study examined the effect of limbal fibroblast conditioned media in a mouse model of limbal stem cell deficiency.
Limbal stem cell deficiency was created in C57/Bl6 mice by performing a limbus to limbus epithelial debridement. The mice were treated topically for 3 weeks with conditioned media derived from human limbal fibroblasts. The control mice were treated with skin fibroblast conditioned media or Dulbecco’s serum-free medium.
The mice treated with limbal fibroblast conditioned media demonstrated substantial growth of corneal type epithelial cells on the corneal surface with less conjunctival goblet cells. By contrast, the control treated corneas were found to be covered primarily by conjunctival type epithelium.
Cell culture media conditioned by limbal fibroblasts appear to contain factor(s) that are therapeutically beneficial in a model of limbal stem cell deficiency. The current results further support the notion that the essential limbal stem cell niche is provided by limbal fibroblasts and suggest a new, non-invasive option in the treatment of limbal stem cell deficiency.
PMCID: PMC3056128  PMID: 21403854
11.  Critical Appraisal of Ex Vivo Expansion of Human Limbal Epithelial Stem Cells 
Current molecular medicine  2010;10(9):841-850.
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-specfic 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.
PMCID: PMC3190238  PMID: 21091422
Epithelium; ex vivo expansion; limbal stem cell deficiency; limbus; ocular surface; reconstruction; stem cells
12.  Ultrastructural Analysis of in vivo Expanded Corneal Epithelium on Amniotic Membrane 
Journal of Korean Medical Science  2006;21(3):544-549.
The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3±1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.
PMCID: PMC2729965  PMID: 16778403
Amnion; Biological Dressings; Amniotic Membrane; Epithelium, Corneal; Tissue Expansion
13.  Expression and Regulation of Cornified Envelope Proteins in Human Corneal Epithelium 
Stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery, termed the cornified envelope. The purpose of this study was to evaluate the presence and distribution of cornified envelope precursors in human corneal epithelium, their expression in human corneal epithelial cell cultures, and the effect of ultraviolet radiation (UVB) and transglutaminase (TG) inhibition on their expression.
Tissue distribution of small proline-rich proteins (SPRRs) and filaggrin and involucrin was studied in human cornea sections by immunofluorescence staining. Primary human corneal epithelial cells (HCECs) from limbal explants were used in cell culture experiments. A single dose of UVB at 20 mJ/cm2 was used to stimulate these cells, in the presence or absence of mono-dansyl cadaverine (MDC), a TG inhibitor. SPRR2 and involucrin protein levels were studied by immunofluorescence staining and Western blot analysis. Gene expression of 12 proteins was investigated by semiquantitative reverse transcription–polymerase chain reaction.
In human cornea tissue, SPRR1, SPRR2, filaggrin, and involucrin protein expression were detected in the central and peripheral corneal and limbal epithelium. In HCECs, SPRR2 and involucrin proteins were detected in the cytosolic fraction, and involucrin levels increased after UVB. Both SPRR2 and involucrin levels accumulated in the presence of MDC. Nine genes including involucrin, SPRR (types 1A, 1B, 2A, 2B, and 3), late envelope protein (LEP) 1 and 16, and filaggrin were expressed by HCECs. SPRR 4, loricrin, and LEP 6 transcripts were not detected. UVB downregulated SPRR (2A, 2B) and LEP 1 transcripts.
Various envelope precursors are expressed in human corneal epithelium and in HCECs, acute UVB stress differentially alters their expression in HCECs. The expression of envelope precursors and their rapid modulation by UVB supports the role of these proteins in the regulation of ocular surface stress. TG function may be relevant in the regulation of soluble precursors in UVB-stimulated corneal epithelium.
