Future directions in managing aniridia-associated keratopathy.

Congenital aniridia is a panocular disorder that is typically characterized by iris hypoplasia and aniridia-associated keratopathy (AAK). AAK results in the progressive loss of corneal transparency and thereby loss of vision. Currently, there is no approved therapy to delay or prevent its progression, and clinical management is challenging because of phenotypic variability and high risk of complications after interventions; however, new insights into the molecular pathogenesis of AAK may help improve its management. Here, we review the current understanding about the pathogenesis and management of AAK. We highlight the biological mechanisms involved in AAK development with the aim to develop future treatment options, including surgical, pharmacological, cell therapies, and gene therapies.

Hsa-miR-150-5p inhibits Wnt-ß-catenin signaling in human corneal epithelial stem cells.

Purpose: In our earlier study, we identified hsa-miR-150-5p as a highly expressed miRNA in enriched corneal epithelial stem cells (CESCs). In this study, we aimed to understand the molecular regulatory function of hsa-miR-150-5p in association with the maintenance of stemness in CESCs. Methods: The target mRNAs of hsa-miR-150-5p were predicted and subjected to pathway analysis to identify targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-150-5p mimic, inhibitor, or scrambled sequence using Lipofectamine 3000. The transfected cells were analyzed to determine (i) their colony-forming potential; (ii) the expression levels of stem cell (SC) markers/transcription factors (ABCG2, NANOG, OCT4, KLF4, and ΔNp63), the differentiation marker (Cx43), and the hsa-miR-150-5p predicted targets (JARID2, INHBA, AKT3, and CTNNB1) by qPCR; and (iii) the expression levels of ABCG2, p63α, Cx43, JARID2, AKT3, p-AKT3, ß-catenin, and active ß-catenin by immunofluorescence staining and/or western blotting. Results: The ectopic expression level of hsa-miR-150-5p increased the colony-forming potential (8.29% ± 0.47%, p < 0.001) with the ability to form holoclone-like colonies compared with the control (1.8% ± 0.47%). The mimic-treated cells had higher expression levels of the SC markers but reduced expression levels of Cx43 and the targets of hsa-miR-150-5p that are involved in the Wnt-ß-catenin signaling pathway. The expression levels of ß-catenin and active ß-catenin in the inhibitor-transfected cells were higher than those in the control cells, and the localized nuclear expression indicated the activation of Wnt signaling. Conclusions: Our results indicate a regulatory role for hsa-miR-150-5p in the maintenance of CESCs by inhibiting the Wnt signaling pathway.

Hsa-miR-143-3p inhibits Wnt-ß-catenin and MAPK signaling in human corneal epithelial stem cells.

Our previous study demonstrated hsa-miR-143-3p as one of the highly expressed miRNAs in enriched corneal epithelial stem cells (CESCs). Hence this study aims to elucidate the regulatory role of hsa-miR-143-3p in the maintenance of stemness in CESCs. The target genes of hsa-miR-143-3p were predicted and subjected to pathway analysis to select the targets for functional studies. Primary cultured limbal epithelial cells were transfected with hsa-miR-143-3p mimic, inhibitor or scrambled sequence using Lipofectamine 3000. The transfected cells were analysed for (i) colony forming potential, (ii) expression of stem cell (SC) markers/ transcription factors (ABCG2, NANOG, OCT4, KLF4, ΔNp63), (iii) differentiation marker (Cx43), (iv) predicted five targets of hsa-miR-143-3p (DVL3, MAPK1, MAPK14, KRAS and KAT6A), (v) MAPK signaling regulators and (vi) Wnt-ß-catenin signaling regulators by qPCR, immunofluorescence staining and/or Western blotting. High expression of hsa-miR-143-3p increased the colony forming potential (10.04 ± 1.35%, p < 0.001) with the ability to form holoclone-like colonies in comparison to control (3.33 ± 0.71%). The mimic treated cells had increased expression of SC markers but reduced expression of Cx43 and hsa-miR-143-3p targets involved in Wnt-ß-catenin and MAPK signaling pathways. The expression of ß-catenin, active ß-catenin and ERK2 in hsa-miR-143-3p inhibitor transfected cells were higher than the control cells and the localized nuclear expression indicated the activation of Wnt and MAPK signaling. Thus, the probable association of hsa-miR-143-3p in the maintenance of CESCs through inhibition of Wnt and MAPK signaling pathways was thus indicated.

Human Oral Mucosal Fibroblasts from Limbal Stem Cell Deficient Patients as an Autologous Feeder Layer for Epithelial Cell Culture.

PURPOSE: To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use. METHODS: HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens-Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD. RESULTS: HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups. CONCLUSIONS: HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Challenges in corneal endothelial cell culture.

Corneal endothelial cells (CECs) facilitate the function of maintaining the transparency of the cornea. Damage or dysfunction of CECs can lead to blindness, and the primary treatment is corneal transplantation. However, the shortage of cornea donors is a significant problem worldwide. Thus, cultured CEC therapy has been proposed and found to be a promising approach to overcome the lack of tissue supply. Unfortunately, CECs in humans rarely proliferate in vivo and, therefore, can be extremely challenging to culture in vitro. Several promising cell isolation and culture techniques have been proposed. Multiple factors affecting the success of cell expansion including donor characteristics, preservation and isolation methods, plating density, media preparation, transdifferentiation and biomarkers have been evaluated. However, there is no consensus on standard technique for CEC culture. This review aimed to determine the challenges and investigate potential options that would facilitate the standardization of CEC culture for research and therapeutic application.

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.

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.

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.

A validated porcine corneal organ culture model to study the limbal response to corneal epithelial injury.

