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1.  The Corneal Epithelial Basement Membrane: Structure, Function, and Disease 
The corneal epithelial basement membrane (BM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma and provide scaffolding during embryonic development but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components—collagens, laminins, heparan sulfate proteoglycans, and nidogens—in addition to other components such as thrombospondin-1, matrilin-2, and matrilin-4 and even fibronectin in some BM. Many studies have focused on characterizing BM due to their potential roles in normal tissue function and disease, and these structures have been well characterized in many tissues. Comparatively few studies, however, have focused on the function of the epithelial BM in corneal physiology. Since the normal corneal stroma is avascular and has relatively low keratocyte density, it is expected that the corneal BM would be different from the BM in other tissues. One function that appears critical in homeostasis and wound healing is the barrier function to penetration of cytokines from the epithelium to stroma (such as transforming growth factor β-1), and possibly from stroma to epithelium (such as keratinocyte growth factor). The corneal epithelial BM is also involved in many inherited and acquired corneal diseases. This review examines this structure in detail and discusses the importance of corneal epithelial BM in homeostasis, wound healing, and disease.
The corneal epithelial basement membrane serves critical functions in development, homeostasis, wound healing, and disease. The basement membrane is composed of many components arranged precisely to serve these critical functions. Variations in the components are noted in the conjunctiva, limbal cornea, and central cornea.
PMCID: PMC3787659  PMID: 24078382
basement membrane; myofibroblasts; corneal epithelium; wound healing
2.  Corneal Limbal Microenvironment Can Induce Transdifferentiation of Hair Follicle Stem Cells into Corneal Epithelial-like Cells 
Stem Cells (Dayton, Ohio)  2009;27(3):642-652.
The aim of this study was to investigate the transdifferentiation potential of murine vibrissa hair follicle (HF) stem cells into corneal epithelial-like cells through modulation by corneal- or limbus-specific microenvironmental factors. Adult epithelial stem cells were isolated from the HF bulge region by mechanical dissection or fluorescence-activated cell sorting using antibodies to α6 integrin, enriched by clonal expansion, and subcultivated on various extracellular matrices (type IV collagen, laminin-1, laminin-5, fibronectin) and in different conditioned media derived from central and peripheral corneal fibroblasts, limbal stromal fibroblasts, and 3T3 fibroblasts. Cellular phenotype and differentiation were evaluated by light and electron microscopy, real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, using antibodies against putative stem cell markers (K15, α6 integrin) and differentiation markers characteristic for corneal epithelium (K12, Pax6) or epidermis (K10). Using laminin-5, a major component of the corneo-limbal basement membrane zone, and conditioned medium from limbal stromal fibroblasts, clonally enriched HF stem and progenitor cells adhered rapidly and formed regularly arranged stratified cell sheets. Conditioned medium derived from limbal fibroblasts markedly upregulated expression of cornea-specific K12 and Pax6 on the mRNA and protein level, whereas expression of the epidermal keratinocyte marker K10 was strongly downregulated. These findings suggest that adult HF epithelial stem cells are capable of differentiating into corneal epithelial-like cells in vitro when exposed to a limbus-specific microenvironment. Therefore, the HF may be an easily accessible alternative therapeutic source of autologous adult stem cells for replacement of the corneal epithelium and restoration of visual function in patients with ocular surface disorders.
PMCID: PMC2729676  PMID: 19074417
Adult stem cells; Cornea; Hair follicle; Transdifferentiation; Epithelial lineages; Limbal stem cell niche
3.  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
4.  A Simple Alkaline Method for Decellularizing Human Amniotic Membrane for Cell Culture 
PLoS ONE  2013;8(11):e79632.
Human amniotic membrane is a standard substratum used to culture limbal epithelial stem cells for transplantation to patients with limbal stem cell deficiency. Various methods were developed to decellularize amniotic membrane, because denuded membrane is poorly immunogenic and better supports repopulation by dissociated limbal epithelial cells. Amniotic membrane denuding usually involves treatment with EDTA and/or proteolytic enzymes; in many cases additional mechanical scraping is required. Although ensuring limbal cell proliferation, these methods are not standardized, require relatively long treatment times and can result in membrane damage. We propose to use 0.5 M NaOH to reliably remove amniotic cells from the membrane. This method was used before to lyse cells for DNA isolation and radioactivity counting. Gently rubbing a cotton swab soaked in NaOH over the epithelial side of amniotic membrane leads to nearly complete and easy removal of adherent cells in less than a minute. The denuded membrane is subsequently washed in a neutral buffer. Cell removal was more thorough and uniform than with EDTA, or EDTA plus mechanical scraping with an electric toothbrush, or n-heptanol plus EDTA treatment. NaOH-denuded amniotic membrane did not show any perforations compared with mechanical or thermolysin denuding, and showed excellent preservation of immunoreactivity for major basement membrane components including laminin α2, γ1-γ3 chains, α1/α2 and α6 type IV collagen chains, fibronectin, nidogen-2, and perlecan. Sodium hydroxide treatment was efficient with fresh or cryopreserved (10% dimethyl sulfoxide or 50% glycerol) amniotic membrane. The latter method is a common way of membrane storage for subsequent grafting in the European Union. NaOH-denuded amniotic membrane supported growth of human limbal epithelial cells, immortalized corneal epithelial cells, and induced pluripotent stem cells. This simple, fast and reliable method can be used to standardize decellularized amniotic membrane preparations for expansion of limbal stem cells in vitro before transplantation to patients.
PMCID: PMC3827346  PMID: 24236148
5.  Alterations of Extracellular Matrix Components and Proteinases in Human Corneal Buttons With INTACS for Post–Laser In Situ Keratomileusis Keratectasia and Keratoconus 
Cornea  2008;27(5):565-573.
To perform an immunohistochemical evaluation of corneas with INTACS for post–laser in situ keratomileusis (LASIK) keratectasia and keratoconus, obtained after corneal transplantation.
Corneas from 1 patient with INTACS for post-LASIK keratectasia and 2 patients with INTACS for keratoconus were obtained within 3 hours after penetrating keratoplasty, and cryostat sections were analyzed by immunostaining for 35 extracellular matrix (ECM) components and proteinases.
In the stroma of all corneas next to an INTACS implant, ECM components typically associated with fibrosis were observed. These included tenascin-C, fibrillin-1, and types III, IV (α1/α2 chains), and XIV collagen. Also, significant deposition of perlecan, nidogen-2, and cellular fibronectin was revealed in the same locations. The keratoconus cases displayed typical Bowman layer breaks and subepithelial fibrosis with deposition of various ECM components. In all cases, some keratocytes around INTACS were positive for specific proteinases associated with stromal remodeling, including cathepsins F and H, matrix metalloproteinase (MMP)-1, MMP-3, and MMP-10. Staining for MMP-7 was variable; MMP-2 and MMP-9 were mostly negative. Patterns of type IV collagen α3, α4, and α6 chains; types VI and VIII collagen; laminin-332,α4, α5,β1, β2, and γ1 laminin chains; vitronectin; thrombospondin-1; urokinase; EMMPRIN; and cathepsins B and L were unchanged around INTACS in all 3 cases compared with normal.
Abnormal accumulation of fibrotic ECM components and proteinases near INTACS suggests ongoing lysis and remodeling of corneal stroma. Specific changes observed in each case may be related to underlying pathology.
PMCID: PMC2746565  PMID: 18520507
INTACS; keratoconus; laser in situ keratomileusis; cornea; extracellular matrix; fibrosis; matrix metalloproteinase; tenascin-C; cathepsin; nidogen
6.  Proteome profiling of wild type and lumican-deficient mouse corneas 
Journal of proteomics  2011;74(10):1895-1905.