PMCID: PMC2906387  PMID: 16639001
14.  Corneal dystrophies 
The term corneal dystrophy embraces a heterogenous group of bilateral genetically determined non-inflammatory corneal diseases that are restricted to the cornea. The designation is imprecise but remains in vogue because of its clinical value. Clinically, the corneal dystrophies can be divided into three groups based on the sole or predominant anatomical location of the abnormalities. Some affect primarily the corneal epithelium and its basement membrane or Bowman layer and the superficial corneal stroma (anterior corneal dystrophies), the corneal stroma (stromal corneal dystrophies), or Descemet membrane and the corneal endothelium (posterior corneal dystrophies). Most corneal dystrophies have no systemic manifestations and present with variable shaped corneal opacities in a clear or cloudy cornea and they affect visual acuity to different degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian mode of inheritance. Different corneal dystrophies are caused by mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFBI, and UBIAD1 genes. Knowledge about the responsible genetic mutations responsible for these disorders has led to a better understanding of their basic defect and to molecular tests for their precise diagnosis. Genes for other corneal dystrophies have been mapped to specific chromosomal loci, but have not yet been identified. As clinical manifestations widely vary with the different entities, corneal dystrophies should be suspected when corneal transparency is lost or corneal opacities occur spontaneously, particularly in both corneas, and especially in the presence of a positive family history or in the offspring of consanguineous parents. Main differential diagnoses include various causes of monoclonal gammopathy, lecithin-cholesterol-acyltransferase deficiency, Fabry disease, cystinosis, tyrosine transaminase deficiency, systemic lysosomal storage diseases (mucopolysaccharidoses, lipidoses, mucolipidoses), and several skin diseases (X-linked ichthyosis, keratosis follicularis spinolosa decalvans). The management of the corneal dystrophies varies with the specific disease. Some are treated medically or with methods that excise or ablate the abnormal corneal tissue, such as deep lamellar endothelial keratoplasty (DLEK) and phototherapeutic keratectomy (PTK). Other less debilitating or asymptomatic dystrophies do not warrant treatment. The prognosis varies from minimal effect on the vision to corneal blindness, with marked phenotypic variability.
PMCID: PMC2695576  PMID: 19236704
15.  Gap Junction Protein Connexin 43 Serves as a Negative Marker for a Stem Cell-Containing Population of Human Limbal Epithelial Cells 
Stem cells (Dayton, Ohio)  2006;24(5):1265-1273.
This study evaluated whether the gap junction protein connexin (Cx) 43 could serve as a negative cell surface marker for human corneal epithelial stem cells. Cx43 expression was evaluated in corneo-limbal tissue and primary limbal epithelial cultures. Immunofluorescent staining and laser scanning confocal microscopy showed that Cx43 was strongly expressed in the corneal and limbal suprabasal epithelial cells, but the basal cells of the limbal epithelium were negative. Cx43 antibody stained mainly large cells but not small cells in primary limbal epithelial cultures. As determined by semiquantitative reverse transcription polymerase chain reaction (PCR) and real-time PCR, Cx43 mRNA was more abundant in the corneal than limbal epithelia, and it was expressed in higher levels in mature limbal epithelial cultures. Using GAP11, a rabbit polyclonal antibody against the Cx32 extracellular loop 2 (151–187), a sequence that is highly homologous in Cx43, the Cx43dim and Cx43bright cells were selected from primary limbal epithelial cultures by fluorescence-activated cell sorting and were evaluated for stem cell properties. These Cx43dim and Cx43bright cells were confirmed by their expression levels of Cx43 protein and mRNA. The Cx43dim cells were found to contain higher percentages of slow-cycling bromodeoxyuridine (BrdU)-label retaining cells and the cells that were positive for stem cell-associated markers p63, ABCG2, and integrin β1 and negative for differentiation markers K3 and involucrin. The Cx43dim cells possessed a greater proliferative potential than Cx43bright cells and nonfractionated cells as evaluated by BrdU incorporation, colony-forming efficiency, and growth capacity. Our findings suggest that human limbal basal cells do not express connexin 43, which could serve as a negative cell surface marker for the stem cell-containing population of human limbal epithelial cells.
PMCID: PMC2906383  PMID: 16424398
Connexin 43; Cornea; Limbus; Epithelia; Stem cells
16.  Human epithelial cell cultures from superficial limbal explants 
Molecular Vision  2011;17:341-354.
To study the kinetics of growth and the phenotype of cells cultured from human limbal explants in a cholera toxin-free medium with no feeder cell layer.
Human organ-cultured corneas were used to prepare limbal explants (full-thickness and superficial limbal explants) and corneal stromal explants. Cell growth kinetics and phenotypes were assessed by cultivating explants in cholera toxin-free Green medium. Epithelial and progenitor cell markers were assessed by immunocytochemistry, flow cytometry, and Reverse Transcription and Polymerase Chain Reaction (RT-PCR).