Limbal epithelial stem cells are required for the maintenance and repair of the corneal epithelial surface. The difficulty in obtaining human corneal tissue for research purposes means that animal models for studying the corneal and limbal epithelium are extremely useful. Porcine corneal tissue represents an attractive experimental model, however, functional analysis of the limbal epithelial cell population is needed to validate the use of this tissue. Single cell clonal analysis revealed that holoclone-generating cells were enriched in the limbus as compared with the central cornea (38.3% vs 8.3%) and that label-retaining cells were also enriched in the limbus and compared with the central cornea (44.7 ± 6.4 vs 4.7 ± 1.5). Furthermore, it was demonstrated that in a 3D-printed organ culture system, porcine tissue was capable of maintaining and healing the corneal epithelium. Ki67 staining of corneal sections revealed that in response to central epithelial wounding, a greater proportion of progenitors in the basal limbal epithelium enter an actively dividing state. The authors present a comprehensively validated model system for studying the interactions between limbal niche factors and limbal epithelial stem cell fate.

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.

Canine Corneal Stromal Cells Have Multipotent Mesenchymal Stromal Cell Properties In Vitro.

The objective of this study was to determine whether corneal stromal cells (CSCs) from the limbal and central corneal stroma in dogs have multipotent mesenchymal stem/stromal cell (MSC) properties, and whether this cell population can be differentiated into keratocyte-like cells (KDCs). Normal, donated, mesocephalic dog corneas were used to isolate CSC in vitro. Immunohistochemistry demonstrated a distinct population of CD90 expressing cells in the anterior stroma throughout the limbal and central cornea. CSC could be cultured from both the limbal and central cornea and the culture kinetics showed a progenitor cell profile. The CSC expressed stem cell markers CD90, CD73, CD105, N-cadherin, and Pax6, while CD34 was negative. Limbal and central CSC differentiated into osteoblasts, chondrocytes, and adipocytes confirming their multipotency. Coculturing allogeneic peripheral blood mononuclear cells (PBMCs) with limbal CSCs did not affect baseline PBMC proliferation indicating a degree of innate immune privilege. Limbal CSC could be differentiated into KDCs that expressed Keratocan, Lumican, and ALDH1A3 and downregulated Pax6 and N-cadherin. In conclusion, canine CSCs have multipotent MSC properties similarly described in humans and could serve as a source of cells for cell therapy and studying corneal diseases.

Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.

Age-related macular degeneration (AMD) remains a major cause of blindness, with dysfunction and loss of retinal pigment epithelium (RPE) central to disease progression. We engineered an RPE patch comprising a fully differentiated, human embryonic stem cell (hESC)-derived RPE monolayer on a coated, synthetic basement membrane. We delivered the patch, using a purpose-designed microsurgical tool, into the subretinal space of one eye in each of two patients with severe exudative AMD. Primary endpoints were incidence and severity of adverse events and proportion of subjects with improved best-corrected visual acuity of 15 letters or more. We report successful delivery and survival of the RPE patch by biomicroscopy and optical coherence tomography, and a visual acuity gain of 29 and 21 letters in the two patients, respectively, over 12 months. Only local immunosuppression was used long-term. We also present the preclinical surgical, cell safety and tumorigenicity studies leading to trial approval. This work supports the feasibility and safety of hESC-RPE patch transplantation as a regenerative strategy for AMD.

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. STATEMENT OF SIGNIFICANCE: For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils and thus subsequently exploited this property in vitro to improve the architecture of engineered RAFT tissue equivalents of the corneal stroma. Existing techniques are extremely lengthy and carry significant risk and cost for GMP manufacture. This rapid and tunable technique takes just 8 h of culture and is therefore ideal for clinical manufacture, creating biomimetic tissue equivalents with both cellular and ECM organization. Thus, cellular self-alignment can be a useful bioengineering tool for the development of organized tissue equivalents in a variety of applications.

Aldehyde dehydrogenase inhibition blocks mucosal fibrosis in human and mouse ocular scarring.

Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.

Human-derived feeder fibroblasts for the culture of epithelial cells for clinical use.

AIM: To investigate human oral mucosal fibroblasts (HOMF) and human limbal fibroblasts (HLF) as alternatives to murine 3T3 feeder fibroblasts currently used to support epithelial cell expansion for the treatment of limbal epithelial stem cell deficiency. METHODS: HLF and HOMF were compared with 3T3s for their ability to support the culture of human limbal epithelial cells and human oral mucosal epithelial cells. RESULTS: HOMF, but not HLF, were equivalent to 3T3s in terms of the number of epithelial population doublings achieved. Human limbal epithelial cells co-cultured with HOMF or 3T3s had similar expression of corneal and putative stem cell markers. CONCLUSION: HOMF are a suitable and safer feeder fibroblast alternative to 3T3s for the production of epithelial cells for clinical use.

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.

Regulatory requirements in the good manufacturing practice production of an epithelial cell graft for ocular surface reconstruction.

In the past decade, stem cell therapy has been increasingly employed for the treatment of various diseases. Subsequently, there has been a great interest in the manufacture of stem cells under good manufacturing practice, which is required by law for their use in humans. The cells for sight Stem Cell Therapy Research Unit, based at UCL Institute of Ophthalmology, delivers somatic cell-based and tissue-engineered therapies to patients suffering from blinding eye diseases at Moorfields Eye Hospital (London, UK). The following article is based on our experience in the conception, design, construction, validation and manufacturing within a good manufacturing practice manufacturing facility based in the UK. As such the regulations can be extrapolated to the 28 members stated within the EU. However, the principles may have a broad relevance outside the EU.

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

PURPOSE: 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 ß1 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.