To elucidate how the deficiency of a major corneal proteoglycan lumican affects corneal homeostasis, we used mass spectrometry to derive the proteome profile of the lumican-deficient and the heterozygous mouse corneas and compared these to the wild type corneal proteome. 2,108 proteins were quantified in the mouse cornea. Selected proteins and transcripts were investigated by western blot and quantitative RT-PCR, respectively. We observed major changes in the composition of the stromal extracellular matrix (ECM) proteins in the lumican-deficient mice. Lumican deficiency altered cellular proteins in the stroma and the corneal epithelium. The ECM changes included increases in fibril forming collagen type I and VI, fibromodulin, perlecan, laminin β2, collagen type IV, nidogen/entactin and anchoring collagen type VII in the Lum+/− and the Lum−/− mouse corneas, while the stromal proteoglycans decorin, biglycan and keratocan were decreased in the Lum−/− corneas. Cellular protein changes included increases in alcohol dehydrogenase, superoxide dismutase and decreases in epithelial cytokeratins 8 and 14. We also detected proteins that are novel to the cornea. The proteomes will provide an insight into the lumican-deficient corneal phenotype of stromal thinning and loss of transparency and a better understanding of pathogenic changes in corneal and ocular dystrophies.
PMCID: PMC3163732  PMID: 21616181
Proteomics; Lumican; Mass spectrometry; iTRAQ; Cornea; Collagen
7.  The Expression And Function Of Netrin-4 In Murine Ocular Tissues 
Experimental Eye Research  2012;96(1):24-35.
Netrin-4, a member of the netrin family, is a potent regulator of embryonic development. It promotes neurite extension and regulates pulmonary airway branching, vasculogenesis patterning, and endothelial proliferation in pathological angiogenesis. The initial characterization of netrin-4 expression was focused on epithelial-derived organs (kidney, lung and salivary gland) and the central nervous system. Ocular development is an ideal system to study netrin-4 expression and function, as it involves both ectodermal (cornea, lens and retina) and mesodermal (sclera and choroid) derivatives and has an extensive and well-characterized angiogenic process. Netrin-4 is expressed in all ocular tissues. It is a prominent component of the basement membranes of the lens and cornea, as well as all three basement membranes of the retina: the inner limiting membrane, vascular basement membranes, and Bruch’s membrane. Netrin-4 is differentially deposited in vascular basement membranes, with more intense anti-netrin-4 reactivity on the arterial side. The retinal microcirculation also expresses netrin-4. In order to test the function of netrin-4 in vivo, we generated a conventional mouse lacking Ntn4 expression. Basement membrane formation in the cornea, lens and retina is undisrupted by netrin-4 deletion, demonstrating that netrin-4 is not a major structural component of these basement membranes. In the Ntn4 homozygous null (Ntn4−/−) cornea, the overall morphology of the cornea, as well as the epithelial, stromal and endothelial stratification are normal; however, epithelial cell proliferation is increased. In the Ntn4−/− retina, neurogenesis appears to proceed normally, as does retinal lamination. In the Ntn4−/− retina, retinal ganglion cell targeting is intact, although there are minor defects in axon fasciculation. In the retinal vasculature of the Ntn4−/− retina, the distribution patterns of astrocytes and the vasculature are largely normal, with the possible exception of increased branching in the deep capillary plexus, suggesting that netrin-4 may act as a negative regulator of angiogenesis. These data, taken together, suggest that netrin-4 is a negative regulator of corneal epithelial cell proliferation and retinal vascular branching in vivo, whereas netrin-4 may be redundant with other members of the netrin family in other ocular tissue development. Ntn4−/− mice may serve as a good model in which to study the role of netrins in vivo of the pathobiologic vascular remodeling in the retina and cornea.
PMCID: PMC3296891  PMID: 22281059
Extracellular matrix; netrin; angiogenesis; cornea; retina; axonal pathfinding
8.  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
Brain research bulletin  2009;81(2-3):262.
Our previous data suggested the involvement of matrix metalloproteinase-10 (MMP-10) and cathepsin F (CTSF) in the basement membrane and integrin changes occurring in diabetic corneas. These markers were now examined in normal human organ-cultured corneas upon recombinant adenovirus (rAV)-driven transduction of MMP-10 and CTSF genes.
Fifteen pairs of normal autopsy human corneas were used. One cornea of each pair was transduced with rAV expressing either CTSF or MMP-10 genes. 1–2 × 108 plaque forming units of rAV per cornea were added to cultures for 48 hr with or without sildenafil citrate. The fellow cornea of each pair received control rAV with vector alone. After 6–10 days incubation without rAV, corneas were analyzed by Western blot or immunohistochemistry, or tested for healing of 5-mm circular epithelial wounds caused by topical application of n-heptanol.
Sildenafil significantly increased epithelial transduction efficiency, apparently through stimulation of rAV endocytosis through caveolae. Corneas transduced with CTSF or MMP-10 genes or their combination had increased epithelial immunostaining of respective proteins compared to fellow control corneas. Staining for diabetic markers integrin α3β1, nidogen-1, nidogen-2, and laminin γ2 chain became weaker and irregular upon proteinase transduction. Expression of phosphorylated Akt was decreased in proteinase-transduced corneas. Joint overexpression of both proteinases led to significantly slower corneal wound healing that became similar to that observed in diabetic ones.
The data suggest that MMP-10 and CTSF may be responsible for abnormal marker patterns and impaired wound healing in diabetic corneas. Inhibition of these proteinases in diabetic corneas may alleviate diabetic keratopathy symptoms.
PMCID: PMC2815249  PMID: 19828126
diabetic cornea; organ culture; MMP-10; cathepsin F; Akt; sildenafil
10.  Induction of Corneal Myofibroblasts by Lens-derived Transforming Growth Factor β1 (TGFβ1): A Transgenic Mouse Model 
Brain research bulletin  2009;81(2-3):287.
Transforming growth factor β(TGFβ) is an important cytokine in corneal development and wound healing. Transgenic mice that express an active form of human TGF β1 driven by a lens-specific promoter were used in the current study to determine the biological effects of lens-derived TGFβ1 on postnatal corneal development and homeostasis.
The postnatal corneal changes in the TGFβ1 transgenic mice were examined by fluorescein labeling and histology. Epithelial/endothelial-to-mesenchymal transition (E/EnMT) in the transgenic mouse cornea was demonstrated by immunostaining for α-smooth muscle actin (α-SMA) and cadherin-11. Expression of E- and N-cadherin in the corneal epithelial and endothelial cells, respectively, was analyzed by in situ hybridization.
Among the established TGF β1 transgenic lines, mice from line OVE853 and OVE917 had normal-sized eyeballs but developed a corneal haze after eyelid opening. Histological examination showed that prenatal corneal development appeared to be normal. However, after postnatal day 7 (P7), the corneal endothelial cells in transgenic line OVE853 began to lose normal cell-cell contact and basement membrane structure. The endothelial layer was eventually absent in the inner surface of the transgenic mouse cornea. The morphological changes in the cornea correlated with abnormal expression of α-SMA, a molecular marker of EMT, and stress fiber formation in myofibroblast-like cells, which initially appeared in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. The E/EnMT in the transgenic mouse cornea was further demonstrated by loss of E- and N-cadherin expression in the corneal epithelial and endothelial cells respectively, and meanwhile increasing expression of cadherin-11 in both corneal epithelium and stroma.
Elevated levels of active TGF β1 in the anterior chamber can lead to myofibroblast formation in the corneal endothelial layer and subsequently in the corneal epithelial and stromal layers. Our data suggest that the levels of biologically active TGFβ in the aqueous humor must be under tight control to maintain corneal homeostasis. TGF β1 is the major cytokine during wound healing. Therefore, our findings also suggest a potential mechanism to explain the loss of corneal endothelial barrier and corneal opacification after intraocular surgery or trauma.
PMCID: PMC2814984  PMID: 19897021
11.  Molecular composition of the peri-islet basement membrane in NOD mice: a barrier against destructive insulitis 
Diabetologia  2008;51(9):1680-1688.
This study examined whether the capsule which encases islets of Langerhans in the NOD mouse pancreas represents a specialised extracellular matrix (ECM) or basement membrane that protects islets from autoimmune attack.
Immunofluorescence microscopy using a panel of antibodies to collagens type IV, laminins, nidogens and perlecan was performed to localise matrix components in NOD mouse pancreas before diabetes onset, at onset of diabetes and after clinical diabetes was established (2–8.5 weeks post-onset).
