Corneal Limbal Epithelial Stem Cells Research Articles

Progress in Transdifferentiation of Autologous Alternative Cell Sources into Corneal Epithelial Cells.

Corneal limbal epithelial stem cells (LESCs) play a crucial role in corneal epithelium regeneration. Severe damage to these cells can result in limbal stem cell deficiency (LSCD), characterized by repeated corneal conjunctivalization, leading to corneal turbidity and scar formation. Restoring functional LESCs and their ecological location are essential for treating LSCD. The goal of this review is to provide researchers and clinicians with key insights into LESCs biology and to conclude the current cell-based therapies advancement in LSCD treatments. Therapeutic cell resources mainly include mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), skin keratinocyte stem cells (SKCs), and oral mucosal epithelial cells (OMECs).

A novel intralamellar semi‐bioresorbable keratoprosthesis—Part B: Surface functionalization and physico‐chemical characterization toward site‐specific cellular activity

AbstractIn this article, site‐specific functionalized keratoprosthesis (KPro) was accomplished to meet epithelial on‐growth on the anterior optical surface, inhibit cell adhesion on the posterior optic surface, and promote cell ingrowth at the peripheral tissue‐haptic‐flange interface toward tissue integration. Gelatin immobilization on PC4 hydrogel surfaces was achieved through acid hydrolysis followed by EDC/NHS chemistry, and polyethylene glycol‐diacrylate (PEGDA)‐modified inter‐penetrating network (IPN) surfaces were prepared using photo‐polymerization of ethylene glycol‐diacrylate (EGDA) for enhancing and inhibiting cellular growth, respectively. The surface chemical structures of modified hydrogels were characterized using attenuated total reflectance‐infrared (ATR‐IR) spectroscopy and a bicinchoninic acid (BCA) protein adsorption assay. Further, suitable differentially functionalized surfaces were prepared and characterized using the universal testing machine (UTM), refractometer, atomic force microscopy (AFM), and contact angle measurements to understand mechanical, optical, surface morphological, and wettability properties, respectively. Finally, in vitro site‐specific cell adhesion, cell inhibition, and cell ingrowth functionalities were tested using isolated goat corneal limbal epithelial stem cells and corneal stromal fibroblast cells, respectively. Overall, the two connective articles represent a successful endeavor to develop a more esthetically pleasing, mechanically stable, tissue‐free hydrogel KPro with site‐specific surface functionality nearly mimicking the cornea's functionality. A further pre‐clinical investigation is necessary to advance this functional novel KPro to reality, especially the tissue integration aspect.

3D bioprinting as a prospective therapeutic strategy for corneal limbal epithelial stem cell deficiency.

Limbal epithelial stem cells (LESCs) are responsible for the maintenance and repair of the corneal surface. Injuries and diseases of the eye may result in a vision condition called limbal stem cell deficiency (LSCD). Without limbal stem cells, the cornea becomes opaque, vascularized, and inflamed. Cultured LESC therapy as a treatment method was first described in 1997, and LESCs cultured from either patients or donors have been used to treat LSCD successfully. However, the main source of cornea for LSCD treatment is from donors, which are too few to meet the demand (less than 1:70 of cases). The global shortage of donor cornea promotes the need for studies exploring corneal limbus alternatives. Many problems still remain unresolved, such as original geometry reconstruction, corneal epithelial regeneration, and ocular optical function restoration. 3D bioprinting has garnered tremendous attention in recent years, and significant advances have been made in fabricating cell-laden scaffolds. These advancements could lead to a promising treatment for LSCD. It is possible that alternative limbus stem cells can be constructed using 3D printing, which, in corneal limbus regeneration, enables personalized corneal implants and fabrication of single- or multilayer corneal limbus equivalents with corneal limbal stem cells. In this review, the progress, applications, and limitations of the most influential works regarding current treatments of LESC deficiency are discussed. The advantages of 3D bioprinting are illustrated, and some first promising steps toward the creation of a functional cornea limbus with 3D bioprinting are discussed. Finally, insights into the prospects and technical challenges facing the future research of 3D bioprinting of corneal limbus alternatives in vivo and in vitro are provided.

Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets

Corneal limbal epithelial stem cell transplantation using cultivated human corneal epithelial cell sheets has been used successfully to treat limbal stem cell deficiencies. Here we report an investigation into the quality of cultivated human corneal epithelial cell sheets using time-lapse imaging of the cell culture process every 20 minutes over 14 days to ascertain the level of cell jamming, a phenomenon in which cells become smaller, more rounded and less actively expansive. In parallel, we also assessed the expression of p63, an important corneal epithelial stem cell marker. The occurrence of cell jamming was variable and transient, but was invariably associated with a thickening and stratification of the cell sheet. p63 was present in all expanding cell sheets in the first 9 days of culture, but it’s presence did not always correlate with stratification of the cell sheet. Nor did p63 expression necessarily persist in stratified cell sheets. An assessment of cell jamming, therefore, can shed significant light on the quality and regenerative potential of cultivated human corneal epithelial cell sheets.

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Iatrogenic limbal stem cell deficiency following drainage surgery for glaucoma

ObjectiveTo describe conjunctival epithelial overgrowth of the cornea after surgery for glaucoma. MethodsThis is a retrospective case series (setting: Moorfields Eye Hospital). Fourteen eyes of 13 patients with suspected limbal stem cell deficiency (LSCD) and corneal conjunctivalization after glaucoma drainage surgery. Conjunctivalization was defined as corneal epithelium that demonstrated late stain after topical application of fluorescein. Patient demographics, clinical features, potential risk factors, treatment, and final visual acuity were recorded. Main outcome measures were potential risk factors for conjunctivalization, complications, and response to treatment. ResultsEleven eyes had multiple procedures involving the limbus, and in 11 eyes mitomycin C (MMC) or 5 fluorouracil had been used as an adjunct to reduce fibrosis. Affected eyes typically had a segment of late stain with fluorescein based at the site of previous glaucoma surgery, but in one eye there was total loss of the corneal epithelial phenotype. All eyes previously had topical treatment for their glaucoma but only 2 had an ocular surface disease associated with LSCD. Most cases were asymptomatic, but in 3 eyes there was visual loss when the abnormal phenotype crossed the visual axis. In these 3 eyes there was recurrent epithelial breakdown, often at the interface between the 2 epithelial phenotypes. In one individual, these symptoms resolved after limbal epithelial transfer from the unaffected contralateral eye. ConclusionsGlaucoma drainage surgery can damage the adjacent corneal limbal epithelial stem cell population. This can be associated with recurrent epithelial breakdown and reduced vision. If there is visual loss, limbal epithelial transplantation is a potential treatment option.

Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method

BackgroundHuman pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs).MethodsMultiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation.ResultsThe defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing.ConclusionsWe propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.

Multimodal imaging quality control of epithelia regenerated with cultured human donor corneal limbal epithelial stem cells

Current imaging techniques for the characterization of differentiated corneal limbal stem cells are destructive and cannot be used in eye bank for monitoring the regenerated epithelium in culture. We presented a minimally invasive, multimodal, marker-free imaging method for the investigation of epithelia regenerated with cultured human donor corneal limbal epithelial stem cells. Two-photon fluorescence and harmonic generation signals were collected from specimens in culture and used for evaluating the structure and morphology of epithelia cultured on two different bio-scaffolds; in addition, donor human corneal tissues were used as controls. The method provided reliable information on the organization of cellular and extracellular components of biomaterial substrates and was highly sensitive to determine differences between the density packing arrangement of epithelial cells of different biomaterials without relying on inferences from exogenous labels. The present minimally invasive standardized quality control methodology can be reliably translated to eye banks and used for monitoring harvested corneal limbal stem cells growth and differentiation in bioengineered materials.

The Favorable Effect of Mesenchymal Stem Cell Treatment on the Antioxidant Protective Mechanism in the Corneal Epithelium and Renewal of Corneal Optical Properties Changed after Alkali Burns.

