P16-A111†Factors affecting the density of corneal endothelial cell cultures obtained from donor corneas.

PURPOSE: The shortage of donor corneas represents a worldwide problem, and corneal endothelial cell (CEC) therapy might be a promising alternative approach. CEC can be implanted alone, which has shown limited efficacy, or with a scaffold that holds the cells together as a monolayer tissue, thus imitating Descemet membrane endothelial keratoplasty. We believe that endothelial cell density (ECD) >2000 cells/mm2, a cut-off value that eye banks use to provide quality tissues for transplantation to surgeons, should also be adopted as a parameter to define the quality of CECs as a new Advanced Therapy Medicinal Product for clinical applications in patients with endothelial dystrophies. METHODS: We isolated and cultured CECs from one or more corneas of elderly age donors with ECDs higher than or below 2000 cells/mm2. CEC cultures were carried out on coated plates and on hydrogels with a preformed basement membrane (from TissueGUARD, Germany). Immunofluorescence with antibodies against ZO-1 was performed to evaluate the ECDs of the CEC graft obtained. RESULTS: Our results suggest that primary cultures with ECDs>2000 cells/mm2 can be obtained on coated plated only when (1) CECs are isolated from one or more corneas of young donors; (2) CECs are isolated and pooled together from at least 2 elderly age donor corneas (if ECD>2000 cells/mm2) or 3 elderly age donor corneas (if ECD<2000 cells/mm2). Secondary cultures are all characterized by low ECDs. Hydrogels have been shown to be able to lead to increased ECDs after their release. CONCLUSION: Our protocol highlights the difficulties in obtaining cultures with ECDs>2000 cells/mm2. Despite being achievable with corneas from young donors, this becomes challenging when corneas from elderly donors are used, i.e., the overall majority of those collected by eye banks, particularly when corneas from elderly age donors with ECD<2000 cells/mm2 are considered as a source. One alternative would be to isolate CECs from more corneas, but this might raise the issue of antigenic stimulation, which could eventually lead to transplantation failure. Our strategy to overcome these challenges is the use of a preformed basement membrane as a scaffold for CECs. However, this challenging approach should be investigated more before proceeding to clinical application.

Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Promote Corneal Wound Repair by Regulating Inflammation and Angiogenesis.

Severe corneal damage leads to complete vision loss, thereby affecting life quality and impinging heavily on the healthcare system. Current clinical approaches to manage corneal wounds suffer from severe drawbacks, thus requiring the development of alternative strategies. Of late, mesenchymal stromal/stem cell (MSC)-derived extracellular vesicles (EVs) have become a promising tool in the ophthalmic field. In the present study, we topically delivered bone-marrow-derived MSC-EVs (BMSC-EVs), embedded in methylcellulose, in a murine model of alkali-burn-induced corneal damage in order to evaluate their role in corneal repair through histological and molecular analyses, with the support of magnetic resonance imaging. Our data show that BMSC-EVs, used for the first time in this specific formulation on the damaged cornea, modulate cell death, inflammation and angiogenetic programs in the injured tissue, thus leading to a faster recovery of corneal damage. These results were confirmed on cadaveric donor-derived human corneal epithelial cells in vitro. Thus, BMSC-EVs modulate corneal repair dynamics and are promising as a new cell-free approach for intervening on burn wounds, especially in the avascularized region of the eye.

Analysis and pharmacological modulation of senescence in human epithelial stem cells.

Human epithelial stem cells (ESCs) are characterized by long-term regenerative properties, much dependent on the tissue of origin and varying during their lifespan. We analysed such variables in cultures of ESCs isolated from the skin, conjunctiva, limbus and oral mucosa of healthy donors and patients affected by ectrodactyly-ectodermal dysplasia-clefting syndrome, a rare genetic disorder caused by mutations in the p63 gene. We cultured cells until exhaustion in the presence or in the absence of DAPT (γ-secretase inhibitor; N-[N-(3, 5-difluorophenacetyl)-L-alanyl]-S-phenylglycine T-butyl ester). All cells were able to differentiate in vitro but exhibited variable self-renewal potential. In particular, cells carrying p63 mutations stopped prematurely, compared with controls. Importantly, administration of DAPT significantly extended the replicative properties of all stem cells under examination. RNA sequencing analysis revealed that distinct sets of genes were up- or down-regulated during their lifetime, thus allowing to identify druggable gene networks and off-the-shelf compounds potentially dealing with epithelial stem cell senescence. These data will expand our knowledge on the genetic bases of senescence and potentially pave the way to the pharmacological modulation of ageing in epithelial stem cells.