Perlecan, a heparan sulphate proteoglycan that is characteristic of basement membranes and has not previously been investigated in islets, was localised in the peri-islet capsule and surrounding intra-islet capillaries. Other components present in the peri-islet capsule included laminin chains α2, β1 and γ1, collagen type IV α1 and α2, and nidogen 1 and 2. Collagen type IV α3–α6 were not detected. These findings confirm that the peri-islet capsule represents a specialised ECM or conventional basement membrane. The islet basement membrane was destroyed in islets where intra-islet infiltration of leucocytes marked the progression from non-destructive to destructive insulitis. No changes in basement membrane composition were observed before leucocyte infiltration.
These findings suggest that the islet basement membrane functions as a physical barrier to leucocyte migration into islets and that degradation of the islet basement membrane marks the onset of destructive autoimmune insulitis and diabetes development in NOD mice. The components of the islet basement membrane that we identified predict that specialised degradative enzymes are likely to function in autoimmune islet damage.
PMCID: PMC2516190  PMID: 18633594
Autoimmunity; Basement membrane; Collagen; Islets; Laminin; Matrix; Nidogen; NOD; Perlecan
12.  Cultured corneal epithelia for ocular surface disease. 
PURPOSE: To evaluate the potential efficacy for autologous and allogeneic expanded corneal epithelial cell transplants derived from harvested limbal corneal epithelial stem cells cultured in vitro for the management of ocular surface disease. METHODS: Human Subjects. Of the 19 human subjects included, 18 (20 procedures) underwent in vitro cultured corneal epithelial cell transplants using various carriers for the epithelial cells to determine the most efficacious approach. Sixteen patients (18 procedures on 17 eyes) received autologous transplants, and 2 patients (1 procedure each) received allogeneic sibling grafts. The presumed corneal epithelial stem cells from 1 patient did not grow in vitro. The carriers for the expanded corneal epithelial cells included corneal stroma, type 1 collagen (Vitrogen), soft contact lenses, collagen shields, and amniotic membrane for the autologous grafts and only amniotic membrane for the allogeneic sibling grafts. Histologic confirmation was reviewed on selected donor grafts. Amniotic membrane as carrier. Further studies were made to determine whether amniotic membrane might be the best carrier for the expanding corneal epithelial cells. Seventeen different combinations of tryspinization, sonication, scraping, and washing were studied to find the simplest, most effective method for removing the amniotic epithelium while still preserving the histologic appearance of the basement membrane of the amnion. Presumed corneal epithelial stem cells were harvested and expanded in vitro and applied to the amniotic membrane to create a composite graft. Thus, the composite graft consisted of the amniotic membrane from which the original epithelium had been removed without significant histologic damage to the basement membrane, and the expanded corneal epithelial stem cells, which had been applied to and had successfully adhered to the denuded amniotic membrane. Animal model. Twelve rabbits had the ocular surface of 1 eye damaged in a standard manner with direct removal of the presumed limbal stem cells, corneal epithelium, and related epithelium, followed by the application of n-heptanol for 60 seconds. After 6 weeks, all damaged eyes were epithelialized and vascularized. Two such treated eyes were harvested without further treatment, to be used for histologic study as damaged controls. The remaining 10 rabbits received composite grafts (consisting of amniotic membrane with expanded allogeneic rabbit corneal epithelial cell transplants) applied to the ocular surface in a standard manner followed by the application of a contact lens. At 16 days following transplantation, 5 of the rabbits were sacrificed and the corneal rims were removed for histologic study. At 28 days, the remaining rabbits were sacrificed and the previously damaged eyes were harvested for histologic and immunohistochemical study. RESULTS: Human subjects. Of the 19 total patients admitted to the study, the presumed corneal epithelial stem cells of 1 patient did not grow in vitro. Of the remaining 18 patients (20 procedures, 19 eyes), 3 patients had unsuccessful results (3 autologous procedures), 1 patient had a partially successful procedure (allogeneic procedure), and 1 patient had a procedure with an undetermined result at present (allogeneic procedure). One unsuccessful patient had entropion/trichiasis and mechanically removed the graft and eventually went into phthisis. The other 2 unsuccessful patients suffered presumed loss of autologous donor epithelium and recurrence of the ocular surface disease (pterygium). The partially successful patient receiving an allogeneic transplant had infectious keratitis delay of his re-epithelialization; he has only minimal visual improvement but has re-epithelialized. The patient receiving the second allogeneic graft lost his donor epithelium at day 4. Additional donor epithelium was reapplied, but the result is undetermined at present. Amniotic membrane as carrier. The in vitro preparation of the amniotic membrane with corneal epithelial stem cell graft overlay was successful.Histology documented removal of the amniotic epithelium and reapplication of corneal epithelial cells. Animal model. The 2 rabbits that had no reparative surgery following standard ocular surface injury had histology and immunopathology consistent with incomplete corneal epithelial stem cell failure with vascularization and scarring of the ocular surface. Light microscopy and immunohistologic staining with AE5 confirmed the conjunctival phenotype of the ocular surface repair but also documented the incomplete model. The allogeneic stern cell transplants had varying results. One rabbit had a suppurative infection and lost the graft. Reparative surgery failed in 2 of the rabbits, failed partially in 3 of the rabbits, was partially successful in 3 others, and was successful in 1 rabbit at 28 days. Histologic and immunopathologic study documented successful growth of corneal epithelium onto the recipient surface. CONCLUSIONS: 1. Presumed corneal epithelial stem cells can be harvested safely from the limbus and expanded successfully in vitro. 2. Expanded corneal epithelial cell cultures can be grown onto various carriers, but currently denuded amniotic membrane seems to be the best carrier for ocular surface repair. 3. Expanded corneal epithelial cell transplants appear to resurface damaged ocular surfaces successfully, but cellular tracking and further confirmation are required. 4. Expanded allogeneic corneal epithelial cell transplants are technically possible and may represent alternative treatment modalities for selected ocular surface problems. 5. These techniques potentially offer a new method of restoring a normal ocular surface while minimizing the threat of damage or depletion to the contralateral or sibling limbal corneal epithelial stem cells. 6. The rabbit model was probably incomplete and should be interpreted with caution. The complete eradication of all corneal epithelial stem cells from any eye is difficult, making confirmation of such work challenging. 7. The results of the rabbit model suggest that allogeneic grafts may restore a nearly normal ocular epithelial surface to certain ocular surface injuries.
PMCID: PMC1298283  PMID: 10703147
13.  Nidogen-1 regulates laminin-1-dependent mammary-specific gene expression 
Journal of cell science  2000;113(Pt 5):849-858.
Nidogen-1 (entactin) acts as a bridge between the extracellular matrix molecules laminin-1 and type IV collagen, and thus participates in the assembly of basement membranes. To investigate the role of nidogen-1 in regulating cell-type-specific gene expression in mammary epithelium, we designed a culture microecosystem in which each component, including epithelial cells, mesenchymal cells, lactogenic hormones and extracellular matrix, could be controlled. We found that primary and established mesenchymal and myoepithelial cells synthesized and secreted nidogen-1, whereas expression was absent in primary and established epithelial cells. In an epithelial cell line containing mesenchymal cells, nidogen-1 was produced by the mesenchymal cells but deposited between the epithelial cells. In this mixed culture, mammary epithelial cells express β-casein in the presence of lactogenic hormones. Addition of either laminin-1 plus nidogen-1, or laminin-1 alone, to mammary epithelial cells induced β-casein production. We asked whether recombinant nidogen-1 alone could signal directly for β-casein. Nidogen-1 did not induce β-casein synthesis in epithelial cells, but it augmented the inductive capacity of laminin-1. These data suggest that nidogen-1 can cooperate with laminin-1 to regulate β-casein expression. Addition of full-length nidogen-1 to the mixed cultures had no effect on β-casein gene expression; however, a nidogen-1 fragment containing the laminin-1 binding domain, but lacking the type IV collagen-binding domain, had a dominant negative effect on β-casein expression. These data point to a physiological role for nidogen-1 in the basement membrane-induced gene expression by epithelial cells.
