Cultivated autologous limbal epithelial cell (CALEC) transplantation: Development of manufacturing process and clinical evaluation of feasibility and safety.

To treat unilateral limbal stem cell (LSC) deficiency, we developed cultivated autologous limbal epithelial cells (CALEC) using an innovative xenobiotic-free, serum-free, antibiotic-free, two-step manufacturing process for LSC isolation and expansion onto human amniotic membrane with rigorous quality control in a good manufacturing practices facility. Limbal biopsies were used to generate CALEC constructs, and final grafts were evaluated by noninvasive scanning microscopy and tested for viability and sterility. Cultivated cells maintained epithelial cell phenotype with colony-forming and proliferative capacities. Analysis of LSC biomarkers showed preservation of "stemness." After preclinical development, a phase 1 clinical trial enrolled five patients with unilateral LSC deficiency. Four of these patients received CALEC transplants, establishing preliminary feasibility. Clinical case histories are reported, with no primary safety events. On the basis of these results, a second recruitment phase of the trial was opened to provide longer term safety and efficacy data on more patients.

Large-Scale Production of Wholly Cellular Bioinks via the Optimization of Human Induced Pluripotent Stem Cell Aggregate Culture in Automated Bioreactors.

Combining the sustainable culture of billions of human cells and the bioprinting of wholly cellular bioinks offers a pathway toward organ-scale tissue engineering. Traditional 2D culture methods are not inherently scalable due to cost, space, and handling constraints. Here, the suspension culture of human induced pluripotent stem cell-derived aggregates (hAs) is optimized using an automated 250 mL stirred tank bioreactor system. Cell yield, aggregate morphology, and pluripotency marker expression are maintained over three serial passages in two distinct cell lines. Furthermore, it is demonstrated that the same optimized parameters can be scaled to an automated 1 L stirred tank bioreactor system. This 4-day culture results in a 16.6- to 20.4-fold expansion of cells, generating approximately 4 billion cells per vessel, while maintaining >94% expression of pluripotency markers. The pluripotent aggregates can be subsequently differentiated into derivatives of the three germ layers, including cardiac aggregates, and vascular, cortical and intestinal organoids. Finally, the aggregates are compacted into a wholly cellular bioink for rheological characterization and 3D bioprinting. The printed hAs are subsequently differentiated into neuronal and vascular tissue. This work demonstrates an optimized suspension culture-to-3D bioprinting pipeline that enables a sustainable approach to billion cell-scale organ engineering.

Vasoactive Intestinal Peptide Promotes Corneal Allograft Survival.

Corneal transplantation is the most prevalent form of tissue transplantation. The success of corneal transplantation mainly relies on the integrity of corneal endothelial cells (CEnCs), which maintain graft transparency. CEnC density decreases significantly after corneal transplantation even in the absence of graft rejection. To date, different strategies have been used to enhance CEnC survival. The neuropeptide vasoactive intestinal peptide (VIP) improves CEnC integrity during donor cornea tissue storage and protects CEnCs against oxidative stress-induced apoptosis. However, little is known about the effect of exogenous administration of VIP on corneal transplant outcomes. We found that VIP significantly accelerates endothelial wound closure and suppresses interferon-γ- and tumor necrosis factor-α-induced CEnC apoptosis in vitro in a dose-dependent manner. In addition, we found that intracameral administration of VIP to mice undergoing syngeneic corneal transplantation with endothelial injury increases CEnC density and decreases graft opacity scores. Finally, using a mouse model of allogeneic corneal transplantation, we found for the first time that treatment with VIP significantly suppresses posttransplantation CEnC loss and improves corneal allograft survival.

Association of the Gutta-Induced Microenvironment With Corneal Endothelial Cell Behavior and Demise in Fuchs Endothelial Corneal Dystrophy.