Autologous simple conjunctival epithelial transplantation for primary pterygium.

PURPOSE: To evaluate the feasibility of a new method of conjunctival transplantation to achieve recovery of the normal conjunctival epithelium over the bare sclera after pterygium excision and prevent its recurrence. METHODS: After excision of the primary pterygium, we performed simple conjunctival epithelial transplantation (SCET) in which we glued an amniotic membrane patch pre-loaded with tiny autologous conjunctival tissue fragments over the scleral defect. Slit-lamp evaluation was performed at 2 and 7-10 days, and then at 1, 3, 6, and 12 months after surgery, together with confocal microscopy at 3, 6, and 12 months. RESULTS: Surgical excision and SCET for nasal primary pterygium were performed in 6 eyes (6 patients). No graft detachment occurred. An inflammatory granuloma was excised without sequelae in one patient 2 months after surgery. No signs of recurrence or sight-threatening complications were recorded at 12 months, and in vivo confocal microscopy showed progressive expansion of the conjunctival cell population and formation of a clear corneal-conjunctival transition. CONCLUSIONS: SCET takes advantage of the ability of the amniotic membrane and conjunctival cells to renew. Outcomes after SCET are comparable to conventional conjunctival flap surgery and can be achieved in less surgical time and with less donor tissue to be removed.

In vitro establishment, validation and characterisation of conjunctival epithelium outgrowth using tissue fragments and amniotic membrane.

BACKGROUND/AIMS: To set up the in vitro conditions for renewal of the conjunctival epithelium using healthy fragments of conjunctival tissue glued over an amniotic membrane. METHODS: We evaluated the capability of conjunctival tissue fragments to generate conjunctival cell outgrowth after seeding them onto amniotic membrane and culture plates; we then assessed conjunctival molecular marker expression by immunofluorescence. We also evaluated the efficiency of glueing the fragments over the amniotic membrane to determine the best setting and the feasibility of shipping preloaded amniotic membranes. RESULTS: Epithelial outgrowth was detected in 65%-80% of conjunctival fragments starting 48-72 hours after glueing, without major differences between type of membrane preparation and fragment size. Within 6-13 days, a full epithelium covered the surface of the amniotic membrane. Specific marker expression (conjunctival epithelium, Muc1, K19, K13; stemness, p63; tight junctions, ZO-1) was detected. Results of the shipping test showed that only 31% of the fragments were still glued over the epithelial side of the membrane within 24 hours compared to more than 90% of fragments stayed attached in the remaining conditions. CONCLUSION: The in vitro regeneration of conjunctival epithelium following outgrowth from conjunctival tissue fragments glued over an amniotic membrane may offer a viable strategy to renew the epithelium in vivo once applied over the ocular surface at the recipient site.

23†Cryopreservation of human amniotic membrane (HAM) for ocular surface reconstruction: a comparison beetween protocols.