PMCID: PMC2933215  PMID: 10671374
Nidogen-1; Entactin; Basement membrane; Extracellular matrix; Tissue-specific gene expression; Epithelial-mesenchymal interaction; Mammary gland
14.  Limbal Stem Cell Transplantation 
Executive Summary
The objective of this analysis is to systematically review limbal stem cell transplantation (LSCT) for the treatment of patients with limbal stem cell deficiency (LSCD). This evidence-based analysis reviews LSCT as a primary treatment for nonpterygium LSCD conditions, and LSCT as an adjuvant therapy to excision for the treatment of pterygium.
Clinical Need: Condition and Target Population
The outer surface of the eye is covered by 2 distinct cell layers: the corneal epithelial layer that overlies the cornea, and the conjunctival epithelial layer that overlies the sclera. These cell types are separated by a transitional zone known as the limbus. The corneal epithelial cells are renewed every 3 to 10 days by a population of stem cells located in the limbus.
Nonpterygium Limbal Stem Cell Deficiency
When the limbal stem cells are depleted or destroyed, LSCD develops. In LSCD, the conjunctival epithelium migrates onto the cornea (a process called conjunctivalization), resulting in a thickened, irregular, unstable corneal surface that is prone to defects, ulceration, corneal scarring, vascularization, and opacity. Patients experience symptoms including severe irritation, discomfort, photophobia, tearing, blepharospasm, chronic inflammation and redness, and severely decreased vision.
Depending on the degree of limbal stem cell loss, LSCD may be total (diffuse) or partial (local). In total LSCD, the limbal stem cell population is completed destroyed and conjunctival epithelium covers the entire cornea. In partial LSCD, some areas of the limbus are unharmed, and the corresponding areas on the cornea maintain phenotypically normal corneal epithelium.
Confirmation of the presence of conjunctivalization is necessary for LSCD diagnosis as the other characteristics and symptoms are nonspecific and indicate a variety of diseases. The definitive test for LSCD is impression cytology, which detects the presence of conjunctival epithelium and its goblet cells on the cornea. However, in the opinion of a corneal expert, diagnosis is often based on clinical assessment, and in the expert’s opinion, it is unclear whether impression cytology is more accurate and reliable than clinical assessment, especially for patients with severe LSCD.
The incidence of LSCD is not well understood. A variety of underlying disorders are associated with LSCD including chemical or thermal injuries, ultraviolet and ionizing radiation, Stevens-Johnson syndrome, multiple surgeries or cryotherapies, contact lens wear, extensive microbial infection, advanced ocular cicatricial pemphigoid, and aniridia. In addition, some LSCD cases are idiopathic. These conditions are uncommon (e.g., the prevalence of aniridia ranges from 1 in 40,000 to 1 in 100,000 people).
Pterygium is a wing-shaped fibrovascular tissue growth from the conjunctiva onto the cornea. Pterygium is the result of partial LSCD caused by localized ultraviolet damage to limbal stem cells. As the pterygium invades the cornea, it may cause irregular astigmatism, loss of visual acuity, chronic irritation, recurrent inflammation, double vision, and impaired ocular motility.
Pterygium occurs worldwide. Incidence and prevalence rates are highest in the “pterygium belt,” which ranges from 30 degrees north to 30 degrees south of the equator, and lower prevalence rates are found at latitudes greater than 40 degrees. The prevalence of pterygium for Caucasians residing in urban, temperate climates is estimated at 1.2%.
Existing Treatments Other Than Technology Being Reviewed
Nonpterygium Limbal Stem Cell Deficiency
In total LSCD, a patient’s limbal stem cells are completely depleted, so any successful treatment must include new stem cells. Autologous oral mucosal epithelium transplantation has been proposed as an alternative to LSCT. However, this procedure is investigational, and there is very limited level 4c evidence1 to support this technique (fewer than 20 eyes examined in 4 case series and 1 case report).
For patients with partial LSCD, treatment may not be necessary if their visual axis is not affected. However, if the visual axis is conjunctivalized, several disease management options exist including repeated mechanical debridement of the abnormal epithelium; intensive, nonpreserved lubrication; bandage contact lenses; autologous serum eye drops; other investigational medical treatments; and transplantation of an amniotic membrane inlay. However, these are all disease management treatments; LSCT is the only curative option.
The primary treatment for pterygium is surgical excision. However, recurrence is a common problem after excision using the bare sclera technique: reported recurrence rates range from 24% to 89%. Thus, a variety of adjuvant therapies have been used to reduce the risk of pterygium recurrence including LSCT, amniotic membrane transplantation (AMT), conjunctival autologous (CAU) transplantation, and mitomycin C (MMC, an antimetabolite drug).
New Technology Being Reviewed
To successfully treat LSCD, the limbal stem cell population must be repopulated. To achieve this, 4 LSCT procedures have been developed: conjunctival-limbal autologous (CLAU) transplantation; living-related conjunctival-limbal allogeneic (lr-CLAL) transplantation; keratolimbal allogeneic (KLAL) transplantation; and ex vivo expansion of limbal stem cells transplantation. Since the ex vivo expansion of limbal stem cells transplantation procedure is considered experimental, it has been excluded from the systematic review. These procedures vary by the source of donor cells and the amount of limbal tissue used. For CLAU transplants, limbal stem cells are obtained from the patient’s healthy eye. For lr-CLAL and KLAL transplants, stem cells are obtained from living-related and cadaveric donor eyes, respectively.
In CLAU and lr-CLAL transplants, 2 to 4 limbal grafts are removed from the superior and inferior limbus of the donor eye. In KLAL transplants, the entire limbus from the donor eye is used.
The recipient eye is prepared by removing the abnormal conjunctival and scar tissue. An incision is made into the conjunctival tissue into which the graft is placed, and the graft is then secured to the neighbouring limbal and scleral tissue with sutures. Some LSCT protocols include concurrent transplantation of an amniotic membrane onto the cornea.
Regulatory Status
Health Canada does not require premarket licensure for stem cells. However, they are subject to Health Canada’s clinical trial regulations until the procedure is considered accepted transplantation practice, at which time it will be covered by the Safety of Human Cells, Tissues and Organs for Transplantation Regulations (CTO Regulations).
Review Strategy
The Medical Advisory Secretariat systematically reviewed the literature to assess the effectiveness and safety of LSCT for the treatment of patients with nonpterygium LSCD and pterygium. A comprehensive search method was used to retrieve English-language journal articles from selected databases.
The GRADE approach was used to systematically and explicitly evaluate the quality of evidence and strength of recommendations.
Summary of Findings
Nonpterygium Limbal Stem Cell Deficiency
The search identified 873 citations published between January 1, 2000, and March 31, 2008. Nine studies met the inclusion criteria, and 1 additional citation was identified through a bibliography review. The review included 10 case series (3 prospective and 7 retrospective).
Patients who received autologous transplants (i.e., CLAU) achieved significantly better long-term corneal surface results compared with patients who received allogeneic transplants (lr-CLAL, P< .001; KLAL, P< .001). There was no significant difference in corneal surface outcomes between the allogeneic transplant options, lr-CLAL and KLAL (P = .328). However, human leukocyte antigen matching and systemic immunosuppression may improve the outcome of lr-CLAL compared with KLAL. Regardless of graft type, patients with Stevens-Johnson syndrome had poorer long-term corneal surface outcomes.
Concurrent AMT was associated with poorer long-term corneal surface improvements. When the effect of the AMT was removed, the difference between autologous and allogeneic transplants was much smaller.
Patients who received CLAU transplants had a significantly higher rate of visual acuity improvements compared with those who received lr-CLAL transplants (P = .002). However, to achieve adequate improvements in vision, patients with deep corneal scarring will require a corneal transplant several months after the LSCT.
No donor eye complications were observed.
Epithelial rejection and microbial keratitis were the most common long-term complications associated with LSCT (complications occurred in 6%–15% of transplantations). These complications can result in graft failure, so patients should be monitored regularly following LSCT.
