Limbal Crypts Research Articles

Cell-Cell and Cell-Matrix Interactions at the Presumptive Stem Cell Niche of the Chick Corneal Limbus.

(1) Background: Owing to its ready availability and ease of acquisition, developing chick corneal tissue has long been used for research purposes. Here, we seek to ascertain the three-dimensional microanatomy and spatiotemporal interrelationships of the cells (epithelial and stromal), extracellular matrix, and vasculature at the corneo-scleral limbus as the site of the corneal stem cell niche of the chicken eye. (2) Methods: The limbus of developing (i.e., embryonic days (E) 16 and 18, just prior to hatch) and mature chicken eyes was imaged using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and the volume electron microscopy technique, serial-block face SEM (SBF-SEM), the latter technique allowing us to generate three-dimensional reconstructions from data sets of up to 1000 serial images; (3) Results: Data revealed that miniature limbal undulations of the embryonic basement membrane, akin to Palisades of Vogt (PoV), matured into distinct invaginations of epithelial cells that extended proximally into a vascularized limbal stroma. Basal limbal epithelial cells, moreover, occasionally exhibited a high nuclear:cytoplasmic ratio, which is a characteristic feature of stem cells. SBF-SEM identified direct cell-cell associations between corneal epithelial and stromal cells at the base of structures akin to limbal crypts (LCs), with cord-like projections of extracellular matrix extending from the basal epithelial lamina into the subjacent stroma, where they made direct contact with stomal cells in the immature limbus. (4) Conclusion: Similarities with human tissue suggest that the corneal limbus of the mature chicken eye is likely the site of a corneal stem cell niche. The ability to study embryonic corneas pre-hatch, where we see characteristic niche-like features emerge, thus provides an opportunity to chart the development of the limbal stem cell niche of the cornea.

The progress in techniques for culturing human limbal epithelial stem cells.

In vitro culture of human limbal epithelial stem cells (hLESCs) is crucial to cell therapy in the treatment of limbal stem cell deficiency, a potentially vision-threatening disease that is characterized by persistent corneal epithelial defects and corneal epithelium conjunctivalization. Traditionally, hLESCs are cultivated based on either limbal tissue explants or single-cell suspensions in culture media containing xenogenous components, such as fetal bovine serum and murine 3T3 feeder cells. Plastic culture dishes and human amniotic membranes are classical growth substrates used in conventional hLESC culture systems. The past few decades have witnessed considerable progress and innovations in hLESC culture techniques to ensure a higher level of biosafety and lower immunogenicity for further cell treatment, including complete removal of xenogenous components from culture media, the application of human-derived feeder cells, and the development of novel scaffolds. Three-dimensional artificial niches and three-dimensional culture techniques have also been established to simulate the real microenvironment of limbal crypts for better cell outgrowth and proliferation. All these progresses ensure that in vitro cultured hLESCs are more adaptable to translational stem cell therapy for limbal stem cell deficiency.

Line-scanning SD-OCT for in-vivo, non-contact, volumetric, cellular resolution imaging of the human cornea and limbus.

In-vivo, non-contact, volumetric imaging of the cellular and sub-cellular structure of the human cornea and limbus with optical coherence tomography (OCT) is challenging due to involuntary eye motion that introduces both motion artifacts and blur in the OCT images. Here we present the design of a line-scanning (LS) spectral-domain (SD) optical coherence tomography system that combines 2 × 3 × 1.7 µm (x, y, z) resolution in biological tissue with an image acquisition rate of ∼2,500 fps, and demonstrate its ability to image in-vivo and without contact with the tissue surface, the cellular structure of the human anterior segment tissues. Volumetric LS-SD-OCT images acquired over a field-of-view (FOV) of 0.7 mm × 1.4 mm reveal fine morphological details in the healthy human cornea, such as epithelial and endothelial cells, sub-basal nerves, as well as the cellular structure of the limbal crypts, the palisades of Vogt (POVs) and the blood microvasculature of the human limbus. LS-SD-OCT is a promising technology that can assist ophthalmologists with the early diagnostics and optimal treatment planning of ocular diseases affecting the human anterior eye.

Stem Cell Niche Microenvironment: Review.