PURPOSE: In the past decades, the human amniotic membrane has been largely applied for several surgical and non-surgical procedures. It has been farther demonstrated that both hAM and cornea share similar patterns of expression of structural components of the basement membrane (like laminin 5 and collagen IV) making hAM an useful tissue for ocular surface reconstruction. Since 1996 in fact, amniotic membrane transplantation has been applied to a large number of ocular surface diseases including Stevens-Johnson syndrome, pterygium, corneal ulceration, ocular surface reconstruction after chemical/thermal burns and in the reconstruction after excision of ocular surface neoplasia. During the previous decades, hAM has achieved a pivotal role in regenerative medicine too.The possibility to preserve human amniotic membrane, without affecting membrane's features, has become pivotal, allowing virological and microbiological analyses to be carried out before grafting. The purpose of the present study is to investigate an easier and cheaper protocol for human amniotic membrane preservation without affecting its properties and structure, ensuring the safety profile of the tissue. We compared the effects on adhesive and structural properties of newer preservation conditions to those obtained with an established, standardized protocol (dimethyl sulfoxide at -160°C). In attempt to simplify and enhance the safety of the procedure, we tested dextran-based freezing medium and a dry condition (no medium) at temperatures of -80°C. METHODS: Five patches of human amniotic membrane were obtained from three different donors. For each donor, five preservation condition were tested: dimethyl sulfoxide at -160°C, dimethyl sulfoxide at -80°C, dextran-based medium at -160°C, dextran-based medium at -80°C and dry freezing at -80°C (no medium). At the end of four months storage period, adhesive properties and structure were analyzed. RESULTS: None of the newer preservation protocols showed differences in adhesive and structural properties of the tissues. The stromal layer always kept its adhesiveness, while both structure and basement membrane were not altered by any the preservation protocol. CONCLUSIONS: Switching from liquid nitrogen cryopreservation to -80°C would reduce manipulation, simplify the procedure, making it also cheaper. The use of dextran-based freezing medium or no medium at all (dry condition) would avoid the potential toxicity of the dimethyl sulfoxide-based freezing media.

22†Renewal of conjunctival epithelium over amniotic membrane to perform autologous simple conjunctival epithelial transplantation (SCET): in vitro validation and results of clinical application for primary pterygium.

BACKGROUND: Transplantation of ex vivo cultured conjunctival cell layers, generated on amniotic membrane or other scaffolds, provides a viable option in treating heterogeneous ocular surface conditions. By comparison, cell therapy is costly, labour-intensive and subject to good manufacturing practice requirements and regulatory approval; no conjunctival cell-based therapy is currently available. Several techniques are available after primary pterygium excision to recover the ocular surface anatomy by restoring healthy conjunctival epithelium and preventing recurrence and complications. However, application of conjunctival free autograft or transpositional flap to cover the bared scleral area is limited when the conjunctiva are to be spared for future glaucoma filtering surgery, in patients with large or double-headed pterygia, in recurrent pterygia, or when the harvesting of donor conjunctival is precluded by scarring. AIM: To develop a simple technique to obtain expansion of the conjunctival epithelium when applied in vivo in diseased eyes. METHODS: We evaluated in vitro the best way of gluing conjunctival fragments over the AM, the efficiency of the fragments to generate conjunctival cell outgrowths, the molecular marker expression, and the feasibility of shipping preloaded AM.We performed simple conjunctival epithelial transplantation (SCET) in which we glued an amniotic membrane patch pre-loaded with autologous conjunctival tissue fragments over the scleral defect after pterygium excision and evaluated the recovery of the normal conjunctival epithelium and the disease recurrence up to 12 months after surgery. RESULTS: 65-80% of fragments generated outgrowth 48-72h after gluing, without differences between type of AM preparation and fragment size. Within 6-13 days, a full epithelium covered the surface of the amniotic membrane. Specific marker expression (Muc1, K19, K13, p63, ZO-1) was detected. The shipping test showed after 24h the 31% of the fragments glued over the AM epithelial side, compared to more than 90% of fragments stayed attached in the remaining conditions (stromal side, stromal without spongy layer, epithelial side without epithelium).Surgical excision and SCET for nasal primary pterygium were performed in 6 eyes/patients. No graft detachment and recurrence occurred within 12 months. In vivo confocal microscopy showed progressive expansion of the conjunctival cell population and formation of a clear cornea-conjunctiva transition. CONCLUSIONS: We established the most suitable conditions for a novel strategy based on in vivo expansion of conjunctival cells from conjunctival fragments glued over the AM. The application of SCET seems to be effective and replicable for the renewal of conjunctiva in patients requiring ocular surface reconstruction.

Genetic Modification of Limbal Stem Cells to Decrease Allogeneic Immune Responses.