The search yielded 152 citations published between January 1, 2000 and May 16, 2008. Six randomized controlled trials (RCTs) that evaluated LSCT as an adjuvant therapy for the treatment of pterygium met the inclusion criteria and were included in the review.
Limbal stem cell transplantation was compared with CAU, AMT, and MMC. The results showed that CLAU significantly reduced the risk of pterygium recurrence compared with CAU (relative risk [RR], 0.09; 95% confidence interval [CI], 0.01–0.69; P = .02). CLAU reduced the risk of pterygium recurrence for primary pterygium compared with MMC, but this comparison did not reach statistical significance (RR, 0.48; 95% CI, 0.21–1.10; P = .08). Both AMT and CLAU had similar low rates of recurrence (2 recurrences in 43 patients and 4 in 46, respectively), and the RR was not significant (RR, 1.88; 95% CI, 0.37–9.5; P = .45). Since sample sizes in the included studies were small, failure to detect a significant difference between LSCT and AMT or MMC could be the result of type II error. Limbal stem cell transplantation as an adjuvant to excision is a relatively safe procedure as long-term complications were rare (< 2%).
GRADE Quality of Evidence
Nonpterygium Limbal Stem Cell Deficiency
The evidence for the analyses related to nonpterygium LSCD was based on 3 prospective and 7 retrospective case series. Thus, the GRADE quality of evidence is very low, and any estimate of effect is very uncertain.
The analyses examining LSCT as an adjuvant treatment option for pterygium were based on 6 RCTs. The quality of evidence for the overall body of evidence for each treatment option comparison was assessed using the GRADE approach. In each of the comparisons, the quality of evidence was downgraded due to serious or very serious limitations in study quality (individual study quality was assessed using the Jadad scale, and an assessment of allocation concealment and the degree of loss to follow-up), which resulted in low- to moderate-quality GRADE evidence ratings (low-quality evidence for the CLAU and AMT and CLAU and MMC comparisons, and moderate-quality evidence for the CLAU and CAU comparison).
Ontario Health System Impact Analysis
Nonpterygium Limbal Stem Cell Deficiency
Since 1999, Ontario’s out-of-country (OOC) program has approved and reimbursed 8 patients for LSCTs and 1 patient for LSCT consultations. Similarly, most Canadian provinces have covered OOC or out-of-province LSCTs. Several corneal experts in Ontario have the expertise to perform LSCTs.
As there are no standard guidelines for LSCT, patients who receive transplants OOC may not receive care aligned with the best evidence. To date, many of the patients from Ontario who received OOC LSCTs received concurrent AMTs, and the evidence from this analysis questions the use of this procedure. In addition, 1 patient received a cultured LSCT, a procedure that is considered investigational. Many patients with LSCD have bilateral disease and therefore require allogeneic transplants. These patients will require systemic and topical immunosuppression for several years after the transplant, perhaps indefinitely. Thus, systemic side effects associated with immunosuppression are a potential concern, and patients must be monitored regularly.
Amniotic membrane transplantation is a common addition to many ocular surface reconstruction procedures, including LSCT. Amniotic membranes are recovered from human placentas from planned, uneventful caesarean sections. Before use, serological screening of the donor’s blood should be conducted. However, there is still a theoretical risk of disease transmission associated with this procedure.
Financial Impact
For the patients who were reimbursed for OOC LSCTs, the average cost of LSCT per eye was $18,735.20 Cdn (range, $8,219.54–$33,933.32). However, the actual cost per patient is much higher as these costs do not include consultations and follow-up visits, multiple LSCTs, and any additional procedures (e.g., corneal transplants) received during the course of treatment OOC. When these additional costs were considered, the average cost per patient was $57,583 Cdn (range, $8,219.54–$130,628.20).
The estimated average total cost per patient for performing LSCT in Ontario is $2,291.48 Cdn (range, $951.48–$4,538.48) including hospital and physician fees. This cost is based on the assumption that LSCT is technically similar to a corneal transplant, an assumption which needs to be verified. The cost does not include corneal transplantations, which some proportion of patients receiving a LSCT will require within several months of the limbal transplant.
Pterygium recurrence rates after surgical excision are high, ranging from 24% to 89%. However, according to clinical experts, the rate of recurrence is low in Ontario. While there is evidence that the prevalence of pterygium is higher in the “pterygium belt,” there was no evidence to suggest different recurrence rates or disease severity by location or climate.
Nonpterygium Limbal Stem Cell Deficiency
Successful LSCTs result in corneal re-epithelialization and improved vision in patients with LSCD. However, patients who received concurrent AMT had poorer long-term corneal surface improvements. Conjunctival-limbal autologous transplantation is the treatment option of choice, but if it is not possible, living-related or cadaveric allogeneic transplants can be used. The benefits of LSCT outweigh the risks and burdens, as shown in Executive Summary Table 1. According to GRADE, these recommendations are strong with low- to very low-quality evidence.
Benefits, Risks, and Burdens – Nonpterygium Limbal Stem Cell Deficiency
Short- and long-term improvement in corneal surface (stable, normal corneal epithelium and decreased vascularization and opacity)
Improvement in vision (visual acuity and functional vision)
Long-term complications are experienced by 8% to 16% of patients
Risks associated with long-term immunosuppression for recipients of allogeneic grafts
Potential risk of induced LSCD in donor eyes
High cost of treatment (average cost per patient via OOC program is $57,583; estimated cost of procedure in Ontario is $2,291.48)
Costs are expressed in Canadian dollars.
GRADE of recommendation: Strong recommendation, low-quality or very low-quality evidence
benefits clearly outweigh risks and burdens
case series studies
strong, but may change if higher-quality evidence becomes available
Conjunctival-limbal autologous transplantations significantly reduced the risk of pterygium recurrence compared with CAU. No other comparison yielded statistically significant results, but CLAU reduced the risk of recurrence compared with MMC. However, the benefit of LSCT in Ontario is uncertain as the severity and recurrence of pterygium in Ontario is unknown. The complication rates suggest that CLAU is a safe treatment option to prevent the recurrence of pterygium. According to GRADE, given the balance of the benefits, risks, and burdens, the recommendations are very weak with moderate quality evidence, as shown in Executive Summary Table 2.
Benefits, Risks, and Burdens – Pterygium
Reduced recurrence; however, if recurrence is low in Ontario, this benefit might be minimal
Long-term complications rare
Increased cost
GRADE of recommendation: Very weak recommendations, moderate quality evidence.
uncertainty in the estimates of benefits, risks, and burden; benefits, risks, and burden may be closely balanced
very weak, other alternatives may be equally reasonable
PMCID: PMC3377549  PMID: 23074512
15.  Enhanced Wound Healing, Kinase and Stem Cell Marker Expression in Diabetic Organ-Cultured Human Corneas Upon MMP-10 and Cathepsin F Gene Silencing 
Diabetic corneas overexpress proteinases including matrix metalloproteinase-10 (M10) and cathepsin F (CF). Our purpose was to assess if silencing M10 and CF in organ-cultured diabetic corneas using recombinant adenovirus (rAV)-driven small hairpin RNA (rAV-sh) would normalize slow wound healing, and diabetic and stem cell marker expression.
Sixteen pairs of organ-cultured autopsy human diabetic corneas (four per group) were treated with rAV-sh. Proteinase genes were silenced either separately, together, or both, in combination (Combo) with rAV-driven c-met gene overexpression. Fellow control corneas received rAV-EGFP. Quantitative RT-PCR confirmed small hairpin RNA (shRNA) silencing effect. Ten days after transfection, 5-mm epithelial wounds were made with n-heptanol and healing time recorded. Diabetic, signaling, and putative stem cell markers were studied by immunofluorescence of corneal cryostat sections.
Proteinase silencing reduced epithelial wound healing time versus rAV–enhanced green fluorescent protein (EGFP) control (23% for rAV-shM10, 31% for rAV-shCF, and 36% for rAV-shM10 + rAV-shCF). Combo treatment was even more efficient (55% reduction). Staining patterns of diabetic markers (α3β1 integrin and nidogen-1), and of activated epidermal growth factor receptor and its signaling target activated Akt were normalized upon rAV-sh treatment. Combo treatment also restored normal staining for activated p38. All treatments, especially the combined ones, increased diabetes-altered staining for putative limbal stem cell markers, ΔNp63α, ABCG2, keratins 15 and 17, and laminin γ3 chain.