The cornea comprises a pool of self-regenerating epithelial cells that are crucial to preserving clarity and visibility. Limbal epithelial stem cells (LESCs), which live in a specialized stem cell niche (SCN), are crucial for the survival of the human corneal epithelium. They live at the bottom of the limbal crypts, in a physically enclosed microenvironment with a number of neighboring niche cells. Scientists also simplified features of these diverse microenvironments for more analysis in situ by designing and recreating features of different SCNs. Recent methods for regenerating the corneal epithelium after serious trauma, including burns and allergic assaults, focus mainly on regenerating the LESCs. Mesenchymal stem cells, which can transform into self-renewing and skeletal tissues, hold immense interest for tissue engineering and innovative medicinal exploration. This review summarizes all types of LESCs, identity and location of the human epithelial stem cells (HESCs), reconstruction of LSCN and artificial stem cells for self-renewal.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells

PurposeSingle cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. MethodsFour human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). ResultsscRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. ConclusionsOur scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

Immunolabelling of usual and putative limbal stem cells markers on flat mounted whole human corneas

AbstractPurposeThe permanent regeneration of the human corneal epithelium involves the proliferation of basal cells of corneal epithelium for compensating for daily desquamation (Majo et al. Nature 2008) and of limbal stem cells (LSCs, Cotsarelis, Cell 1989) located in limbal crypts that can be stimulated in case of important epithelial lesion. Many markers of LSCs had been described in the literature but some are controversial, particularly for their specificity.AimTo evaluate the specificity of the main LSC markers proposed in the literature and map their expression on the surface of the cornea.MethodsReferenced or potential markers of LCSs were tested on cross‐sections of fresh human corneas (body donation to Sciences, time between death‐retrieval‐fixation <21 hr) using a standard technique of immunohistochemistry: ABCB5, ΔNp63, P63, ABCG2, Pax6, CK15, CK17, N‐cadherin, Connexin 43, EGFR, vimentin, Sox2, OCT 4. Secondly, antibodies that showed positivity on cross sections were used for immunolabelling flat‐mounted whole cornea to map their expression.ResultsNone of 13 markers was exclusively located at the basal layer of the limbal epithelium. Nevertheless, CK15 appears to be the most promising marker because of its very strong staining in the limbal basal cell layer. However, its staining was also found, less intense, in basal layer of conjunctival epithelium and medium layers of limbal epithelium. On flat‐mounted corneas, CK15 was expressed in a circular ring corresponding to the anatomical limbus, but CK15+ cells were not restricted within the Vogt palisades (or limbal crypts on cross sections).ConclusionsA marker of cells located exclusively in limbal crypts or Vogt palisades and presumed to be the true LSCs is still missing. CK15 seems the most specific markers for limbal basal cells but not restricted to specific niches.

The Sheep Cornea: Structural and Clinical Characteristics

ABSTRACT Purpose The aim of this study was to perform qualitative and quantitative analyses to characterize the corneas of young, healthy sheep. Materials and methods Eight healthy male sheep, 10 months to 1 year of age, were included as experimental subjects. Central corneal thickness was measured using a handheld pachymeter, and an Easygraph corneal topographer provided topographic maps. Microstructural imaging of corneal layers was achieved by using the Heidelberg Retina Tomograph III Rostock Corneal Module in vivo corneal microscope (IVCM). An Ocular Response Analyzer (ORA) provided quantitative measurements of intraocular pressure (IOP), corneal hysteresis (CH), and corneal resistance factor. Tissue histology and immunohistochemistry were carried out to obtain detail on the corneal layers. Results Light microscopy and immunohistochemical labeling revealed a stratified epithelium, a limbus with numerous limbal crypts, a thick basement membrane, a thin Bowman’s layer, a thick corneal stroma with a dense population of keratocytes, and a thick, hyper-reflective Descemet’s membrane. Using IVCM, the cell density of the basal layer was noted to be significantly higher than that of other epithelial cell types. The density of keratocytes was significantly higher (P value = 0.0223) in the anterior compared to the posterior stroma. The endothelial cells were organized in a characteristic honeycomb pattern. The mean and standard deviation values for central corneal pachymetry were 623.14 ± 19.5 μm and 616.37 ± 34.87 μm for the left and right eyes, respectively. ORA-derived mean values for IOPcc and CH for the left and right eyes were 14.93 ± 1.73 mm Hg and 15.16 ± 2.02 mm Hg and 3.56 ± 0.72 mm Hg and 3.73 ± 0.49 mm Hg, respectively. Conclusions The anatomical and clinical characteristics of the sheep cornea, as outlined in this study, make the sheep a suitable and relevant model for corneal research. This study provides researchers with important data on the suitability of sheep as a model for ophthalmic experiments.