The successful epithelial cell growth rates from full thickness limbal explant and superficial limbal explant tissues were 41 and 86%, respectively (p=0.0001). The mean cell area and the percentage of small cells in superficial and full-thickness explant cultures were, respectively, 317 µm2 and 429 µm2, and 8.9% and 1.7% (p<0.001). The percentage of positive cells in superficial and full-thickness limbal explant cultures as assessed by immunocytochemistry were the following: broad spectrum cytokeratins (cytokeratins 4, 5, 6, 8, 10, 13, and 18 [MNF116]), 82%/37% (p=0.01); cytokeratin 3 (CK3), 74%/25% (p=0.009); cytokeratin 19 (CK19), 46%/25% (p=0.19); vimentin, 56%/53% (p=0.48); delta N p63α, 54%/0% (p<0.001); and ABCG2, 5%/0% (p=0.1). Flow cytometry showed a higher percentage of small cells, a higher percentage of MNF116+ cells, and stronger expression of progenitor-associated markers in superficial than in full-thickness explant cultures. For superficial limbal explant cultures, analysis of the expression profiles for various mRNAs at the end of 21 days of culture showed high levels of expression of the mRNAs encoding CK3, vimentin, and CK19. The expression of mRNA of delta N p63α and ABCG2 was weaker. Cultures obtained from full-thickness limbal explants featured no expression of mRNA of CK19, delta N p63α, and ABCG2, whereas mRNAs encoding CK3 and vimentin were detected. Human corneal stromal explants cultured with the same medium featured late cell growth, large mean cell area (2,529 µm2), no expression of cytokeratins, delta N p63α, and ABCG2, and high expression of vimentin.
Superficial limbal explants appear to be superior to full-thickness limbal explants for growing human limbal epithelial cells. Preparation of explants using surgical facilities (i.e., operating microscope and microsurgical blades) led to a dramatic increase in the percentage of successful cultures, higher epithelial cell growth, decreased fibroblast contamination, and better preservation of limbal epithelial progenitors.
PMCID: PMC3033435  PMID: 21297898
17.  Functional reconstruction of rabbit corneal epithelium by human limbal cells cultured on amniotic membrane 
Molecular vision  2003;9:635-643.
To investigate the phenotype of fetal and adult human limbal cells cultured on human amniotic membrane and the ability of cultured adult human limbal cells to repair limbal stem cell deficiency in a rabbit model.
Human adult and fetal limbal cells were isolated and cultured either on plastic plates or on human amniotic membrane. Connexin43, p63, and keratins 3 and 12 (K3 and K12) were detected by immunofluorescence and RT-PCR. Limbal stem cell deficiency was established in rabbits using chemical ablation and mechanical debridement. Cultured adult human limbal cells were transplanted onto rabbit corneas one month after injury, then fixed and imbedded in paraffin forty days later. Immunofluorescent staining of human-nuclear antigen, p63, K3, and connexin43 identified human-specific cells, progenitor cells, and differentiated corneal epithelial cells, respectively.
Adult and fetal cultured limbal cells appeared similar in morphology. RT-PCR results showed that cells cultured from the human adult and fetal limbal area expressed both p63 and K12, whereas cells from central adult epithelium expressed K12 only. Immunofluorescent staining showed that more cells were p63 positive when cultured on human amniotic membrane than on plastic. Double staining for p63 and connexin43 showed some p63-positive cells co-expressing connexin43. After transplantation of adult human limbal cells cultured on human amniotic membrane, injured rabbit corneas were completely reconstructed exhibiting epithelial integrity, improved corneal clarity, and little or no neovascularization. The majority of repopulated epithelial cells expressed anti-human nuclear antibody. Cells expressing p63 occurred throughout the new epithelium.
During healing, expression of p63 is not limited to epithelial stem cells but may also mark transient amplifying progenitor cells. Culture on human amniotic membrane suppresses differentiation of limbal epithelial cells and promotes the proliferation of p63 expressing cells. Amniotic membrane-cultured human limbal cells fully reconstructed rabbit corneas having limbal stem cell deficiency, with human cells providing most of the cells of the new epithelium. Expression p63 is distributed throughout the reconstructed tissue.
