Anatomical features and cell-cell interactions in the human limbal epithelial stem cell niche

Epithelial stem cells of the ocular surface are essential for the maintenance of corneal transparency and therefore for vision. Human corneal/limbal epithelial stem cells (LESCs) are believed to reside in the limbus, the interface between the peripheral cornea and neighboring conjunctiva. A specific anatomical microenvironment called the niche regulates the proliferative and differentiation potential of LESCs and their daughter cells. This review covers multiple structural and functional aspects of the human limbal epithelial stem cell niche, including: anatomical features of the niche, composition of the local extracellular matrix, soluble factors and signaling pathways, cell-to-cell interactions with surrounding stromal niche cells and melanocytes

Limbal Stem Cells and Corneal Epithelial Regeneration: Current Status and Prospectives

The clear cornea functions like a window that controls the entry of light for visual information and plays a protective role. The failure of appropriate repair following corneal injury results in loss of corneal function. The limbal region of the cornea is thought to serve as a unique reservoir of corneal epithelial stem cells where limbal stem cells (LSC) contributed to the regeneration of corneal epithelium. The deficiency of LSC (LSCD) results in the failed regeneration of corneal epithelium following injuries. In this review, we discuss the current knowledge of LSC and LSC-based transplantation for regeneration of corneal epithelium. We will first review the latest development of corneal structures. Next we will introduce the concept of LSC and the associated debates. Third, we will review different LSC-based transplantation methods for LSCD treatment and compare their advantages and disadvantages. Finally, we will discuss the improvements of regeneration of corneal epithelium.published_or_final_versio

Changes in Corneal Basal Epithelial Phenotypes in an Altered Basement Membrane

To examine the corneal epithelial phenotype in an altered basement membrane.Corneas from 9 patients with symptoms of continuous unstable corneal curvature (CUCC) were harvested by penetrating keratoplasty and subjected to histology examination and immunohistochemical staining with transactivating and N-terminally truncated pP63 transcript (ΔNp63), cytokeratin 3 (Krt3), ATP-binding cassette sub-family G member 2 (ABCG2), connexin 43 (CX43), p38 mitogen-activated protein kinases (p38MAPK), activating protein 2 (TFAP2), and extracellular signal-regulated kinase (Erk1/2) monoclonal antibodies. Positive immunostaining with ABCG2, p38MAPK, and TFAP2 monoclonal antibodies was observed in the basal epithelial cells of CUCC patients, and CX43 and ΔNp63 were detected in the full-thickness epithelial cells of CUCC patients.Our results indicate that alteration of the corneal basement membrane induces a de-differentiation-like phenotype in corneal basal epithelial cells

The limbus: Structure and function

Limbal function is a key determinant of corneal epithelial integrity. Lineage tracing studies in mice have highlighted that the centripetal movement of epithelial progenitors from the limbus drives both the steady-state maintenance of the corneal epithelium and its regeneration following injury. It is well established that this is facilitated by a population of limbal epithelial stem cells within the limbus. It is becoming increasingly apparent that the behaviour of these stem cells and their ability to respond to the needs of the tissue are closely linked to their immediate microenvironment – the stem cell niche. Increasing understanding of the structural features of this niche and the signalling networks that they coordinate is required to enhance the therapeutic application of these cells in the treatment of limbal stem cell deficiency. Importantly, an improved characterisation of the hierarchy of limbal epithelial progenitors using both new and old putative markers will enable a greater appreciation for the effects of many of these limbal niche factors on stem cell fate

Characterisation of the porcine corneal epithelial stem/progenitor cell niche

It is widely accepted that the surface epithelium of the cornea is maintained by a population of stem cells that resides in the corneoscleral limbus. However, the extracellular matrix and surrounding cells that maintain this stem cell population and constitute the niche of these cells are not fully understood. The aim of this thesis was to further characterise the extracellular matrix, specifically the chondroitin sulphate/dermatan sulphate distribution, and cellular components of the limbal epithelial stem cell niche using a porcine model. This was done using immunohistochemistry, transmission electron microscopy, scanning electron microscopy and creating 3D tissue reconstructions via serial block-face scanning electron microscopy (SBFSEM) and X-ray micro-computed tomography (microCT). This thesis was the first to investigate using microCT to image the limbal/corneal epithelium and found the porcine cornea has one or more continuous limbal troughs that span across all limbal quadrants, as opposed to small, discrete limbal crypts as found in humans. These elongated projections of the limbal epithelium into the stroma contain basal epithelial cells positive for the putative limbal stem cell markers cytokeratin 19 and ABCB5. The stroma immediately subjacent to the limbal trough is rich in the chondroitin sulphate sulphation motifs recognised by antibodies 6C3 and 3B3(+), as well as hyaluronic acid. The distribution of sulphated proteoglycans varies between the stroma and epithelial basement membranes of the porcine central cornea versus the limbus. SBFSEM reconstructions demonstrate direct cell-cell contact between stromal niche cells and limbal basal epithelial cells in the porcine cornea, the third mammalian species to show this. This thesis has shown marked differences in the limbal stem cell niche of the porcine eye from the human, but also confirming some shared characteristics. The newly identified limbal trough forms part of this niche, playing host to putative stem cells, where forms of chondroitin sulphate are in close proximity to these cells and stromal-epithelial cell interaction exists. These factors may play a role in stem cell maintenance in vivo in the porcine cornea and also could have uses in the culturing of limbal stem cells and future niche reconstruction

Human corneal stromal stem cells support limbal epithelial cells cultured on RAFT tissue equivalents.