Spectral-domain Optical Coherence Tomography in Limbal Stem Cell Deficiency. A Case-Control Study

To evaluate the efficacy of optical coherence tomography (OCT) as a noncontact method for imaging the ocular surface in limbal stem cell deficiency (LSCD) and normal eyes. Retrospective case-control study. Setting: Institutional. Twenty-two eyes with LSCD (study group, 22 patients) and 10 normal eyes (control group, 10 patients). Spectral-domain (SD)-OCT and confocal microscopy in both the limbal and central corneal zones. Pachymetry data from the central cornea, presence of the palisades of Vogt, limbal crypts, and clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium assessed on cross sections parallel and perpendicular to the limbus and en face sections of the limbal region. Parallel, perpendicular, and en face limbal scores were calculated by adding results of the 4 limbal quadrants. Both the difference between the minimal and the maximal epithelial thicknesses and the epithelial thickness standard deviation were significantly higher in the study group (mean, 47μm/10μm) compared with the control group (mean, 8μm/2μm). The parallel, perpendicular, and en face limbal scores were significantly lower in the study group (0.1/0.6/0.2) compared with the control group (7.4/4.8/3.5). Poorer visual acuity was significantly associated with higher standard deviation and difference between minimal and maximal corneal epithelial thicknesses (rs,+0.81/+0.77) and lower parallel, perpendicular, and en face limbal scores (-0.82/-0.73/-0.82). SD-OCT of both the central cornea and limbus with various section orientations is a valuable imaging modality allowing noninvasive and rapid overall precise assessment of both normal and LSCD eyes.

In-vivo imaging of the palisades of Vogt and the limbal crypts with sub-micrometer axial resolution optical coherence tomography.

A research-grade OCT system was used to image in-vivo and without contact with the tissue, the cellular structure and microvasculature of the healthy human corneo-scleral limbus. The OCT system provided 0.95 µm axial and 4 µm (2 µm) lateral resolution in biological tissue depending on the magnification of the imaging objective. Cross-sectional OCT images acquired tangentially from the inferior limbus showed reflective, loop-like features that correspond to the fibrous folds of the palisades of Vogt (POV). The high OCT resolution allowed for visualization of individual cells inside the limbal crypts, capillaries extending from the inside of the POV's fibrous folds and connecting to a lateral grid of micro-vessels located in the connective tissue directly below the POV, as well as reflections from individual red blood cells inside the capillaries. Difference in the reflective properties of the POV was observed among subjects of various pigmentation levels of the POV. Morphological features observed in the high resolution OCT images correlated well with histology. The ability to visualize the limbal morphology and microvasculature in-vivo at cellular level can aid the diagnostics and treatment of limbal stem cell dysfunction and dystrophies.

A method for quantifying limbal stem cell niches using OCT imaging

AimsTo evaluate the efficacy of Fourier domain-optical coherence tomography (FD-OCT) in imaging and quantifying the limbal palisades of Vogt and to correlate these images with histological findings.MethodsThe superior and inferior...

Generating minicorneal organoids from human induced pluripotent stem cells.

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures.

Recreating the Human Limbal Epithelial Stem Cell Niche with Bioengineered Limbal Crypts

ABSTRACTPurpose: Creation of an in vitro model incorporating specific features that characterize a particular stem niche would allow researchers to study stem cell behavior in a more physiological environment.Materials and methods: We have developed a tissue engineering process (RAFT) that rapidly and reliably creates bioengineered limbal crypts (BLCs) in the surface of collagen-based tissue equivalents (TEs). These BLCs mimic the three-dimensional topography of the limbal crypts (LCs), located in the limbal region of the human cornea, which are home to a population of limbal epithelial stem cells (LESCs).Results: Human limbal epithelial (hLE) cells occupying our BLCs expressed putative LESC markers such as ΔNp63α and Bmi1 and produced basement membrane proteins such as laminin β1 and laminin γ3; expression patterns are very similar to those seen in native LCs. Human limbal stromal cells elongate and align along the edge of native LCs and in our RAFT TEs, human limbal fibroblasts (hLFs) also appeared to exhibit this alignment and elongation behavior in response to the BLC topography.Conclusions: We have demonstrated that we can maintain an immature population of hLE cells and aligned stromal cells in our BLCs to mimic some elements of the complexity of the human LESC niche.

Three‐dimensional structure of the mammalian limbal stem cell niche

PurposeAlthough the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its 3D architecture remains incomplete.MethodsThe LSC niche was explored on freshly excised corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full‐field optical coherence microscopy (FFOCM).ResultsLimbal crypt features were detected in 90% of human corneoscleral rims, extending between the palisades of Vogt as radial (74%) and/or rounded (23%) forms, often branching off to or becoming interconnected by sub‐scleral radially or circumferentially oriented crypts (56%). [kg1] Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss‐crossing invaginations beneath the sclera. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of limbal volume in humans, 9% in pigs and 7% in mice. FFOCM combined with fluorescence showed presence of p63‐α+ cells and cytokeratin‐3 + cells in the limbal crypts. LSC density increased with percentage limbal volume occupied by crypts. Colony Forming Efficiency increased with limbal crypt volume. [kg1] See how to rephrase all this.ConclusionsCrypt architecture in the three species appears associated with eye exposure to light. Clone production correlated strongly with the limbal crypt volume in humans indicating that limbal crypts constitute a niche for adult limbal stem cells. FFOCM imaging could assist in assessment of the richness of the limbal crypts and targeted biopsy for cell culture.

Three-dimensional structure of the mammalian limbal stem cell niche

Although the existence of the limbal stem cell (LSC) niche is accepted, precise knowledge of its three-dimensional (3D) architecture remains incomplete. The LSC niche was explored on freshly excised and organ-cultured corneoscleral rims from human donors (n = 47), pigs (n = 15) and mice (n = 27) with full-field optical coherence microscopy (FFOCM). Limbal crypt features were detected in 90% of organ-cultured human corneoscleral rims, extending between the palisades of Vogt as radially oriented rectangular (74% of eyes) and/or rounded (23% of eyes) forms, often branching off to, or becoming interconnected by, sub-scleral radially or circumferentially oriented crypts (in 56% of eyes). Mean crypt volume represented 16% of sampled limbal volume on the vertical axis and 8% on the horizontal axis. In pigs, palisades were finer and crypts wider with relatively uniform distribution around the eye, and radial orientation, connecting to numerous narrow criss-crossing invaginations beneath the scleral surface. In mice, only a circumferential limbal trough was detected. Mean crypt volume represented 13% of sampled limbal volume in humans and 9% in pigs. FFOCM combined with fluorescence, and confocal fluorescence microscopy, showed presence of p63-α+ cells and cytokeratin-3+ cells in the limbal crypts. To assess colony forming efficiency (CFE), limbal epithelial cells were cultured at low density with mitomycin-arrested 3T3 feeders. CFE increased with limbal crypt volume and was not significantly decreased in organ-cultured cornea, despite degradation of the epithelial roof, suggesting that stem cells remain protected at the base of crypts during organ culture. CFE in human samples was significantly greater than in pig, and CFE in mouse was zero. Crypt architecture in the three species appears associated with eye exposure to light. LSC density increased with percentage limbal volume occupied by crypts.

Limbal melanocytes support limbal epithelial stem cells in 2D and 3D microenvironments

Human limbal epithelial stem cells (LESCs) are essential for the maintenance of the corneal epithelium of the ocular surface. LESCs are located within limbal crypts between the palisades of Vogt in the limbus; the interface between the peripheral cornea and conjunctiva. The limbal crypts have been proposed as a LESC niche owing to their support of epithelial cells, which can form holoclone colonies in vitro. Closely associated with the limbal crypts is a concentrated population of melanocytes. The anatomical location and close proximity to putative LESC suggests that melanocytes might play a role in maintenance of these stem cells in the niche. The aim of this study was to assess the ability of human limbal melanocytes (hLM) to support the expansion of human limbal epithelial cells (LECs) in vitro as an indicator of functional cell–cell interaction.After observing that hLM co-localize with clusters of compact epithelial cells in the native limbal crypts, hLM were isolated from crypt-rich cadaveric limbal biopsies and used as feeders for the culture of LECs. Interestingly, LECs grown on mitotically active hLM were able to generate large epithelial colonies that contained small and compact cells with morphological stem cell characteristics. Immunocytochemistry revealed that LECs expanded on hLM were positive for the expression of the putative stem cell markers CK15, Bmi-1 and p63α and negative for the marker of terminal cell differentiation CK3. LECs and hLM were finally co-cultured on RAFT (real architecture for 3D tissue) collagen tissue equivalents. In 3D co-cultures, hLM promoted multi-layering of the epithelial sheet in which basal cells were maintained in an undifferentiated state.Taken together, these observations suggest melanocytes could play an important role in the maintenance of LESCs in the native human limbal stem cell niche.