Importance: The number and size of guttae increase over time in Fuchs endothelial corneal dystrophy (FECD); however, the association between these physical parameters and disease pathogenesis is unclear. Objective: To determine the role of guttae in corneal endothelial cell function. Design, Settings, and Participants: In an in vitro model, cells from a human corneal endothelial cell line, HCENC-21T, were seeded on decellularized normal (n = 30) and FECD (n = 70) endothelial basement (Descemet) membranes (DMs). Normal human corneas were sent to our laboratory from 3 sources. The study took place at the Schepens Eye Research Institute, Massachusetts Eye and Ear, Boston, and was performed from September 2015 to July 2017. Normal DMs were obtained from 3 different tissue banks and FECD-DMs were obtained from patients undergoing endothelial keratoplasty in 2 departments. Main Outcomes and Measures: Endothelial cell shape, growth, and migration were assessed by live-cell imaging, and gene expression analysis as a function of guttae diameter was assessed by laser capture microscopy. Results: Mean (SD) age of normal-DMs donors was 65.6 (4.4) years (16 women [53%]), and mean (SD) age of FECD-DMs donors was 68.9 (10.6) years (43 women [61%]). Cells covered a greater area (mean [SD], 97.7% [8.5%]) with a greater mean (SD) number of cells (2083 [153] cells/mm2) on the normal DMs compared with the FECD DMs (72.8% [11%]; P = .02 and 1541 [221] cells/mm2 221/mm2; P = .01, respectively). Differences in endothelial cell growth over guttae were observed on FECD DMs depending on the guttae diameter. Guttae with a mean (SD) diameter of 10.5 (2.9) µm did not impede cell growth, whereas those with a diameter of 21.1 (4.9) µm were covered only by the cell cytoplasm. Guttae with the largest mean (SD) diameter, 31.8 (3.8) µm, were not covered by cells, which instead surrounded them in a rosette pattern. Moreover, cells adjacent to large guttae upregulated αSMA, N-cadherin, Snail1, and NOX4 genes compared with ones grown on normal DMs or small guttae. Furthermore, large guttae induced TUNEL-positive apoptosis in a rosette pattern, similar to ex vivo FECD specimens. Conclusions and Relevance: These findings highlight the important role of guttae in endothelial cell growth, migration, and survival. These data suggest that cell therapy procedures in FECD might be guided by the diameter of the host guttae if subsequent clinical studies confirm these laboratory findings.

NQO1 downregulation potentiates menadione-induced endothelial-mesenchymal transition during rosette formation in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a genetic and oxidative stress disorder of post-mitotic human corneal endothelial cells (HCEnCs), which normally exhibit hexagonal shape and form a compact monolayer compatible with normal corneal functioning and clear vision. FECD is associated with increased DNA damage, which in turn leads to HCEnC loss, resulting in the formation rosettes and aberrant extracellular matrix (ECM) deposition in the form of pro-fibrotic guttae. Since the mechanism of ECM deposition in FECD is currently unknown, we aimed to investigate the role of endothelial-mesenchymal transition (EMT) in FECD using a previously established cellular in vitro model that recapitulates the characteristic rosette formation, by employing menadione (MN)-induced oxidative stress. We demonstrate that MN treatment alone, or a combination of MN and TGF-ß1 induces reactive oxygen species (ROS), cell death, and EMT in HCEnCs during rosette formation, resulting in upregulation of EMT- and FECD-associated markers such as Snail1, N-cadherin, ZEB1, and transforming growth factor-beta-induced (TGFßI), respectively. Additionally, FECD ex vivo specimens displayed a loss of organized junctional staining of plasma membrane-bound N-cadherin, with corresponding increase in fibronectin and Snail1 compared to ex vivo controls. Addition of N-acetylcysteine (NAC) downregulated all EMT markers and abolished rosette formation. Loss of NQO1, a metabolizing enzyme of MN, led to greater increase in intracellular ROS levels as well as a significant upregulation of Snail1, fibronectin, and N-cadherin compared to normal cells, indicating that NQO1 regulates Snail1-mediated EMT. This study provides first line evidence that MN-induced oxidative stress leads to EMT in corneal endothelial cells, and the effect of which is further potentiated when redox cycling activity of MN is enhanced by the absence of NQO1. Given that NAC inhibits Snail-mediated EMT, this may be a potential therapeutic intervention for FECD.

Limbal Cysts: A Subset Exhibiting Cornea-Specific Cytokeratins.