The aim of this study was to examine whether mesenchymal stem cells (MSCs) and/or corneal limbal epithelial stem cells (LSCs) influence restoration of an antioxidant protective mechanism in the corneal epithelium and renewal of corneal optical properties changed after alkali burns. The injured rabbit corneas (with 0.25 N NaOH) were untreated or treated with nanofiber scaffolds free of stem cells, with nanofiber scaffolds seeded with bone marrow MSCs (BM-MSCs), with adipose tissue MSCs (Ad-MSCs), or with LSCs. On day 15 following the injury, after BM-MSCs or LSCs nanofiber treatment (less after Ad-MSCs treatment) the expression of antioxidant enzymes was restored in the regenerated corneal epithelium and the expressions of matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS), α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), and vascular endothelial factor (VEGF) were low. The central corneal thickness (taken as an index of corneal hydration) increased after the injury and returned to levels before the injury. In injured untreated corneas the epithelium was absent and numerous cells revealed the expressions of iNOS, MMP9, α-SMA, TGF-β1, and VEGF. In conclusion, stem cell treatment accelerated regeneration of the corneal epithelium, restored the antioxidant protective mechanism, and renewed corneal optical properties.

The use of an IL-1 receptor antagonist peptide to control inflammation in the treatment of corneal limbal epithelial stem cell deficiency.

Corneal limbal stem cell deficiency (LSCD) may be treated using ex vivo limbal epithelial stem cells (LESCs) derived from cadaveric donor tissue. However, continuing challenges exist around tissue availability, inflammation, and transplant rejection. Lipopolysaccharide (LPS) or recombinant human IL-1β stimulated primary human keratocyte and LESC models were used to investigate the anti-inflammatory properties of a short chain, IL-1 receptor antagonist peptide for use in LESC sheet growth to control inflammation. The peptide was characterized using mass spectroscopy and high performance liquid chromatography. Peptide cytotoxicity, patterns of cell cytokine expression in response to LPS or IL-1β stimulation, and peptide suppression of this response were investigated by MTS/LDH assays, ELISA, and q-PCR. Cell differences in LPS stimulated toll-like receptor 4 expression were investigated using immunocytochemistry. A significant reduction in rIL-1β stimulated inflammatory cytokine production occurred following LESC and keratocyte incubation with anti-inflammatory peptide and in LPS stimulated IL-6 and IL-8 production following keratocyte incubation with peptide (1 mg/mL) (P < 0.05). LESCs produced no cytokine response to LPS stimulation and showed no TLR4 expression. The peptide supported LESC growth when adhered to a silicone hydrogel contact lens indicating potential use in improved LESC grafting through suppression of inflammation.

SiRNA Silencing of the Mutant Keratin 12 Allele in Corneal Limbal Epithelial Cells Grown From Patients With Meesmann's Epithelial Corneal Dystrophy

The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles. Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC). The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression. Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.

Cultivation and characterization of corneal limbal epithelial stem cells on lens capsule in animal material‐free medium

AbstractPurpose To develop simple, reproducible and animal‐materials free method for cultivating and differentiating human limbal stem cells (LSCs) into corneal epithelium on human lens capsule (LC) for clinical transplantation.Methods Limbal tissues (2x2x0,25 mm) were harvested from cadavers (44 males and 33 females, age 70,5+9,3 years) within 12 hours of biologic death. All tissue collection complied with the guidelines of the Helsinki Declaration and was approved by the Regional Ethical Committee. Cell viability was measured with the MTT and annexin‐FITC/propidium iodide assays. Characterization of the growing cells was performed by RT‐qPCR and immunfluorescence stainings accordingly.Results Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (&gt;97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were tested for positivity for markers of stemness (P63, ABCG2, CK19, ITGA9, VIM), proliferation (Ki‐67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non‐hematopoietic, ‐endothelial and ‐mesenchymal stem cell phenotype, while the cell adhesion molecules, integrins and lectin‐based surface carbohydrate profiling showed a specific pattern on the LESCs.Conclusion We report a method for isolating and expanding cornea LESCs from cadavers or alternatively from autologous donors capable of producing viable cell outgrowth on LC for possible treatment of LESC deficiency.

Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) Transplantation for the Treatment of Corneal Limbal Epithelial Stem Cell Deficiency

Total bilateral corneal limbal epithelial stem cell deficiency (LSCD) cannot be treated with the surgical transplantation of autologous limbus or cultured autologous limbal epithelium. Transplantation of allogenic limbal epithelium is possible but requires immunosuppressive treatments. Cultured autologous oral mucosal epithelial cell sheet (CAOMECS) is a transparent, resistant, viable, and rapidly bioadhesive cell sheet, cultured with the UpCell-Insert technology (CellSeed, Inc., Tokyo, Japan), which allows for grafting onto the patient's corneal stroma without suturing. It has therefore been proposed as an alternative treatment for LSCD. The objectives were to assess the safety and efficacy of CAOMECS, using a prospective Gehan's design. Safety was measured in terms of ocular adverse events during the study period, and efficacy was measured using a composite criterion based on epithelial defect, punctate epithelial keratopathy, conjunctival epithelium on the cornea, number of vascular pediculi, and vessel activity. CAOMECS was found to be safe and effective. In total, 26 eyes of 25 patients received a graft. Two patients experienced serious adverse events classified as not product related. Twenty-five patients were included in the efficacy analysis, as one patient was lost to follow-up. The treatment was found to be effective in 16 of 25 patients at 360 days after grafting. Of the 23 patients who completed follow-up at 360 days, 22 had no ulcers, and 19 showed a decrease in the severity of the punctate epithelial keratopathy. CAOMECS is a well-tolerated and safe tissue-engineered product. These results suggest its efficacy for reconstructing the ocular surface in patients with total bilateral corneal LSCD.

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

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

Culture of Corneal Limbal Epithelial Stem Cells: Experience From Benchtop to Bedside in a Tertiary Care Hospital in India

To standardize the technique of ex vivo culture of limbal epithelial stem cells (LESCs) using locally available adapted methods and evaluate the outcome of transplantation in patients with limbal stem cell deficiency. Limbal tissue specimens were isolated from cadaveric corneoscleral rims, living related donors, or contralateral eye of the patients. Harvested tissue was cultured on denuded human amniotic membrane (dHAM) using various techniques to stabilize dHAM. The optimization of in vitro culture conditions was achieved by modifications in culture media (culture media 1 and 2). The LESCs were cultured in both types of media for 2 weeks, and growth patterns were observed. Expanded cells were further characterized by immunocytochemistry (K3/12, K19, and ABCG2) and reverse transcriptase polymerase chain reaction (K12, Cx43, Pax6, ABCG2, p63, and K19). The cultivated epithelium was transplanted in 50 patients with total and partial limbal stem cell deficiencies. Stabilization of dHAM was successfully achieved using coverslips. The outgrowth was observed within 1-3 days of culture using both types of culture media (P = 0.20), but cultures in culture medium 1 reached confluency faster than cultures in culture medium 2 (P = 0.0004). Histopathological analysis showed multilayer formation and immunostaining, and reverse transcriptase polymerase chain reaction data confirmed the expression of both stem cell markers (K19, p63, and ABCG2) and differentiation markers (K3, K12, and Cx43). Patients who had undergone limbal stem cell transplantation showed a stable ocular surface with improved visual acuity over a long-term follow-up period. LESCs were successfully cultured using locally available adapted methods, and their clinical benefits verified by transplantation.

Ex VivoConstruction of an Artificial Ocular Surface by Combination of Corneal Limbal Epithelial Cells and a Compressed Collagen Scaffold Containing Keratocytes

We have investigated the use of a laminin-coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane (AM). Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibers within the compressed gel were dense, homogeneous, and similar in structure to those within denuded AM. Limbal epithelial cells were successfully expanded upon the compressed collagen, resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology, and cell stratification. Similar protein expression profiles for cytokeratin 3 (CK3) and CK14 and no significant difference in CK12 mRNA expression levels by real-time polymerase chain reaction were also observed. This study provides the first line of evidence that a laminin-coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification, and differentiation to a degree that is comparable to the leading conventional scaffold, denuded AM.