Limbal stem cell (LSC) transplantation is the only efficient treatment for patients affected by LSC deficiency (LSCD). Allogeneic LSC transplantation is one of the most successful alternative for patients with bilateral LSCD. Nevertheless, the high variability of the human leukocyte antigens (HLA) remains a relevant obstacle to long-term allogeneic graft survival. This study characterized the immunologic properties of LSCs and proposed a genetic engineering strategy to reduce the immunogenicity of LSCs and of their derivatives. Hence, LSC HLA expression was silenced using lentiviral vectors encoding for short hairpin (sh) RNAs targeting ß2-microglobulin (ß2M) or class II major histocompatibility complex transactivator (CIITA) to silence HLA class I and II respectively. Beside the constitutive expression of HLA class I, LSCs showed the capability to upregulate HLA class II expression under inflammatory conditions. Furthermore, LSCs demonstrated the capability to induce T-cell mediated immune responses. LSCs phenotypical and functional characteristics are not disturbed after genetic modification. However, HLA silenced LSC showed to prevent T cell activation, proliferation and cytotoxicity in comparison to fully HLA-expressing LSCs. Additionally; HLA-silenced LSCs were protected against antibody-mediated cellular-dependent cytotoxicity. Our data is a proof-of-concept of the feasibility to generate low immunogenic human LSCs without affecting their typical features. The use of low immunogenic LSCs may support for long-term survival of LSCs and their derivatives after allogeneic transplantation.

A new standardized immunofluorescence method for potency quantification (SMPQ) of human conjunctival cell cultures.

The aim of this study is to set up a standardized and reproducible method to determine the potency (= stem cell content) of human conjunctival cell cultures by means of immunofluorescence-based analyses. This will help the development of new Advanced Therapy Medicinal Products (ATMPs) to use in future cell therapy clinical studies when fewer cells are available to perform the quality controls. To achieve this purpose, a reference standard was investigated and the expression levels of ΔNp63α (considered as a marker of conjunctival stem cells) was correlated to cell size. The limbal hTERT cells were used as reference standard to define the expression value of ΔNp63α. The mean intensity value of limbal hTERT cells ranging between 15 and 20 µm in diameter was used to distinguish between ΔNp63α bright and not bright cells. As ΔNp63α bright expression was mainly seen in the smaller cell size group (10-15 µm), we defined as conjunctival stem cells (= potency) those cells which were bright and with sizes between 10 and 15 µm. Assays on cells from clonal analyses were used to validate the method, as they do allow to observe a decrease in potency (Holoclones > Meroclones > Paraclones). The stem cell content of conjunctival grafts was found to be 11.3% ± 5.0 compared to 21.9% ± 0.6, 9.0% ± 8.1 and 0% from Holoclones, Meroclones and Paraclones, respectively. This new method, here named as Standardized Method for Potency Quantification, will allow to detect the potency in conjunctival cell cultures, thus obtaining a quality control assay responding to the GMP standards required for ATMP release.

Bioinspired Precision Engineering of Three-Dimensional Epithelial Stem Cell Microniches.

Maintenance of the epithelium relies on stem cells residing within specialized microenvironments, known as epithelial crypts. Two-photon polymerization (2PP) is a valuable tool for fabricating 3D micro/nanostructures for stem cell niche engineering applications. Herein, biomimetic gelatin methacrylate-based constructs, replicating the precise geometry of the limbal epithelial crypt structures (limbal stem cell "microniches") as an exemplar epithelial niche, are fabricated using 2PP. Human limbal epithelial stem cells (hLESCs) are seeded within the microniches in xeno-free conditions to investigate their ability to repopulate the crypts and the expression of various differentiation markers. Cell proliferation and a zonation in cell phenotype along the z-axis are observed without the use of exogenous signaling molecules. Significant differences in cell phenotype between cells located at the base of the microniche and those situated towards the rim are observed, demonstrating that stem cell fate is strongly influenced by its location within a niche and the geometrical details of where it resides. This study provides insight into the influence of the niche's spatial geometry on hLESCs and demonstrates a flexible approach for the fabrication of biomimetic crypt-like structures in epithelial tissues. This has significant implications for regenerative medicine applications and can ultimately lead to implantable synthetic "niche-based" treatments.

Optimized Protocol for Regeneration of the Conjunctival Epithelium Using the Cell Suspension Technique.