Two cases of limbal cysts lined by nonkeratinizing epithelium were studied with a panel of cytokeratins. One was a long-standing lesion in a 30-year-old man, whereas the other was excised from a 40-year-old man following pterygium surgery. Each cyst was immunostained with a panel of cytokeratins that were specific exclusively and separately for corneal and conjunctival epithelia. The epithelial lining of each cyst was CK12 positive for corneal epithelium and CK13 negative for conjunctival epithelium. It is hypothesized that a subset of corneoscleral cysts contain corneal epithelium, probably derived from a type of limbal stem cell differentiation.

Activation of mitophagy leads to decline in Mfn2 and loss of mitochondrial mass in Fuchs endothelial corneal dystrophy.

Human corneal endothelial cells (HCEnCs) are terminally differentiated cells that have limited regenerative potential. The large numbers of mitochondria in HCEnCs are critical for pump and barrier function required for corneal hydration and transparency. Fuchs Endothelial Corneal Dystrophy (FECD) is a highly prevalent late-onset oxidative stress disorder characterized by progressive loss of HCEnCs. We previously reported increased mitochondrial fragmentation and reduced ATP and mtDNA copy number in FECD. Herein, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitochondrial depolarization decreased mitochondrial mass and Mfn2 levels, which were rescued with mitophagy blocker, bafilomycin, in FECD. Moreover, electron transport chain complex (I, V) decrease in FECD indicated deficient mitochondrial bioenergetics. Transmission electron microscopy of FECD tissues displayed an increased number of autophagic vacuoles containing degenerated and swollen mitochondria with cristolysis. An elevation of LC3-II and LAMP1 and downregulation of Mfn2 in mitochondrial fractions suggested that loss of fusion capacity targets fragmented mitochondria to the pre-autophagic pool and upregulates mitophagy. CCCP-induced mitochondrial fragmentation leads to Mfn2 and LC3 co-localization without activation of proteosome, suggesting a novel Mfn2 degradation pathway via mitophagy. These data indicate constitutive activation of mitophagy results in reduction of mitochondrial mass and abrogates cellular bioenergetics during degeneration of post-mitotic cells of ocular tissue.

Hepatocyte Growth Factor Suppresses Inflammation and Promotes Epithelium Repair in Corneal Injury.

Corneal injuries are among the major causes of ocular morbidity and vision impairment. Optimal epithelial wound healing is critical for the integrity and transparency of the cornea after injury. Hepatocyte growth factor (HGF) is a mitogen and motility factor that primarily regulates epithelial cell function. Herein, we investigate the effect of HGF on proliferation of corneal epithelial cells (CECs) in inflamed conditions both in vitro and in vivo. We demonstrate that HGF not only promotes CEC proliferation in homeostatic conditions but also reverses the anti-proliferative effect of the inflammatory environment on these cells. Furthermore, using a mouse model of ocular injury, we show that HGF treatment suppresses ocular inflammation and actively augments CEC proliferation, leading to improved and accelerated corneal epithelial repair. These findings have potential translational implications and could provide a framework for the development of novel HGF-based therapies for corneal epithelial defects.

Restoration of Corneal Transparency by Mesenchymal Stem Cells.

Transparency of the cornea is indispensable for optimal vision. Ocular trauma is a leading cause of corneal opacity, leading to 25 million cases of blindness annually. Recently, mesenchymal stem cells (MSCs) have gained prominence due to their inflammation-suppressing and tissue repair functions. Here, we investigate the potential of MSCs to restore corneal transparency following ocular injury. Using an in vivo mouse model of ocular injury, we report that MSCs have the capacity to restore corneal transparency by secreting high levels of hepatocyte growth factor (HGF). Interestingly, our data also show that HGF alone can restore corneal transparency, an observation that has translational implications for the development of HGF-based therapy.

Existence of Neural Crest-Derived Progenitor Cells in Normal and Fuchs Endothelial Dystrophy Corneal Endothelium.

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma, aging, and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein, we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging, propensity to form spheres, and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2, OCT4, LGR5, TP63 (p63), as well as neural crest marker genes PSIP1 (p75(NTR)), PAX3, SOX9, AP2B1 (AP-2ß), and NES, generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2, ß-III tubulin, and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion, we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies.

Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

Science and Art of Cell-Based Ocular Surface Regeneration.

The potential cause of blindness worldwide includes diseases of the cornea, ocular surface (limbal stem cell deficiency, allergic conjunctivitis, dry eye diseases), and retinal diseases. The presence of stem cells (limbal stem cells) in the basal region of the limbus makes it an important tool for the ocular regeneration and also in maintaining the transparency of eye by replacing the corneal epithelium continuously. Various surgical modalities have been developed like cultured limbal epithelial transplantation, cultured oral mucosal epithelial transplantation, simple limbal epithelial transplantation, etc., utilizing the cell-based regenerative properties to treat limbal disorder. Cell-based therapies for ocular repair and regeneration comprise a major hope by therapies involving the mesenchymal stem cells, embryonic stem cells, and limbal stem cells for the restoration of vision in individuals whose ocular tissue has been irreversibly damaged by disease or trauma. This review explores critical needs in human disease mainly the ocular problem where cell-based therapeutics is exceptionally well suited and also the use of animal models, various artificial scaffolds, as well as advancement in clinical technique to challenge the current demand to overcome corneal blindness.

Mesenchymal stem cells home to inflamed ocular surface and suppress allosensitization in corneal transplantation.

PURPOSE: To investigate whether systemically injected syngeneic mesenchymal stem cells (MSCs) can home to the transplanted cornea, suppress induction of alloimmunity, and promote allograft survival. METHODS: Mesenchymal stem cells were generated from bone marrow of wild-type BALB/c or GFP (green fluorescent protein)+ C57BL/6 mice, and 1×10(6) cells were intravenously injected to allografted recipients 3 hours after surgery. Mesenchymal stem cells homing to the cornea were examined at day 3 post transplantation by immunohistochemistry. MHC (major histocompatibility complex) II+CD11c+ cells were detected in the cornea and lymph nodes (LNs) 14 days post transplantation using flow cytometry. Cytokine expression of bone marrow-derived dendritic cells (BMDCs) was determined using real-time PCR. ELISPOT assay was used to assess indirect and direct host T cell allosensitization, and graft survival was evaluated by slit-lamp biomicroscopy weekly up to 8 weeks. RESULTS: Intravenously injected GFP+ MSCs were found in abundance in the transplanted cornea, conjunctiva, and LNs, but not in the ungrafted (contralateral) tissue. The frequencies of mature MHC II+CD11c+ antigen-presenting cells (APCs) were substantially decreased in the corneas and draining LNs of MSC-injected allograft recipients compared to control recipients. Maturation and function of in vitro cultured BMDCs were decreased when cocultured with MSCs. Draining LNs of MSC-injected allograft recipients showed lower frequencies of IFNγ-secreting Th1 cells compared to the control group. Allograft survival rate was significantly higher in MSC-injected recipients compared to non-MSC-injected recipients. CONCLUSIONS: Our data demonstrate that systemically administered MSCs specifically home to the inflamed ocular surface and promote allograft survival by inhibiting APC maturation and induction of alloreactive T cells.

Comparative transcriptomic analysis of cultivated limbal epithelium and donor corneal tissue reveals altered wound healing gene expression.

PURPOSE: The improved surgical outcomes associated with transplantation of cultivated amniotic membrane expanded limbal epithelium (AMLE) compared to traditional donor methods has led to substantial adoption of this technique for treatment of limbal stem cell deficiency. METHODS: The mRNA expression profiles of AMLE and CE were assayed using microarrays. Transcripts with a 1.5-fold change in either direction in addition to a Bonferroni adjusted P value < 0.05 were considered to be differentially expressed. Expression changes detected by microarray profiling and important corneal-limbal markers were assessed using quantitative real-time PCR (qRT-PCR) and immunofluorescence staining. RESULTS: A total of 487 probe sets (319 upregulated and 168 downregulated) were found to be differentially expressed between AMLE and CE. Enrichment analysis revealed significant overrepresentation of multiple biological processes (e.g., response to wounding, wound healing, and regulation of cell morphogenesis) within the differentially expressed gene list. The expression of a number of genes that were upregulated (ABCG2, S100A9, ITGA5, TIMP2, FGF5, PDGFC, SEMA3A) and downregulated (KLF4, P63α) in AMLE was confirmed using qRT-PCR. Immunofluorescence confirmed that AMLE cultures were P63α, ABCG2, CK3, CK12, and E-cadherin (E-cad) positive. CONCLUSIONS: In this study, we have shown that genes associated with wound healing processes are upregulated in AMLE. These gene expression changes may contribute to corneal restoration and the positive outcomes associated with transplantation.