Ex Vivo Expansion and Transplantation of Limbal Epithelial Stem Cells

To determine, using objective measures, the outcome of ex vivo cultured limbal epithelial stem cell (LESC) transplantation performed in compliance with good manufacturing practice using a novel culture system without 3T3 feeder cells. Prospective, noncomparative, interventional case series. Ten eyes of 10 patients with profound LESC deficiency arising from chemical injury (4 eyes), aniridia (3 eyes), ectodermal dysplasia (1 eye), Reiger's anomaly with Pax6 haploinsufficiency (1 eye), and unknown cause (1 eye). Allogeneic (7 eyes) or autologous (3 eyes) corneal LESCs were cultured on human amniotic membrane. Tissue was transplanted to the recipient eye after superficial keratectomy. Impression cytology and confocal microscopy were performed 6 months after surgery with clinical follow-up to 13 months. Success was defined as an improvement in the defined clinical parameters of LESC deficiency, an improvement in visual acuity, the restoration of a more normal corneal phenotype on impression cytology, and the appearance of a regular hexagonal basal layer of cells on corneal confocal microscopy. Clinical parameters of LESC deficiency (loss of epithelial transparency, superficial corneal vascularization, epithelial irregularity, and epithelial breakdown), visual acuity, impression cytology and cytokeratin expression profiles, and in vivo confocal corneal confocal microscopy. The success rate using this technique was 60% (autografts 33%, allografts 71%). All patients with a successful outcome experienced an improvement in visual acuity of >/=2 lines Snellen acuity. Preoperatively, CK3+ and CK19+ cells accounted for 12+/-2.4% (mean +/- standard error of the mean) and 80+/-2.15% of cells, respectively, whereas postoperatively these accounted for 69+/-6.43% (P<0.0001) and 30+/-6.34% (P<0.0001) of cells, respectively. Goblet cells accounted for 8+/-1.19% of cells preoperatively and 1+/-0.35% of cells postoperatively (P<0.0001). These data demonstrate that it is possible to culture LESCs ex vivo in compliance with good manufacturing practice regulations. A set of objective outcome measures that confirm the efficiency of this technique in treating LESC deficiency is described. The widespread use of such standardized and objective outcome measures would facilitate a comparison between the different culture methods in use.

In Vivo Corneal Confocal Microscopic Findings of Palisades of Vogt and Its Underlying Limbal Stroma

To demonstrate the corneal confocal microscopic findings of limbal palisades of Vogt and its underlying limbal stroma. Two unrelated healthy subjects (a 56-year-old man and a 40-year-old man) with prominent palisades of Vogt were enrolled in this study. A detailed examination with confocal microscopy was performed in addition to a routine slit-lamp biomicroscopy. Cell sizes of epithelial basal layers of both the central and limbal (beneath the palisades of Vogt) were measured and statistically analyzed. In both subjects, confocal microscopy in the region of the palisades of Vogt revealed normal appearance of corneal superficial layers. However, in some images, undulant basal epithelial layer was observed. In the superficial stromal layer adjacent to the corneal epithelium, we noted corneal nerves that seemed to terminate at the epithelial basal layers. The mid-stromal layers showed a highly reflective spatter-like pattern as well as numerous dark striae in a branching pattern. The corneal endothelial layer seemed normal. The average cell size of epithelial basal cells beneath the palisades of Vogt was significantly smaller than those of the central cornea (P = 0.015 and 0.005 in cases 1 and 2, respectively). In vivo corneal confocal microscopy is useful in observing limbal stromal microstructures. Further investigations of pathologic corneal limbus may be useful in elucidating the mechanisms contributing to corneal limbal epithelial stem cell deficiency.