PURPOSE: To develop autologous tissue-engineered conjunctival epithelial sheets to be used as advanced therapy medicinal products for severe ocular surface disorders involving the conjunctiva. METHODS: Methods used aimed at 1) mapping the conjunctiva for identification of the stem cell location, 2) establishing proper cell culturing conditions, 3) identifying the proper scaffold, and 4) characterizing the conjunctival grafts better. For these purposes, immunostaining and PAS staining, serial cultivation of cells, and quantitative polymerase chain reaction ([INCREMENT]Np63α and MUC5AC) were performed. RESULTS: The inferior fornix represents the ideal area where to take the conjunctival biopsies from, with at least +3.58% of clonogenic colonies and higher percentages of stem cells compared with other areas, as confirmed by [INCREMENT]Np63α expression levels (6.79% ± 1.18%). The standard culture conditions are necessary when cells are cultured on bare plastic, while animal-free media can be used for conjunctival cell culture on the scaffold. Fibrin glue represents the ideal scaffold for production of epithelial conjunctival grafts because it allows physiological expression of the main conjunctival cell markers, with K19 as the ideal one (98.5% ± 0.5% positive cells). The presence of goblet cells (6.3% ± 1.3%) and expression of the stem cell marker [INCREMENT]Np63α (1.65% ± 0.35% positive cells) were also assessed. CONCLUSIONS: Our findings pave the way for ex vivo cultivation of conjunctival epithelial cells onto a scaffold using the cell suspension technique by means of animal-free media. This would allow us to obtain conjunctival grafts for clinical purposes, thus giving a therapeutic option to patients with conjunctival diseases refractory to current therapies.

3D Microfabricated Scaffolds and Microfluidic Devices for Ocular Surface Replacement: a Review.

In recent years, there has been increased research interest in generating corneal substitutes, either for use in the clinic or as in vitro corneal models. The advancement of 3D microfabrication technologies has allowed the reconstruction of the native microarchitecture that controls epithelial cell adhesion, migration and differentiation. In addition, such technology has allowed the inclusion of a dynamic fluid flow that better mimics the physiology of the native cornea. We review the latest innovative products in development in this field, from 3D microfabricated hydrogels to microfluidic devices.

Towards xeno-free cultures of human limbal stem cells for ocular surface reconstruction.

Isolated limbal epithelial stem cells (LESCs) were cultured with or without a 3T3 murine fibroblast feeder-layer (FL) in 4 different culture media on culture plates or on denuded human amniotic membrane (AM) support and fibrin gel support: (1) control medium supplemented with fetal bovine serum; (2) control medium supplemented with the synthetic serum "XerumFree™ XF205" (XF); (3) CnT-20 medium supplemented with "XerumFree™ XF205" (CnT-XF) and (4) CnT-20 medium supplemented with human AB serum (CnT-AB). The three xenogeneic media were compared to standard condition (control + FL) and parameters assessed included cell morphology, proliferative potential, number of passages, assessment of clonogenic and abortive colonies, life span, ∆Np63α expression and epithelial morphology on AM. During serial cultivation of LESCs, most of the tested xeno-free media supported similar numbers of cell passages, total colony number, cumulative cell doublings (CCD) rates and expression of ∆Np63α compared to control. The conditions cultivated with a FL showed a non-statistically significant higher number of cell passages and CCD rates before senescence when compared to the same conditions cultured without FL. Except for the control medium, only XF medium enabled the growth of cells on AM. The expression of ∆Np63α was comparable in all the cultures grown onto AM, when compared to the controls on fibrin gel. In conclusion, the xeno-free media enabled LESC culture both on plastic and on denuded human AM. Despite the analyses were carried out in a statistically low number of samples and need re-assessment in a larger cohort, our results suggest that the production of a completely xeno-free LESC graft could be beneficial for future clinical applications.

Safety outcomes and long-term effectiveness of ex vivo autologous cultured limbal epithelial transplantation for limbal stem cell deficiency.

