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

Optimization of Optical and Mechanical Properties of Real Architecture for 3-Dimensional Tissue Equivalents: Towards Treatment of Limbal Epithelial Stem Cell Deficiency

Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The ‘optimal’ RAFT TE was thin, transparent but still handleable and was produced using 0.6 ml of 3 mg/ml collagen. Degradation rates of the ‘optimal’ RAFT TE and HAM were similar. hLE achieved confluency on ‘optimal’ RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency

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

The impact of biomechanics on corneal endothelium tissue engineering

The integrity of innermost layer of the cornea, the corneal endothelium, is key to sustaining corneal transparency. Therefore, disease or injury causing loss or damage to the corneal endothelial cell population may threaten vision. Transplantation of corneal tissue is the standard treatment used to replace malfunctioning corneal endothelial cells. However, this surgery is dependent upon donor tissue, which is limited in supply. Hence, tissue engineers have attempted to construct alternative transplantable tissues or cell therapies to alleviate this problem. Nevertheless, the intrinsic non-dividing nature of corneal endothelial cells continues to foil scientists in their attempts to yield large numbers of cells in the laboratory for use in such novel therapies. Interestingly, the contribution of the biomechanical properties of the underlying extracellular matrix (ECM) on cell division, tissue development and maintenance has been extensively investigated in other many cell types. However, the impact of biomechanics on corneal endothelial cell behaviour is relatively unexplored. Here, we describe contemporary tissue engineering solutions aimed at circumventing donor tissue scarcity. We review the ECM structure and biomechanical features of corneal endothelial cells. We discuss the alterations of ECM in endothelial disease development and progression and point out the role of ECM in developing a tissue-engineered corneal endothelium. We highlight the main biomechanical cues, including topographical and mechanical features, that impact cellular behaviors. Finally, we discuss the influence of biomechanical cues on cell and tissue development, and how corneal endothelial cells response to individual biomechanical stimuli in tissue engineering, which have implications for designing an engineered endothelium and maintaining cell function

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

Mini-Review: Limbal Stem Cells Deficiency in Companion Animals: Time to Give Something Back?

Experimental animals have been used extensively in the goal of developing sight-saving therapies for humans. One example is the development of transplantation of cultured limbal epithelial stem cells (LESC) to restore vision following ocular surface injury or disease. With clinical trials of cultured LESC therapy underway in humans and a potential companion animal population suffering from similar diseases, it is perhaps time to give something back. Comparatively to humans, what is known about the healthy limbus and corneal surface physiology of companion animals is still very little. Blinding corneal diseases in animals such as symblepharon in cats with Feline Herpes Virus-1 infections require a basic understanding of the functional companion animal limbus and corneal stem cells. Our understanding of many other vision threatening conditions such as scarring of the cornea post-inflammation with lymphocytic-plasmacytic infiltrate in dogs (aka chronic superficial keratitis) or pigment proliferation with Pigmentary Keratitis of Pugs would benefit from a better understanding of the animal cornea in health and disease. This is also vital when new therapeutic approaches are considered. This review will explore the current challenges and future research directions that will be required to increase our understanding of corneal diseases in animals and consider the potential development and delivery of cultured stem cell therapy to veterinary ocular surface patients

Oral Mucosa Tissue Equivalents for the Treatment of Limbal Stem Cell Deficiency

Cultured limbal and oral epithelial cells have been successfully used to treat patients with limbal stem cell deficiency (LSCD). The most common culture method for these cell therapies utilizes amniotic membrane as a cell support and/or murine 3T3s as feeder fibroblasts. The aim of this study is to refine the production of autologous oral mucosal cell therapy for the treatment of LSCD. Real architecture for 3D tissue (RAFT) is used as an alternative cell culture support. In addition, oral mucosal cells (epithelial and fibroblast) are used as autologous alternatives to donor human limbal epithelial cells (HLE) and murine 3T3s. The following tissue equivalents are produced and characterized: first, for patients with bilateral LSCD, an oral mucosa tissue equivalent consisting of human oral mucosal epithelial cells on RAFT supported by human oral mucosal fibroblasts (HOMF). Second, for patients with unilateral LSCD, HLE on RAFT supported by HOMF. For both tissue equivalent types, features of the cornea are observed including a multi-layered epithelium with small cells with a stem cell like phenotype in the basal layer and squamous cells in the top layers, and p63α and PAX6 expression. These tissue equivalents may therefore be useful in the treatment of LSCD