PMCID: PMC2877914  PMID: 14685149
18.  A re-evaluation of corneal development. 
The corneal epithelium is initially a continuation of the surface ectoderm, but later on in development appears to arise from the superficial cells of the corneal stroma. The corneal epithelium varies in thickness depending on the status of the lids, viz either fused or open. When the lids are fused, the epithelium is only 2 to 3 layers in depth. When the lids are separated, a basement membrane is distinguishable and the epithelium is 4 to 5 layers in depth. Bowman's membrane develops from processes of the superficial mesenchymal cells of the stroma that become thickened and are arranged in the long axis of the corneal surface. The corneal stroma develops from in situ mesenchymal tissue and does not migrate from the limbal mesenchymal tissue. The attenuation of the central cornea, early in development, is due to the impingement of the lens against the developing cornea. The central constriction of the cornea has led previous observers to believe that the stroma migrates from the peripheral limbal area towards the center of the cornea. Descemet's membrane arises from processes of the deep mesenchymal cells of the corneal stroma. These processes thicken and become arranged in the long axis of the posterior surface of the cornea. The membrane is initially cellular, and well-defined septae are noted between the cells. With maturity, Descemet's membrane becomes a homogeneous structure. The endothelium is derived from the mesenchymal cells of the posterior stroma. These cells are initially cuboidal but then become flattened. During the early development of the cornea, the tunica vasculosa lentis may play an important role. It is suggested that the anterior chamber is maintained early on by a transudate from the vessels of the tunica vasculosa lentis. The vessels of the tunica vasculosa lentis are compressed by the vanguard of the optic cup against the equator of the lens. With regression of this vascular system, there is a simultaneous development of aqueous humor. The hydrodynamic force of aqueous production assists corneal molding by a vis a tergo affect. Pari passu with this hydrodynamic force, the corneal stromal fibers increase in length and width. The structures involved in aqueous humor production, viz the ciliary epithelium, and the aqueous humor drainage, viz the filtration angle, trabecular meshwork and aqueous veins, develop contemporaneously as the tunica vasculosa lentis regresses. The limiting and fixed stabilizing site is at the limbus, the site of insertion of the rectus muscles. At these sites a dimpling occurs as the cornea is enlarging.
PMCID: PMC1298807  PMID: 2979018
19.  Bilateral, anterior stromal ring opacity of the cornea 
AIMS/BACKGROUND—To describe a bilateral, mid peripheral, ring-shaped corneal opacity, not resembling any known corneal degeneration, dystrophy, or other disorder, and occurring without ocular or systemic disease.
METHODS—Ophthalmic examination, haematological screening, and ultrasound biomicroscopy.
RESULTS—A 25 year old man showed grey-white, granular opacities in both corneas, with an 8 mm diameter ring configuration, and a V-shaped distribution in the anterior stroma. The surrounding corneal stroma was clear, and the tear film, the epithelium and its basement membrane, Descemet's membrane, and the endothelium were normal. Evidence of systemic disease was not found. Family members did not show corneal abnormalities.
CONCLUSION—A bilateral corneal ring opacity may occur in healthy, asymptomatic, young people. These corneal rings may result from depositions of unknown origin, or possibly a rare corneal dystrophy.

 Keywords: cornea; corneal opacity; corneal rings
PMCID: PMC1722592  PMID: 9713059
20.  Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction 
To investigate the clinical outcomes of cultivated corneal limbal epithelial transplantation (CLET) using human amniotic membrane for corneal limbal stem-cell deficiency.
Prospective, noncomparative case series. Eighteen patients (19 eyes) with severe ocular surface diseases were chosen to undergo CLET using human amniotic membrane. Twelve eyes received auto-CLET, and seven eyes received allo-CLET. Clinical outcomes of corneal surface epithelialization, conjunctivalization, inflammation, visual acuity, graft status, and complications were observed.
Corneal epithelium cultivated on amniotic membrane (two to four layers) was positive for molecular markers p63, ABCG2, CK3, and CK12. The mean patient age was 44.7 ± 15.2 years. A successful clinical outcome, defined as corneal epithelialization without central conjunctivalization or severe inflammation, was obtained in 14 (73.7%) of 19 eyes (mean follow-up 26.1 ± 13.5 months; range 6–47). A histopathologic success, defined as absence of goblet cells at the central cornea, was achieved in 12 (63.2%) eyes. Clinical failures occurred in five (26.3%) of 19 eyes, and histopathologic failures occurred in seven (36.8%) of 19 eyes. Survival analysis at 1 year showed that the clinical success rate was 77.9% and the pathological success rate was 72.3%. Fourteen of 19 (73.7%) eyes had visual acuity improvements after CLET. Six cases underwent penetrating keratoplasty; five of these grafts remained clear after 20.4 ± 6.9 months (range, 12–31) of follow-up. Complications included infectious keratitis (three cases) and recurrent symblepharon (one case). All complicated cases had lid abnormalities. Factors affecting the final clinical outcomes were lid abnormalities, abnormal corneal stromal beds, and complications.