PURPOSE: To evaluate the safety and effectiveness of ex vivo autologous cultured limbal stem cell transplantation (CLET). METHODS: We reviewed the clinical records of 59 consecutive patients treated with 65 CLETs. Efficacy was graded 1 year after surgery as successful, partially successful or failed. A safety analysis was performed considering side effects and complications that were recorded during the first year after CLET and those reported later than 1 year, including the events related to subsequent treatments. RESULTS: The mean post-CLET follow-up was 6.0±4.1 years. 69% of CLETs had either one or more adverse events (AEs), or adverse drug reactions (ADRs), within 1 year of surgery, with inflammation being the most common (42%), followed by corneal epithelium defects/disepithelialisation (31%), and blood coagula under the fibrin (24%). One year after surgery, 41% of the 59 primary CLET procedures were successful, 39% partially successful and 20% failed. The most common ADRs recorded for the primary unsuccessful CLETs were ulcerative keratitis, melting/perforation, and epithelial defects/disepithelialisation. Six failed CLETs required reconstructive penetrating keratoplasty (PK). Among CLETs with a favourable outcome, 13 underwent corrective PK (mean 4.8±3.4 years), and thereafter seven eyes maintained integrity of the corneal epithelium, five showed corneal surface failure, and one had recurrent epithelial defects. Corneal graft rejection episodes were reported in 71% and 58% of patients following corrective or reconstructive PK, respectively. Seven primary CLETs with a favourable outcome worsened thereafter, and the overall 3-year long-term effectiveness was 68%. CONCLUSIONS: This study addresses important issues regarding possible risks associated with disarray of the ocular surface homeostasis following autologous CLET in patients with limbal stem cell deficiency, despite the fact that the majority of patients experienced a favourable long-term benefit.

Preservation of Ocular Epithelial Limbal Stem Cells: The New Frontier in Regenerative Medicine.

Significant advances have been made in the field of ocular regenerative medicine. Promising stem cell-based therapeutic strategies have been translated into the clinical practice over the last few decades. These new stem cell-based therapies offer the possibility of permanently restoring corneal epithelium in patients with severe disabling and blinding ocular surface disease. The European Union has already classified stem cell-based therapies as "medicinal products". Therefore, manipulation is strictly regulated according to the defined conditions of good manufacturing practice, with the production of stem cell therapeutics at only accredited production sites authorized by the national regulatory agencies. In this regard, as first medical products are licensed for commercial use in Europe enabling a more widespread access to a stem cell-based therapy, the need for safe, validated and reproducible techniques for ex vivo cultured tissue preservation and distribution are coming to the forefront of research. However, these provide various new challenges for biobanking industry such as the retention of viability, good functionality of stem cells and sterility issues. This chapter provides an overview of the current advances in the field of corneal/limbal epithelial stem cell culture preservation techniques using either hypothermic storage or cryopreservation methods, that were used in different culturing steps (from stem cell isolation to the ex vivo epithelial graft preparation), with the reported impact on the post-thawing product recovery.

Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures.

PURPOSE: To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC). METHODS: Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67. RESULTS: Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM. CONCLUSION: Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.

Corneal Epithelial Stem Cells Repopulate the Donor Area within 1 Year from Limbus Removal for Limbal Autograft.

PURPOSE: To determine whether limbal epithelial stem cells (LESCs) repopulate the site harvested for limbal autograft transplantation (LAT), the expression of LESCs markers was evaluated in bioptic specimens obtained from the donor area 12 months or more after surgery. DESIGN: Interventional case series. PARTICIPANTS: Patients who underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. METHODS: Corneal limbal explants were obtained from 2 sites, the harvested area and the untouched control area, in the donor eyes of 6 patients who previously underwent LAT for unilateral acquired limbal stem cell deficiency after chemical burn. Limbal epithelial stem cells were isolated, and cellular, immunohistochemistry, and histologic parameters were assessed to compare differences between LESCs isolated from harvested or control sites. MAIN OUTCOME MEASURES: Presence of LESCs 1 year or more after LAT. RESULTS: Specific markers (p63, Ki67, K12), percentage of LESCs, cell doubling, and number of passages in culture did not differ significantly between harvested and control sites. However, the distinctive structure of the palisades of Vogt was found only in 2 of 6 harvested sites. CONCLUSIONS: Limbal epithelial stem cells repopulate the donor site as early as 1 year after limbus removal for LAT. Autologous transplantation of conjunctiva and limbus are safe procedures and can be performed in cases that cannot be treated by simple grafting of LESCs cultured ex vivo.