Small hairpin RNA silencing of proteinases overexpressed in diabetic corneas enhanced corneal epithelial and stem cell marker staining and accelerated wound healing. Combined therapy with c-met overexpression was even more efficient. Specific corneal gene therapy has a potential for treating diabetic keratopathy.
Adenovirus-driven shRNA silencing of select proteinases upregulated in diabetic corneas restored normal wound healing time, the expression of diabetes-altered markers including limbal stem cell markers, and patterns of activated EGFR and Akt in human diabetic corneal organ cultures. The maximum effect was obtained combining proteinase shRNA with c-met overexpression.
PMCID: PMC3867183  PMID: 24255036
diabetic cornea; limbal stem cell; wound healing; MMP-10; cathepsin F; EGFR; Akt; p-38; keratin; organ culture; c-met; gene therapy
16.  Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice 
Cell and Tissue Research  2009;339(3):613-624.
Despite the mouse being an important laboratory species, little is known about changes in its extracellular matrix (ECM) during follicle and corpora lutea formation and regression. Follicle development was induced in mice (29 days of age/experimental day 0) by injections of pregnant mare’s serum gonadotrophin on days 0 and 1 and ovulation was induced by injection of human chorionic gonadotrophin on day 2. Ovaries were collected for immunohistochemistry (n=10 per group) on days 0, 2 and 5. Another group was mated and ovaries were examined on day 11 (n=7). Collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2 and perlecan were present in the follicular basal lamina of all developmental stages. Collagen type XVIII was only found in basal lamina of primordial, primary and some preantral follicles, whereas laminin α2 was only detected in some preantral and antral follicles. The focimatrix, a specialised matrix of the membrana granulosa, contained collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. In the corpora lutea, staining was restricted to capillary sub-endothelial basal laminas containing collagen type IV α1 and α2, laminin α1, β1 and γ1 chains, nidogens 1 and 2, perlecan and collagen type XVIII. Laminins α4 and α5 were not immunolocalised to any structure in the mouse ovary. The ECM composition of the mouse ovary has similarities to, but also major differences from, other species with respect to nidogens 1 and 2 and perlecan.
PMCID: PMC2831189  PMID: 20033213
Follicle; Corpus luteum; Extracellular matrix; Collagen; Laminin; Perlecan; Nidogen; Mouse (CBAxC57BL/6F1)
17.  Reconstruction of a human hemicornea through natural scaffolds compatible with the growth of corneal epithelial stem cells and stromal keratocytes 
Molecular Vision  2009;15:2084-2093.
To reconstruct a human hemicornea in vitro by means of limbal stem cells cultured onto human keratoplasty lenticules (HKLs) and to obtain a natural corneal graft for clinical applications.
Limbal stem cells were seeded onto HKLs with or without the presence of feeder layers of lethally irradiated 3T3-J2 cells and compared with the current “gold standard” scaffold, i.e., the fibrin glue. The effects of the scaffold on the preservation of stemness and/or induction of differentiation pathways were investigated through analysis of a variety of markers, including p63 and ΔNp63α for stemness, 14-3-3σ for early differentiation, keratins 3, 14, 12, and 19 to determine cell phenotype, and α6, β1, and β4 integrins to evaluate interactions with the stroma. Integrity of the stroma was assessed through analysis of keratan sulfate, CD-34 and aldehyde dehydrogenase 3A1 (ALDH3A1) (for keratocytes), visual system homeobox 1 (VSX1), and alpha-smooth muscle actin (α-SMA) (for fibroblasts and myofibroblasts). The structural properties of the reconstructed “hemicornea” were investigated through scanning electron microscopy. To evaluate the preservation of the stemness potential, cells were trypsinized from each scaffold and clonogenic/proliferative characteristics analyzed.
Limbal stem cells expanded onto HKLs gave rise to a stratified squamous keratinized epithelium morphologically similar to that of normal corneas. The resulting corneal epithelium was characterized by basal expression of p63 and ΔNp63α, while expression of 14-3-3σ, keratin 3, and keratin 12 was found in the upper cell layers. The basal cuboidal epithelial cells were anchored to the basement membrane and expressed keratin 14 and α6, β1, and β4 integrins. In the stroma of HKLs, keratocytes maintained the biosynthetic and phenotypic appearances typical of resting/quiescent cells and expressed keratan sulfate, CD-34, and ALDH3A1. Fibroblastic transformation was observed with the appearance of VSX1 and α-SMA. Scanning electron microscopy analysis showed that HKLs maintained their native conformation with collagen fibrils interconnected to the network and parallel to the corneal surface. HKLs did not alter the clonogenic/proliferative capacity of limbal stem cells. No differences were seen when HKL was compared to fibrin glue, one of the scaffolds currently used for limbal stem cell transplantation.
Our findings demonstrate that HKL could be a suitable scaffold for corneal epithelial stem cells as they were shown to proliferate, express differentiation markers, and bind to the underlying stroma with no alterations in clonogenic potential. HKLs have some advantages over currently used scaffolds, such as the possibility to allow cell growth with no feeder layers, to be freeze dried, and to preserve the integrity and viability of stromal keratocytes. The development of a tissue-engineered “hemicornea” might offer new therapeutic perspectives to patients affected by total limbal stem cell deficiency with stromal scarring.
PMCID: PMC2765239  PMID: 19862337
18.  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
19.  Imaging the Intact Mouse Cornea Using Coherent Anti-Stokes Raman scattering (CARS) 
The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon–hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents.
Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed.
Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers.
The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques.
Coherent anti-Stokes Raman scattering was used to image cells of the corneal epithelium, stroma, and endothelium in freshly isolated mouse tissue. Three-dimensional structural information was deciphered without the need for histologic processing.
PMCID: PMC3736760  PMID: 23821187
cornea; corneal stroma; coherent anti-Stokes Raman scattering (CARS)
20.  Gene Structure and Functional Analysis of the Mouse Nidogen-2 Gene: Nidogen-2 Is Not Essential for Basement Membrane Formation in Mice 
Molecular and Cellular Biology  2002;22(19):6820-6830.
Nidogens are highly conserved proteins in vertebrates and invertebrates and are found in almost all basement membranes. According to the classical hypothesis of basement membrane organization, nidogens connect the laminin and collagen IV networks, so stabilizing the basement membrane, and integrate other proteins. In mammals two nidogen proteins, nidogen-1 and nidogen-2, have been discovered. Nidogen-2 is typically enriched in endothelial basement membranes, whereas nidogen-1 shows broader localization in most basement membranes. Surprisingly, analysis of nidogen-1 gene knockout mice presented evidence that nidogen-1 is not essential for basement membrane formation and may be compensated for by nidogen-2. In order to assess the structure and in vivo function of the nidogen-2 gene in mice, we cloned the gene and determined its structure and chromosomal location. Next we analyzed mice carrying an insertional mutation in the nidogen-2 gene that was generated by the secretory gene trap approach. Our molecular and biochemical characterization identified the mutation as a phenotypic null allele. Nidogen-2-deficient mice show no overt abnormalities and are fertile, and basement membranes appear normal by ultrastructural analysis and immunostaining. Nidogen-2 deficiency does not lead to hemorrhages in mice as one may have expected. Our results show that nidogen-2 is not essential for basement membrane formation or maintenance.
PMCID: PMC135501  PMID: 12215539
21.  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
22.  Collagen Cross-Linking Using Riboflavin and Ultraviolet-A for Corneal Thinning Disorders 
Executive Summary
The main objectives for this evidence-based analysis were to determine the safety and effectiveness of photochemical corneal collagen cross-linking with riboflavin (vitamin B2) and ultraviolet-A radiation, referred to as CXL, for the management of corneal thinning disease conditions. The comparative safety and effectiveness of corneal cross-linking with other minimally invasive treatments such as intrastromal corneal rings was also reviewed. The Medical Advisory Secretariat (MAS) evidence-based analysis was performed to support public financing decisions.