CLET can successfully restore ocular surface damage in most cases with corneal limbal stem cell deficiency.
PMCID: PMC3460717  PMID: 23055668
limbal deficiency; limbal transplantation; corneal epithelial stem-cell transplantation; cultivated corneal epithelial stem cells
21.  Corneal nerve architecture in a donor with unilateral epithelial basement membrane dystrophy 
Ophthalmic research  2013;49(4):185-191.
Epithelial basement membrane dystrophy (EBMD) is by far the most common corneal dystrophy. In this study, we used a newly developed method of immunofluorescence staining and imaging to study the entire corneal nerve architecture of a donor with unilateral EBMD.
Two fresh eyes from a 56-year-old male donor were obtained; the right eye of the donor was diagnosed with EBMD and the left was normal. After slit lamp examination, the corneas were immunostained with anti-β-tubulin III antibody. Images were recorded by a fluorescent microscope equipped with a Photometrics digital camera using MetaVue imaginig software.
The left cornea appeared normal as observed by slit lamp and stereomicroscope, but the right eye had numerous irregular geographic patches in the basement membrane. Immunofluorescence showed no difference in the stromal nerve distribution between the two eyes, but there were areas without innervations in the EBMD cornea. Subbasal nerve fibers also showed tortuous courses and fewer divisions. There was a significant decrease in the density of subbasal nerve fibers and the number of terminals in the right eye.
We show for the first time detailed nerve architecture in an EBMD cornea. Our results suggest that EBMD-induced abnormalities of basement membrane altered epithelial nerve architecture and decreased nerve density, contributing to the pathology of the disease.
PMCID: PMC3971924  PMID: 23306594
Epithelial basement membrane dystrophy; corneal innervations; recurrent erosion syndrome; immunofluorescence of corneal nerves
22.  SSEA4 Is a Potential Negative Marker for the Enrichment of Human Corneal Epithelial Stem/Progenitor Cells 
This study shows the first evidence that SSEA4 is highly expressed in differentiated corneal epithelial cells and could be used as a negative marker to enrich the isolation of limbal stem cells.
To examine the expression of stage-specific embryonic antigen-4 (SSEA4) in the epithelium of the human ocular surface and characterize SSEA4+ and SSEA4− limbal epithelial cells.
SSEA4 expression in the human cornea and limbus was examined by RT-PCR and immunohistochemistry. SSEA4+ and SSEA4− cells were then separated by using magnetic beads. The phenotypes of these two cell populations were evaluated on the basis of cell size, clonogenic assay, and expression of putative limbal stem cell (LSC) and corneal epithelial differentiation markers.
SSEA4 was expressed in all layers of the corneal and anterior limbal epithelia. Discrete clusters of SSEA4+ cells were present in the central and posterior limbal epithelia. SSEA4+ cells accounted for an average of 40% of the total limbal epithelial cells. The SSEA4− population contained five times more small cells (≤11 μm in diameter) than did the SSEA4+ population. The expression levels of the putative LSC markers ABCG2, ΔNp63α, and cytokeratin (K)14 were significantly higher in the SSEA4− population than in the SSEA4+ population. The SSEA4− cells also expressed a significantly higher level of N-cadherin, but a lower level of the differentiation marker K12. The colony-forming efficiency in the SSEA4− population was 25.2% (P = 0.04) and 1.6-fold (P < 0.05) higher than in the unsorted population and the SSEA4+ population, respectively.
SSEA4 is highly expressed in differentiated corneal epithelial cells, and SSEA4− limbal epithelial cells contain a higher proportion of limbal stem/progenitor cells. SSEA4 could be used as a negative marker to enrich the isolation of LSCs.
PMCID: PMC3175983  PMID: 21685344
23.  Down-regulation of Notch signaling during corneal epithelial proliferation 
Molecular Vision  2008;14:1041-1049.
We evaluated the expression and activation of Notch pathway genes in the adult human and murine corneal epithelium during proliferation.
The expression of Notch pathway genes in the limbal and central human corneal epithelium was compared by reverse transcription polymerase chain reaction (RT–PCR). Their expression pattern was examined by immunofluorescence and in situ hybridization. The temporal expression of Notch1 during murine wound healing was assessed by RT–PCR. Notch activity was determined using western blot for the Notch intracellular domain (NotchIC). The expression of Hes1 was evaluated in cell culture.