Regenerative medicine for diabetes: differentiation of human pluripotent stem cells into functional ß-cells in vitro and their proposed journey to clinical translation.

Diabetes is a group of metabolic diseases, rising globally at an alarming rate. Type 1 (juvenile diabetes) is the autoimmune version of diabetes where the pancreas is unable to produce insulin, whereas type 2 (adult onset diabetes) is caused due to insulin resistance of the cells. In either of the cases, elevated blood glucose levels are observed which leads to progressive comorbidity like renal failure, cardiovascular disease, retinopathy, etc. Metformin, sulphonyl urea group of drugs, as well as insulin injections are the available therapies. In advanced cases of diabetes, the drug alone or drug in combination with insulin injections are not able to maintain a steady level of blood glucose. Moreover, frequent insulin injections are rather cumbersome for the patient. So, regenerative medicine could be a permanent solution for fighting diabetes. Islet transplantation has been tried with a limited amount of success on a large population of diabetics because of the shortage of cadaveric pancreas. Therefore, the best proposed alternative is regenerative medicine involving human pluripotent stem cell (hPSC)-derived beta islet transplantation which can be obtained in large quantities. Efficient protocols for in vitro differentiation of hPSC into a large number of sustained insulin-producing beta cells for transplantation will be considered to be a giant leap to address global rise in diabetic cases. Although most of the protocols mimic in vivo pancreatic development in humans, considerable amount of lacuna persists for near-perfect differentiation strategies. Moreover, beta islets differentiated from hPSC have not yet been successfully translated under clinical scenario.

Differentiation potential of limbal fibroblasts and bone marrow mesenchymal stem cells to corneal epithelial cells.

The cornea is covered by a stratified epithelium that is renewed by stem cells located in the peripheral region of the cornea known as the limbus. This stroma of the limbus contains stromal keratocytes that, when expanded in culture, are termed limbal fibroblasts (LFs). It is thought that LFs exhibit similar characteristics to bone marrow mesenchymal stem cells (BM MSCs) and help maintain the epithelial stem cell phenotype in the limbal region. In this study, we aimed at reprogramming stage-specific embryonic antigen-4 (SSEA4+) LFs and BM MSCs into corneal epithelial lineage using a three-dimensional culture system and embryonic stem cell medium. After enrichment, SSEA4+ cells showed a higher level of stem cell marker expression such as Sox2, Oct4, Nanog, Rex1, ABCG2, and TRA-1-60, and colony-forming efficiency than did SSEA4- cells. SSEA4+, as compared to SSEA4- cells, had a greater propensity to form spheres that, in turn, were induced into ectodermal lineage and further differentiated into functional corneal epithelium. Results show that LFs were similar to BM MSCs in marker profiles, and together with the differences noted between SSEA4+ and SSEA4- cells, point to LFs' being tissue-specific MSCs. However, LFs showed a greater potential for differentiation into corneal epithelium, indicating the potential importance of tissue-specific adult progenitors in their reprogramming capacity into cells of interest. This study opens a new avenue for investigating the molecular mechanism involved in maintaining a limbal stem cell niche and thus a potentially important clinical application to treat corneal epithelial stem cell loss.

Demodex-associated Bacillus proteins induce an aberrant wound healing response in a corneal epithelial cell line: possible implications for corneal ulcer formation in ocular rosacea.

