Mapping the burden of severe forms of epidermolysis bullosa - Implications for patient management.

Background: The pathophysiological processes underlying the phenotypic spectrum of severe forms of epidermolysis bullosa (EB) are complex and poorly understood. Objective: To use burden mapping to explore relationships between primary pathomechanisms and secondary clinical manifestations in severe forms of EB (junctional and dystrophic EB [JEB/DEB]) and highlight strengths and weaknesses in evidence regarding the contribution of different pathways. Methods: Literature searches were performed to identify evidence regarding the pathophysiological and clinical aspects of JEB/DEB. Identified publications and clinical experience were used to construct burden maps to visually communicate plausible connections and their relative importance by subtype. Results: Our findings suggest that most of the clinical consequences of JEB/DEB may result from an abnormal state and/or faulty skin remodeling driven by a vicious cycle of delayed wound healing, predominantly mediated through inflammation. The quantity and quality of evidence varies by individual manifestations and disease subtype. Limitations: The burden maps are provisional hypotheses requiring further validation and are limited by the published evidence base and subjectivity in clinical opinion. Conclusions: Delayed wound healing appears to be a key driver of the burden of JEB/DEB. Further studies are warranted to understand the role of inflammatory mediators and accelerated wound healing in patient management.

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.

Reappearance of limbal pigmentation post-simple limbal epithelial transplant.

We report the repigmentation at the limbus in patients who underwent simple limbal epithelial transplant (SLET) for uniocular chemical injury. The first case is of an 8-year-old child who presented with grade 4 chemical injury, with limbal stem cell deficiency (LSCD) corresponding to 6 o' clock till 11 o' clock. He was managed by amniotic membrane graft in the acute stage and SLET after 6 months of the initial injury. The second case is of a 15-year-old female who presented with lime injury, which had resulted in 6 o' clock of limbal involvement (10 o' clock till 4 o' clock). The patient was managed on similar lines with amniotic membrane graft (AMG) in the acute phase and SLET after 6 months of injury. The ocular surface was stable in both the patients post-SLET. The effected limbus showed pigmentation at 8 months of follow-up which eventually became distinct and remained stable. We speculate that the pigmentation at limbus could be attributed to proliferation and movement of melanocytes from limbal biopsy in SLET. These may be capable of supporting the proliferation of limbal epithelial cells and modulation of corneal wound healing.

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, interactions with surrounding stromal niche cells and melanocytes.

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.

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.

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.