The expression of Notch1 and Jagged1 was higher in the human limbal epithelium while the expression of Hes1 and Hes5 was higher in the central cornea. Expression of Notch1, Jagged1, and Hes1 was found predominantly in the basal and immediate suprabasal cells. During neonatal corneal development, NotchIC was detected in occasional cells at P10 while at P15 and P90, it was found in the basal and early suprabasal layers. NotchIC was found to be lower in the limbal compared to central corneal epithelium. The expression of Notch1 was lower at 24 h post-wounding but was completely restored in six days. The levels of NotchIC were decreased at 24 h post-wounding and after application of topical phorbol myristate. In vitro, the expression of Hes1 was higher in confluent cells maintained under high calcium conditions.
The inverse correlation between Notch signaling and the proliferative status of the corneal epithelium is consistent with the idea that Notch plays a role in corneal epithelial differentiation.
PMCID: PMC2426716  PMID: 18552977
24.  Isolation of adult progenitor cells with neuronal potential from rabbit corneal epithelial cells in serum- and feeder layer-free culture conditions 
Molecular Vision  2010;16:1712-1719.
To isolate progenitor cells from rabbit corneal epithelial cells (CEC) in serum- and feeder layer-free culture conditions and to compare the self-renewal capacity of corneal epithelial progenitor cells obtained from the central and limbal regions of the cornea.
Tissue samples of New Zealand white rabbit corneas were dissected from the limbal and central regions to obtain CEC for sphere-forming culture, in which the cells formed spheres in serum-free medium containing growth factors. The number of primary and secondary sphere colonies and the size of the primary spheres were compared between the limbal and central regions. To promote differentiation, isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin. The expression of epithelial, neural, and mesenchymal mRNAs was examined in the sphere colonies and their progeny by immunocytochemistry and/or the reverse transcription–polymerase chain reaction (RT–PCR). Adherent differentiated cells from the sphere colonies were also examined morphologically.
Primary spheres were isolated from both the limbal and central regions of the cornea. The rate of primary sphere formation by CEC from the limbal region (55.6±10.6/10,000 cells) was significantly higher than that by cells from the central cornea (43.1±7.2/10,000 cells, p=0.0028), but there was no significant difference in the size of primary spheres derived from both regions. The self-renewal capacity of cells from the limbal region was higher than that of cells from the central region, as evidenced by the significantly higher secondary sphere formation rate of limbal cells (38.7±8.5/10,000 cells) in comparison with that for central cells (31.3±5.7/10,000 cells, p=0.013). The primary sphere colonies expressed bromodeoxyuridine (BrdU), a 63-kDa protein (p63), p75 neurotrophin receptor (p75NTR), and nestin, whereas their progeny expressed cytokeratin 3, cytokeratin 12, vimentin, α-smooth muscle actin, microtubule-associated protein 2, and neuron-specific enolase on immunocytochemical analysis. These markers were confirmed by RT–PCR.
Our findings indicate that limbal CEC contain more progenitor cells with a stronger self-renewal capacity than cells from the central region. These progenitor cells differentiate into the epithelial lineage, and can also produce neuronal protein.
PMCID: PMC2927443  PMID: 20806049
25.  A serum- and feeder-free technique of culturing human corneal epithelial stem cells on amniotic membrane 
Molecular Vision  2009;15:1294-1302.
To describe a simple technique of cultivating human corneal epithelial stem cells using an Epilife® culture medium under serum- and feeder-free conditions.
Cadaveric donor limbal corneal epithelial cells were cultured on denuded amniotic membranes using an explant technique that was free of serum and feeder cells in the Epilife® medium containing a growth supplement of defined composition. These cells were assessed by phase contrast microscope. The expressions of the proposed corneal epithelial stem cell markers (p63, ATP-binding cassette member 2 (ABCG2), and cytokeratin 15 and 19) and differentiation markers (cytokeratin 3, 12, connexin 43, and p75) were analyzed using reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemical staining.
Successful cultures were obtained, resulting in a monolayer to double layer cell sheets with a cobblestone-like morphology. RT-PCR and immunocytochemistry disclosed an expression of both putative limbal stem cell (LSC) markers and differentiation-associated markers in the cultured cells. Most of the cultured corneal epithelial cells that were immunopositive for putative LSC markers were smaller, more uniform, and closer to the limbal explant than cells positively stained with differentiation-associated markers.
A serum- and feeder-free culture system using Epilife® medium may grow human corneal epithelial equivalents, minimizing the risk of contamination during culture. The technique may also be useful for the clinical application of limbal stem cell culture.
PMCID: PMC2704912  PMID: 19578552

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