PURPOSE: The aim of the work presented here was to establish the response of a corneal epithelial cell line (hTCEpi) to protein extracted from a bacterium (Bacillus oleronius) previously isolated from a Demodex mite from a rosacea patient. METHODS: The response of the corneal epithelial cell line to Bacillus proteins was measured in terms of alterations in cell migration and invasiveness. Changes in the expression of metalloproteinase genes and proteins were also assessed. RESULTS: The results indicated increased cell migration (14.5-fold, P = 0.001) as measured using 8-µm PET inserts (BD Falcon) in a transwell assay and invasiveness (1.7-fold, P = 0.003) as measured using 8-µm Matrigel (BD Biocoat) invasion inserts in a 24-well plate assay format, following exposure to the Bacillus proteins. Cells exposed to the Bacillus protein showed a dose-dependent increase in expression of genes coding for matrix metalloprotease (MMP)-3 (61-fold) and MPP-9 (301-fold). This dose-dependent increase in gene expression was also reflected in elevated levels of MMP-9 protein (1.34-fold, P = 0.033) and increased matrix metalloprotease activity (1.96-fold, P = 0.043) being present in the culture supernatant. Cells also displayed reduced levels of ß-integrin (1.25-fold, P = 0.01), indicative of increased motility and elevated levels of vinculin (2.7-fold, P = 0.0009), suggesting altered motility. CONCLUSIONS: The results indicate that exposure of corneal epithelial cells to Bacillus proteins results in an aberrant wound healing response as visualized using a scratch wound assay. These results suggest a possible link between the high density of Demodex mites on the eyelashes of ocular rosacea patients and the development of corneal ulcers.

Dependence of corneal stem/progenitor cells on ocular surface innervation.

PURPOSE: Neurotrophic keratopathy (NK) is a corneal degeneration associated with corneal nerve dysfunction. It can cause corneal epithelial defects, stromal thinning, and perforation. However, it is not clear if and to which extent epithelial stem cells are affected in NK. The purpose of this study was to identify the relationship between corneolimbal epithelial progenitor/stem cells and sensory nerves using a denervated mouse model of NK. METHODS: NK was induced in mice by electrocoagulation of the ophthalmic branch of the trigeminal nerve. The absence of corneal nerves was confirmed with ß-III tubulin immunostaining and blink reflex test after 7 days. ATP-binding cassette subfamily G member 2 (ABCG2), p63, and hairy enhancer of split 1 (Hes1) were chosen as corneolimbal stem/progenitor cell markers and assessed in denervated mice versus controls by immunofluorescent microscopy and real-time PCR. In addition, corneolimbal stem/progenitor cells were detected as side population cells using flow cytometry, and colony-forming efficiency assay was performed to assess their function. RESULTS: ABCG2, p63, and Hes1 immunostaining were significantly decreased in denervated eyes after 7 days. Similarly, the expression levels of ABCG2, p63, K15, Hes1, and N-cadherin transcripts were also significantly decreased in denervated eyes. Stem/progenitor cells measured as side population from NK mice were decreased by approximately 75% compared with normals. In addition, the authors found a significant (P = 0.038) reduction in colony-forming efficiency of stem/progenitor cells harvested from denervated eyes. CONCLUSIONS: Corneolimbal stem/progenitor cells are significantly reduced after depletion of sensory nerves. The data suggest a critical role of innervation in maintaining stem cells and/or the stem cell niche.

Comparative analysis of human-derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of human limbal and oral epithelium.

Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iß1 and p63 (putative stem cell markers) in all culture conditions. The level of Iß1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.

Immunohistochemical and immunofluorescence procedures for protein analysis.

Immunohistochemistry (IHC) and immunofluorescence (IF) involve the binding of an antibody to a cellular or tissue antigen of interest and then visualisation of the bound product by fluorescence/with the 3,3'-diaminobenzidine (DAB) chromogen detection system. With increasing numbers of available antibodies against cellular epitopes, IHC and IF are very useful diagnostic tools as well as a means to guide specific therapies that target a particular antigen on cell/tissue samples.There are several IHC and IF staining methods that can be employed depending on the type of specimen under study, the degree of sensitivity required, and the cost considerations. The following is a basic "generic" method for localising proteins and other antigens by direct, indirect, IHC and IF. The method relies on proper fixation of tissue/cells to retain cellular distribution of antigen and to preserve cellular morphology. Details of reagents required are outlined. Consideration is also given to artefacts and other potential pitfalls and thus means to avoid them.