Spatial Distribution of Niche and Stem Cells in Ex Vivo Human Limbal Cultures

Stem cells at the limbus mediate corneal epithelial regeneration and regulate normal tissue homeostasis. Ex vivo cultured limbal epithelial transplantations are being widely practiced in the treatment of limbal stem cell deficiency. In this report, we examined whether the limbal niche cells that nurture and regulate epithelial stem cells coexist in ex vivo limbal cultures. We also compared the inherent differences between explant and suspension culture systems in terms of spatial distribution of niche cells and their effect on epithelial stem cell proliferation, migration, and differentiation in vitro. We report that the stem cell content of both culture systems was similar, explaining the comparable clinical outcomes reported using these two methods. We also showed that the niche cells get expanded in culture and the nestin-positive cells migrate at the leading edges to direct epithelial cell migration in suspension cultures, whereas they are limited to the intact niche in explant cultures. We provide evidence that C/EBPδ-positive, p15-positive, and quiescent, label-retaining, early activated stem cells migrate at the leading edges to regulate epithelial cell proliferation in explant cultures, and this position effect is lost in early suspension cultures. However, in confluent suspension cultures, the stem cells and niche cells interact with each another, migrate in spiraling patterns, and self-organize to form three-dimensional niche-like compartments resembling the limbal crypts and thereby reestablish the position effect. These 3D-sphere clusters are enriched with nestin-, vimentin-, S100-, and p27-positive niche cells and p15-, p21-, p63α-, C/EBPδ-, ABCG2-, and Pax6-positive quiescent epithelial stem cells.

Localisation of Epithelial Cells Capable of Holoclone Formation In Vitro and Direct Interaction with Stromal Cells in the Native Human Limbal Crypt

Limbal epithelial stem cells (LESCs) are essential to maintain the transparent ocular surface required for vision. Despite great advances in our understanding of ocular stem cell biology over the last decade, the exact location of the LESC niche remains unclear. In the present study we have used in vitro clonal analysis to confirm that limbal crypts provide a niche for the resident LESCs. We have used high-resolution imaging of the basal epithelial layer at the limbus to identify cells with a morphology consistent with stem cells that were only present within the basal layer of the limbal crypts. These cells are proximal to limbal stromal cells suggesting direct cell-to-cell interaction. Serial block-face scanning electron microscopy (SBFSEM) confirmed that the putative LESCs are indeed in direct contact with cells in the underlying stroma, a contact that is facilitated by focal basement membrane interruptions. Limbal mesenchymal cells previously identified in the human limbus collocate in the crypt-rich limbal stromal area in the vicinity of LESCs and may be involved in the cell-to-cell contact revealed by SBFSEM. We also observed a high population of melanocytes within the basal layer of the limbal crypts. From these observations we present a three dimensional reconstruction of the LESC niche in which the stem cell is closely associated and maintained by both dendritic pigmented limbal melanocytes and elongated limbal stromal cells.

Human corneal epithelial subpopulations: oxygen-dependentex vivoexpansion and transcriptional profiling