Subject of the Evidence-Based Analysis
The primary treatment objective for corneal cross-linking is to increase the strength of the corneal stroma, thereby stabilizing the underlying disease process. At the present time, it is the only procedure that treats the underlying disease condition. The proposed advantages for corneal cross-linking are that the procedure is minimally invasive, safe and effective, and it can potentially delay or defer the need for a corneal transplant. In addition, corneal cross-linking does not adversely affect subsequent surgical approaches, if they are necessary, or interfere with corneal transplants. The evidence for these claims for corneal cross-linking in the management of corneal thinning disorders such as keratoconus will be the focus of this review.
The specific research questions for the evidence review were as follows:
Technical: How technically demanding is corneal cross-linking and what are the operative risks?
Safety: What is known about the broader safety profile of corneal cross-linking?
Effectiveness - Corneal Surface Topographic Affects:
What are the corneal surface remodeling effects of corneal cross-linking?
Do these changes interfere with subsequent interventions, particularly corneal transplant known as penetrating keratoplasty (PKP)?
Effectiveness -Visual Acuity:
What impacts does the remodeling have on visual acuity?
Are these impacts predictable, stable, adjustable and durable?
Effectiveness - Refractive Outcomes: What impact does remodeling have on refractive outcomes?
Effectiveness - Visual Quality (Symptoms): What impact does corneal cross-linking have on vision quality such as contrast vision, and decreased visual symptoms (halos, fluctuating vision)?
Effectiveness - Contact lens tolerance: To what extent does contact lens intolerance improve after corneal cross-linking?
Vision-Related QOL: What is the impact of corneal cross-linking on functional visual rehabilitation and quality of life?
Patient satisfaction: Are patients satisfied with their vision following the procedure?
Disease Process:
What impact does corneal cross-linking have on the underling corneal thinning disease process?
Does corneal cross-linking delay or defer the need for a corneal transplant?
What is the comparative safety and effectiveness of corneal cross-linking compared with other minimally invasive treatments for corneal ectasia such as intrastromal corneal rings?
Clinical Need: Target Population and Condition
Corneal ectasia (thinning) disorders represent a range of disorders involving either primary disease conditions, such as keratoconus (KC) and pellucid marginal corneal degeneration, or secondary iatrogenic conditions, such as corneal thinning occurring after laser in situ keratomileusis (LASIK) refractive surgery.
Corneal thinning is a disease that occurs when the normally round dome-shaped cornea progressively thins causing a cone-like bulge or forward protrusion in response to the normal pressure of the eye. The thinning occurs primarily in the stroma layers and is believed to be a breakdown in the collagen process. This bulging can lead to irregular astigmatism or shape of the cornea. Because the anterior part of the cornea is responsible for most of the focusing of the light on the retina, this can then result in loss of visual acuity. The reduced visual acuity can make even simple daily tasks, such as driving, watching television or reading, difficult to perform.
Keratoconus is the most common form of corneal thinning disorder and involves a noninflammatory chronic disease process of progressive corneal thinning. Although the specific cause for the biomechanical alterations in the corneal stroma is unknown, there is a growing body of evidence suggesting that genetic factors may play an important role. Keratoconus is a rare disease (< 0.05% of the population) and is unique among chronic eye diseases because it has an early onset, with a median age of 25 years. Disease management for this condition follows a step-wise approach depending on disease severity. Contact lenses are the primary treatment of choice when there is irregular astigmatism associated with the disease. Patients are referred for corneal transplants as a last option when they can no longer tolerate contact lenses or when lenses no longer provide adequate vision.
Keratoconus is one of the leading indications for corneal transplants and has been so for the last 3 decades. Despite the high success rate of corneal transplants (up to 20 years) there are reasons to defer it as long as possible. Patients with keratoconus are generally young and a longer-term graft survival of at least 30 or 40 years may be necessary. The surgery itself involves lengthy time off work and postsurgery, while potential complications include long-term steroid use, secondary cataracts, and glaucoma. After a corneal transplant, keratoconus may recur resulting in a need for subsequent interventions. Residual refractive errors and astigmatism can remain challenges after transplantation, and high refractive surgery and regraft rates in KC patients have been reported. Visual rehabilitation or recovery of visual acuity after transplant may be slow and/or unsatisfactory to patients.
Description of Technology/Therapy
Corneal cross-linking involves the use of riboflavin (vitamin B2) and ultraviolet-A (UVA) radiation. A UVA irradiation device known as the CXL® device (license number 77989) by ACCUTECH Medical Technologies Inc. has been licensed by Health Canada as a Class II device since September 19, 2008. An illumination device that emits homogeneous UVA, in combination with any generic form of riboflavin, is licensed by Health Canada for the indication to slow or stop the progression of corneal thinning caused by progressive keratectasia, iatrogenic keratectasia after laser-assisted in situ keratomileusis (LASIK) and pellucid marginal degeneration. The same device is named the UV-X® device by IROCMedical, with approvals in Argentina, the European Union and Australia.
UVA devices all use light emitting diodes to generate UVA at a wavelength of 360-380 microns but vary in the number of diodes (5 to 25), focusing systems, working distance, beam diameter, beam uniformity and extent to which the operator can vary the parameters. In Ontario, CXL is currently offered at over 15 private eye clinics by refractive surgeons and ophthalmologists.
The treatment is an outpatient procedure generally performed with topical anesthesia. The treatment consists of several well defined procedures. The epithelial cell layer is first removed, often using a blunt spatula in a 9.0 mm diameter under sterile conditions. This step is followed by the application of topical 0.1% riboflavin (vitamin B2) solution every 3 to 5 minutes for 25 minutes to ensure that the corneal stroma is fully penetrated. A solid-state UVA light source with a wavelength of 370 nm (maximum absorption of riboflavin) and an irradiance of 3 mW/cm2 is used to irradiate the central cornea. Following treatment, a soft bandage lens is applied and prescriptions are given for oral pain medications, preservative-free tears, anti-inflammatory drops (preferably not nonsteroidal anti-inflammatory drugs, or NSAIDs) and antibiotic eye drops. Patients are recalled 1 week following the procedure to evaluate re-epithelialization and they are followed-up subsequently.
Evidence-Based Analysis Methods
A literature search was conducted on photochemical corneal collagen cross-linking with riboflavin (vitamin B2) and ultraviolet-A for the management of corneal thinning disorders using a search strategy with appropriate keywords and subject headings for CXL for literature published up until April 17, 2011. The literature search for this Health Technology Assessment (HTA) review was performed using the Cochrane Library, the Emergency Care Research Institute (ECRI) and the Centre for Reviews and Dissemination. The websites of several other health technology agencies were also reviewed, including the Canadian Agency for Drugs and Technologies in Health (CADTH) and the United Kingdom’s National Institute for Clinical Excellence (NICE). The databases searched included OVID MEDLINE, MEDLINE IN-Process and other Non-Indexed Citations such as EMBASE.
As the evidence review included an intervention for a rare condition, case series and case reports, particularly for complications and adverse events, were reviewed. A total of 316 citations were identified and all abstracts were reviewed by a single reviewer for eligibility. For those studies meeting the eligibility criteria, full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search.
Inclusion Criteria
English-language reports and human studies
patients with any corneal thinning disorder
reports with CXL procedures used alone or in conjunction with other interventions
original reports with defined study methodology
reports including standardized measurements on outcome events such as technical success, safety effectiveness, durability, vision quality of life or patient satisfaction
systematic reviews, meta-analyses, randomized controlled trials, observational studies, retrospective analyses, case series, or case reports for complications and adverse events
Exclusion Criteria
nonsystematic reviews, letters, comments and editorials
reports not involving outcome events such as safety, effectiveness, durability, vision quality or patient satisfaction following an intervention with corneal implants
reports not involving corneal thinning disorders and an intervention involving CXL
Summary of Evidence Findings
In the Medical Advisory Secretariat evidence review on corneal cross-linking, 65 reports (16 case reports) involving 1403 patients were identified on the use of CXL for managing corneal thinning disorders. The reports were summarized according to their primary clinical indication, whether or not secondary interventions were used in conjunction with CXL (referred to as CXL-Plus) and whether or not it was a safety-related report.