Personalized Stem Cell Therapy to Correct Corneal Defects Due to a Unique Homozygous-Heterozygous Mosaicism of Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

UNLABELLED: : Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches. SIGNIFICANCE: This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.

Biobanking of Human Retinas: The Next Big Leap for Eye Banks?

Retinal degenerative diseases are one of the main clinical causes of incurable and severe visional impairment. Thus, extensive research effort is put into the development of new causal therapeutic options. Promisingly, a number of studies showed regenerative capacity in specific retinal regions (the ciliary epithelium, retinal pigmented epithelium, iris, and Müller glia cells). However, most recent research studies are based on animal models or in vitro cultured cells, probably because of the limited availability of human posterior eye tissues (vitreous, retina, and choroid). To address this, we showed in our previous reports that eye banks with large numbers of globes collected yearly could set up biorepositories/biobanks where these precious tissues are isolated, quality controlled, and finally stored for scientists and clinicians wanting to access human tissues and test their own hypotheses. These precious human posterior eye tissues could be used for further research purposes, epidemiological studies, and target validation of newly developed drugs. In addition, this could be a promising and challenging option to retrieve potential retinal stem and progenitor cells from different parts of the retina and could be a breakthrough in the future delivery of ex vivo prepared customized (histocompatible) retinal tissue on scaffolds for transplantation purposes. In this Perspective, we will consider how the biorepositories could influence the future strategies for retinal stem cell therapies.

In Vivo Confocal Microscopy 1 Year after Autologous Cultured Limbal Stem Cell Grafts.

PURPOSE: To correlate clinical, impression cytologic, and in vivo confocal microscopy findings on the corneal surface after cultured limbal stem cell transplantation. DESIGN: Prospective, interventional, noncomparative, masked case series. PARTICIPANTS: Thirteen patients with limbal stem cell deficiency after unilateral (9 eyes) or bilateral (2 eyes) chemical burn, liquid nitrogen injury (1 eye), or herpes simplex virus infection (1 eye). METHODS: Limbal cells were harvested from healthy or less affected eyes, cultured on 3T3 cells and fibrin glue, and transplanted to the patient's injured eye. Patients underwent clinical examination and impression cytologic examination of the central cornea before and 1 year after intervention. In vivo confocal microscopy scans were obtained in all corneal quadrants after 1 year. The interexamination agreement was established by calculation of the Cohen's κ coefficient. MAIN OUTCOME MEASURES: Results of surgery were assessed considering clinical signs (successful: restoration of transparent, avascular, and stable corneal epithelium without neovascularization in central corneal surface; partially successful: recurrence of superficial neovascularization; failed: recurrent epithelial defects, pannus, and inflammation), phenotype of cells covering the corneal surface (conjunctivalized corneal surface: cytokeratin 12 [cK12]-negative and mucin 1 [MUC1]-positive cells; mixed epithelium: cK12-positive and MUC1-positive cells; corneal epithelium: cK12-positive and MUC1-negative cells), and cell morphologic features (corneal epithelium: multilayered polygonal and flat cells with hyperreflective nuclei; conjunctival epithelium: stratified cuboidal or polygonal cells, hyperreflective cytoplasm, and barely defined borders; epithelial transition: transition of epithelial cells from the cornea to the conjunctiva over the corneal surface). RESULTS: We found a moderate to substantial degree of concordance between confocal microscopy and clinical evaluation (κ = 0.768) and between confocal microscopy and impression cytologic analysis (κ = 0.629). Confocal microscopy showed that 46.2% of patients exhibited corneal epithelium in the central and peripheral cornea, 30.8% showed an irregular mixed corneal and conjunctival epithelium, and 23.0% showed conjunctival epithelium. Palisades of Vogt were absent in all (100.0%) patients, and the cornea-conjunctiva epithelial transition localized approximately 1 mm internally on the cornea. CONCLUSIONS: Confocal microscopy provides objective measures of the corneal epithelium and may significantly improve the evaluation of outcomes after cultured limbal stem cell graft.