Corneal epithelium is being regenerated throughout life by limbal epithelial stem cells (LESCs) believed to be located in histologically defined stem cell niches in corneal limbus. Defect or dysfunctional LESCs result in limbal stem cell deficiency (LSCD) causing pain and decreased visual acuity. Since the first successful treatment of LSCD by transplantation of ex vivo expanded LESCs in 1997, many attempts have been carried out to optimize culture conditions to improve outcome of surgery. To date, progress in this field of bioengineering is substantially hindered not only by the lack of specific biomarkers of LESCs, but also by the lack of a precise molecular characterization of in situ epithelial subpopulations. The aim of this dissertation (Bath 2013a) was to optimize culture systems with regard to the environmental oxygen concentration for selective ex vivo expansion of LESCs and to analyse in situ subpopulations in human corneal epithelium using a combination of laser capture microdissection and RNA sequencing for global transcriptomic profiling. We compared dissociation cultures, using either expansion on γ-irradiated NIH/3T3 feeder cells in serum-rich medium or expansion directly on plastic in serum-free EpiLife medium, using a range of physiologically relevant oxygen concentrations (2%, 5%, 10%, 15% and 20%). Using immunocytochemistry and advanced fluorescence microscopy, cells were characterized according to growth, cell cycle distribution, colony-forming efficiency, phenotypes, and cytomorphometry. Limbal epithelial cells expanded in 2% O2 exhibited slow growth, low fraction of cells in S/G2, high CFE, high expression of stem cell markers ABCG2 and p63α, and low fraction of differentiation marker CK3 resembling an LESC phenotype. The effect of hypoxia to maintain LESCs in culture was not dependent on the system used for propagation (Bath et al. 2013b). Laser capture microdissection was used to isolate cellular subpopulations in situ from the spatially defined differentiation pathway in human corneal epithelium according to an optimized protocol for maintenance of expression profiles. Isolated total RNA from basal limbal crypts (BLCs), superficial limbal crypts (SLCs), paracentral/central cornea and limbal stroma was amplified and converted to fragmented cDNA libraries for use in deep paired-end next-generation sequencing. Global transcriptional profiling was carried out using bioinformatics. The location of primitive cells in BLCs, migratory and activated cells in SLCs, and differentiated cells in paracentral/central cornea was evident from mapping of significantly upregulated genes in each compartment to the Gene Ontology (GO). Interestingly, many GO terms in BLCs were also involved in neurogenic processes, whereas many GO terms in SLCs were related to vasculature. Mapping upregulated genes in BLCs to pathway annotations in Kyoto Encyclopaedia of Genes and Genomes described many active pathways as signalling and cancer-associated pathways. We supply extensive information on possible novel biomarkers, reveal insight into both active pathways and novel regulators of LESCs such as Lrig1 and SOX9, and provide an immense amount of data for the future exploration (Bath et al. 2013c). Selective ex vivo expansion of LESCs in hypoxia and the comprehensive molecular characterization of corneal epithelial subpopulations in situ are expected to be beneficial for the future treatment for LSCD by cultured limbal epithelial transplantation.

Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture

Limbal epithelial stem cells are responsible for the maintenance of the human corneal epithelium and these cells reside in a specialised stem cell niche. They are located at the base of limbal crypts, in a physically protected microenvironment in close proximity to a variety of neighbouring niche cells. Design and recreation of elements of various stem cell niches have allowed researchers to simplify aspects of these complex microenvironments for further study in vitro. We have developed a method to rapidly and reproducibly create bioengineered limbal crypts (BLCs) in a collagen construct using a simple one-step method. Liquid is removed from collagen hydrogels using hydrophilic porous absorbers (HPAs) that have custom moulded micro-ridges on the base. The resulting topography on the surface of the thin collagen constructs resembles the dimensions of the stromal crypts of the human limbus. Human limbal epithelial cells seeded onto the surface of the constructs populate these BLCs and form numerous layers with a high proportion of the cells lining the crypts expressing putative stem cell marker, p63α. The HPAs are produced using a moulding process that is flexible and can be adapted depending on the requirements of the end user. Creation of defined topographical features using this process could be applicable to numerous tissue-engineering applications where varied 3-dimensional niche architectures are required.

Transcriptional profiling of human corneal epithelium using laser capture microdissection and massive parallel sequencing

AbstractThis study was conducted to perform global transcriptomic profiling and biomarker discovery on undisturbed human corneal epithelial subpopulations using a novel approach combining laser capture microdissection (LCM) and RNA sequencing. LCM facilitated the harvest of four cellular compartments along the corneal epithelial differentiation pathway: Stroma, basal limbal crypt (BLC), superficial limbal crypt (SLC), and cornea. Compartments were analyzed using massive parallel sequencing and bioinformatics. Specificity of LCM was confirmed by imaging, expression of epithelial markers, and gene ontology analysis of biological processes. Interestingly, many significant upregulated genes in SLC mapped to processes involved in regulation of vasculature like sFLT1. In contrast, BLC had many upregulated genes mapping to neurogenic and developmental processes. The primitive nature of BLC was confirmed by KEGG pathway analysis. We present full gene lists of unique expressed genes in both BLC and cornea representing candidate biomarkers. Possible novel regulators of limbal epithelial stem cells (LESCs) include Lrig1 and SOX9. The presented molecular insight in LESC biology is expected to be beneficial for both research and clinical translation.