The safety review was based on information from the cohort studies evaluating effectiveness, clinical studies evaluating safety, treatment response or recovery, and published case reports of complications. Complications, such as infection and noninfectious keratitis (inflammatory response), reported in case reports, generally occurred in the first week and were successfully treated with topical antibiotics and steroids. Other complications, such as the cytotoxic effects on the targeted corneal stroma, occurred as side effects of the photo-oxidative process generated by riboflavin and ultraviolet-A and were usually reversible.
The reports on treatment effectiveness involved 15 pre-post longitudinal cohort follow-up studies ranging from follow-up of patients’ treated eye only, follow-up in both the treated and untreated fellow-eye; and follow-up in the treated eye only and a control group not receiving treatment. One study was a 3-arm randomized control study (RCT) involving 2 comparators: one comparator was a sham treatment in which one eye was treated with riboflavin only; and the other comparator was the untreated fellow-eye. The outcomes reported across the studies involved statistically significant and clinically relevant improvements in corneal topography and refraction after CXL. In addition, improvements in treated eyes were accompanied by worsening outcomes in the untreated fellow-eyes. Improvements in corneal topography reported at 6 months were maintained at 1- and 2-year follow-up. Visual acuity, although not always improved, was infrequently reported as vision loss. Additional procedures such as the use of intrastromal corneal ring segments, intraocular lenses and refractive surgical practices were reported to result in additional improvements in topography and visual acuity after CXL.
Considerations for Ontario Health System
The total costs of providing CXL therapy to keratoconus patients in Ontario was calculated based on estimated physician, clinic, and medication costs. The total cost per patient was approximately $1,036 for the treatment of one eye, and $1,751 for the treatment of both eyes. The prevalence of keratoconus was estimated at 4,047 patients in FY2011, with an anticipated annual incidence (new cases) of about 148 cases. After distributing the costs of CXL therapy for the FY2011 prevalent keratoconus population over the next 3 years, the estimated average annual cost was approximately $2.1 million, of which about $1.3 million would be physician costs specifically.
Corneal cross-linking effectively stabilizes the underlying disease, and in some cases reverses disease progression as measured by key corneal topographic measures. The affects of CXL on visual acuity are less predictable and the use of adjunct interventions with CXL, such as intrastromal corneal ring segments, refractive surgery, and intraocular lens implants are increasingly employed to both stabilize disease and restore visual acuity. Although the use of adjunct interventions have been shown to result in additional clinical benefit, the order, timing, and risks of performing adjunctive interventions have not been well established.
Although there is potential for serious adverse events with corneal UVA irradiation and photochemical reactions, there have been few reported complications. Those that have occurred tended to be related to side effects of the induced photochemical reactions and were generally reversible. However, to ensure that there are minimal complications with the use of CXL and irradiation, strict adherence to defined CXL procedural protocols is essential.
Keratoconus, corneal cross-linking, corneal topography, corneal transplant, visual acuity, refractive error.
PMCID: PMC3377552  PMID: 23074417
23.  Targeted Deletion of AP-2α Leads to Disruption in Corneal Epithelial Cell Integrity and Defects in the Corneal Stroma 
The present study was undertaken to create a conditional knockout of AP-2α in the corneal epithelium.
A line of mice expressing Cre-recombinase specifically in the early lens placode was crossed with mice in which the AP-2α allele is flanked by two loxP sites. The resultant Le-AP-2α mutants exhibited a targeted deletion of AP-2α in lens placode derivatives, including the differentiating corneal epithelium.
The Le-AP-2α mutant mice were viable and had a normal lifespan. The adult corneal epithelium exhibited a variation in the number of stratified epithelial layers, ranging from 2 to 10 cell layers. A substantial decrease in expression of the cell–cell adhesion molecule, E-cadherin, was observed in all layers of the Le-AP-2α mutant corneal epithelium. The basement membrane, or Bowman's layer, was thinner in the mutant cornea and in many regions was discontinuous. These defects corresponded with altered distribution of laminin and entactin, and to a lesser degree, type IV collagen. The Le-AP-2α mutant cornea also exhibited stromal defects, including disrupted organization of the collagen lamellae and accumulation of fibroblasts beneath the epithelium that showed increased immunoreactivity for proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), p-Smad2, and TGF-β2.
In the absence of AP-2α, the corneal epithelium exhibits altered cell adhesion and integrity and defects in its underlying basement membrane. These defects likely caused the alterations in the corneal stroma.
PMCID: PMC2517422  PMID: 16186342
24.  Corneal stromal changes following reconstruction by ex vivo expanded limbal epithelial cells in rabbits with total limbal stem cell deficiency 
The British Journal of Ophthalmology  2003;87(12):1509-1514.
Aim: To study corneal stromal changes and the presence of myofibroblasts after transplantation of ex vivo expanded limbal epithelium.
Methods: A state of limbal deficiency was induced in 16 rabbits. After transplantation with autologous ex vivo expanded limbal epithelium on amniotic membrane (AM), their clinical outcomes were classified as success, partial success or failure according to surface smoothness, stromal clarity, and vascularisation. Clinical outcomes were correlated with phenotypic outcomes of corneal, conjunctival, or mixed epithelium, defined by expression of K3 keratin or MUC5AC. Immunostaining was performed with antibodies against collagen IV, fibronectin, and α-smooth muscle actin (α-SMA) to assess stromal wound remodelling.
Results: Rabbits were sacrificed after a mean follow up of 10 (SD 3.3) months. Collagen IV, expressed in the basement membrane of all three groups, was found in the stroma of the partial success, but not in that of the success or the failure. Fibronectin was absent in the success and the failure, but expressed in the stroma of the partial success. α-SMA was expressed in superficial stroma of the partial success, but suppressed in areas with AM remnants.
Conclusion: Restoration of a clear and transparent cornea is associated with a normal corneal epithelium and complete wound remodelling. In contrast, wound healing remains active and incomplete in conjunctivalised corneas, which remain opaque with myofibroblasts.
PMCID: PMC1920578  PMID: 14660463
amniotic membrane; epithelial cells; limbus; limbal stem cell deficiency; wound healing; rabbits
25.  High resolution immunoelectron microscopic localization of functional domains of laminin, nidogen, and heparan sulfate proteoglycan in epithelial basement membrane of mouse cornea reveals different topological orientations 
The Journal of Cell Biology  1988;107(4):1599-1610.
Thin and ultrathin cryosections of mouse cornea were labeled with affinity-purified antibodies directed against either laminin, its central segments (domain 1), the end of its long arm (domain 3), the end of one of its short arms (domain 4), nidogen, or low density heparan sulfate proteoglycan. All basement membrane proteins are detected by indirect immunofluorescence exclusively in the epithelial basement membrane, in Descemet's membrane, and in small amorphous plaques located in the stroma. Immunoelectron microscopy using the protein A-gold technique demonstrated laminin domain 1 and nidogen in a narrow segment of the lamina densa at the junction to the lamina lucida within the epithelial basement membrane. Domain 3 shows three preferred locations at both the cellular and stromal boundaries of the epithelial basement membrane and in its center. Domain 4 is located predominantly in the lamina lucida and the adjacent half of the lamina densa. The low density heparan sulfate proteoglycan is found all across the basement membrane showing a similar uniform distribution as with antibodies against the whole laminin molecule. In Descemet's membrane an even distribution was found with all these antibodies. It is concluded that within the epithelial basement membrane the center of the laminin molecule is located near the lamina densa/lamina lucida junction and that its long arm favors three major orientations. One is close to the cell surface indicating binding to a cell receptor, while the other two are directed to internal matrix structures. The apparent codistribution of laminin domain 1 and nidogen agrees with biochemical evidence that nidogen binds to this domain.
PMCID: PMC2115247  PMID: 2459133

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