Human limbal epithelial cells (HLE) and corneal stromal stem cells (CSSC) reside in close proximity in vivo in the corneal limbal stem cell niche. However, HLE are typically cultured in vitro without supporting niche cells. Here, we re-create the cell-cell juxtaposition of the native environment in vitro, to provide a tool for investigation of epithelial-stromal cell interactions and to optimize HLE culture conditions for potential therapeutic application. RAFT (Real Architecture For 3D Tissue) tissue equivalents (TEs) were used as a 3-dimensional substrate for co-culturing HLE and CSSC. Our results demonstrate that a monolayer of HLE that maintained expression of p63α, ABCB5, CK8 and CK15 (HLE markers), formed on the surface of RAFT TEs within 13 days of culture. CSSC remained in close proximity to HLE and maintained expression of mesenchymal stem cell markers. This simple technique has a short preparation time of only 15 days with the onset of HLE layering and differentiation observed. Furthermore, co-cultivation of HLE with another niche cell type (CSSC) directly on RAFT TEs, eliminates the requirement for animal-derived feeder cells. RAFT TEs may be useful for future therapeutic delivery of multiple cell types to restore the limbal niche following ocular surface injury or disease

Pathophysiology of aniridia-associated keratopathy: Developmental aspects and unanswered questions

Aniridia, a rare congenital disease, is often characterized by a progressive, pronounced limbal insufficiency and ocular surface pathology termed aniridia-associated keratopathy (AAK). Due to the characteristics of AAK and its bilateral nature, clinical management is challenging and complicated by the multiple coexisting ocular and systemic morbidities in aniridia. Although it is primarily assumed that AAK originates from a congenital limbal stem cell deficiency, in recent years AAK and its pathogenesis has been questioned in the light of new evidence and a refined understanding of ocular development and the biology of limbal stem cells (LSCs) and their niche. Here, by consolidating and comparing the latest clinical and preclinical evidence, we discuss key unanswered questions regarding ocular developmental aspects crucial to AAK. We also highlight hypotheses on the potential role of LSCs and the ocular surface microenvironment in AAK. The insights thus gained lead to a greater appreciation for the role of developmental and cellular processes in the emergence of AAK. They also highlight areas for future research to enable a deeper understanding of aniridia, and thereby the potential to develop new treatments for this rare but blinding ocular surface disease

Characterisation of corneas following different time and storage methods for their use as a source of stem-like limbal epithelial cells

The transplantation of expansions of limbal epithelial stem cells (LESC) remains one of the most efficient therapies for the treatment of limbal stem cell deficiency (LSCD) to date. However, the available donor corneas are scarce, and the corneas conserved for long time, under hypothermic conditions (after 7 days) or in culture (more than 28 days), are usually discarded due to poor viability of the endothelial cells. To establish an objective criterion for the utilisation or discarding of corneas as a source of LESC, we characterized, by immunohistochemistry analysis, donor corneas conserved in different conditions and for different periods of time. We also studied the potency of LESCs isolated from these corneas and maintained in culture up to 3 cell passages. We hoped that the study of markers of LESCs present in both the corneoscleral histological sections and the cell cultures would show the adequacy of the methods used for cell isolation and how fit the LESC enrichment of the obtained cell populations to be expanded was. Thus, the expressions of markers of the cells residing in the human limbal and corneal epithelium (cytokeratin CK15 and CK12, vimentin, Collagen VII, p63 alpha, ABCG2, Ki67, Integrin beta 4, ZO1, and melan A) were analysed in sections of corneoscleral tissues conserved in hypothermic conditions for 2-9 days with post-mortem time (pmt) 8 h or for 1 day with pmt 16 h, and in sclerocorneal rims maintained in an organ culture medium for 29 days. Cell populations isolated from donor corneoscleral tissues were also assessed based on these markers to verify the adequacy of isolation methods and the potential of expanding LESCs from these tissues. Positivity for several putative stem cell markers such as CK15 and p63 alpha was detected in all corneoscleral tissues, although a decrease was recorded in the ones conserved for longer times. The barrier function and the ability to adhere to the extracellular matrix were maintained in all the analysed tissues. In limbal epithelial cell cultures, a simultaneous decrease in the melan A melanocyte marker and the putative stem cell markers was detected, suggesting a close relationship between the melanocytes and the limbal stem cells of the niche. Holoclones stained with putative stem cell markers were obtained from long-term, hypothermic, stored sclerocorneal rims. The results showed that the remaining sclerocorneal rims after corneal transplantation, which were conserved under hypothermic conditions for up to 7 days and would have been discarded at a first glance, still maintained their potential as a source of LESC cultures.This research study has been funded by grants from the Department of Heath of the Basque Government (RIS3, 2019222049) and the University of the Basque Country UPV/EHU-Instituto Clinico Quirurgico de Oftalmologia ICQO (US19/18). C. R-V. has received fellowship support from the University of the Basque Country UPV/EH

Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface

The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea's transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET) present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD