Cryo-Electrospinning Generates Highly Porous Fiber Scaffolds Which Improves Trabecular Meshwork Cell Infiltration.

Human trabecular meshwork is a sieve-like tissue with large pores, which plays a vital role in aqueous humor outflow. Dysfunction of this tissue can occur, which leads to glaucoma and permanent vision loss. Replacement of trabecular meshwork with a tissue-engineered device is the ultimate objective. This study aimed to create a biomimetic structure of trabecular meshwork using electrospinning. Conventional electrospinning was compared to cryogenic electrospinning, the latter being an adaptation of conventional electrospinning whereby dry ice is incorporated in the fiber collector system. The dry ice causes ice crystals to form in-between the fibers, increasing the inter-fiber spacing, which is retained following sublimation. Structural characterization demonstrated cryo-scaffolds to have closer recapitulation of the trabecular meshwork, in terms of pore size, porosity, and thickness. The attachment of a healthy, human trabecular meshwork cell line (NTM5) to the scaffold was not influenced by the fabrication method. The main objective was to assess cell infiltration. Cryo-scaffolds supported cell penetration deep within their structure after seven days, whereas cells remained on the outer surface for conventional scaffolds. This study demonstrates the suitability of cryogenic electrospinning for the close recapitulation of trabecular meshwork and its potential as a 3D in vitro model and, in time, a tissue-engineered device.

Hematopoietic cell-based and non-hematopoietic cell-based strategies for immune tolerance induction in living-donor renal transplantation: A systematic review.

INTRODUCTION: Despite its use to prevent acute rejection, lifelong immunosuppression can adversely impact long-term patient and graft outcomes. In theory, immunosuppression withdrawal is the ultimate goal of kidney transplantation, and is made possible by the induction of immunological tolerance. The purpose of this paper is to review the safety and efficacy of immune tolerance induction strategies in living-donor kidney transplantation, both chimerism-based and non-chimerism-based. The impact of these strategies on transplant outcomes, including acute rejection, allograft function and survival, cost, and immune monitoring, will also be discussed. MATERIALS AND METHODS: Databases such as PubMed, Scopus, and Web of Science, as well as additional online resources such as EBSCO, were exhaustively searched. Adult living-donor kidney transplant recipients who developed chimerism-based tolerance after concurrent bone marrow or hematopoietic stem cell transplantation or those who received non-chimerism-based, non-hematopoietic cell therapy using mesenchymal stromal cells, dendritic cells, or regulatory T cells were studied between 2000 and 2021. Individual sources of evidence were evaluated critically, and the strength of evidence and risk of bias for each outcome of the transplant tolerance study were assessed. RESULTS: From 28,173 citations, 245 studies were retrieved after suitable exclusion and duplicate removal. Of these, 22 studies (2 RCTs, 11 cohort studies, 6 case-control studies, and 3 case reports) explicitly related to both interventions (chimerism- and non-chimerism-based immune tolerance) were used in the final review process and were critically appraised. According to the findings, chimerism-based strategies fostered immunotolerance, allowing for the safe withdrawal of immunosuppressive medications. Cell-based therapy, on the other hand, frequently did not induce tolerance except for minimising immunosuppression. As a result, the rejection rates, renal allograft function, and survival rates could not be directly compared between these two groups. While chimerism-based tolerance protocols posed safety concerns due to myelosuppression, including infections and graft-versus-host disease, cell-based strategies lacked these adverse effects and were largely safe. There was a lack of direct comparisons between HLA-identical and HLA-disparate recipients, and the cost implications were not examined in several of the retrieved studies. Most studies reported successful immunosuppressive weaning lasting at least 3 years (ranging up to 11.4 years in some studies), particularly with chimerism-based therapy, while only a few investigators used immune surveillance techniques. The studies reviewed were often limited by selection, classification, ascertainment, performance, and attrition bias. CONCLUSIONS: This review demonstrates that chimerism-based hematopoietic strategies induce immune tolerance, and a substantial number of patients are successfully weaned off immunosuppression. Despite the risk of complications associated with myelosuppression. Non-chimerism-based, non-hematopoietic cell protocols, on the other hand, have been proven to facilitate immunosuppression minimization but seldom elicit immunological tolerance. However, the results of this review must be interpreted with caution because of the non-randomised study design, potential confounding, and small sample size of the included studies. Further validation and refinement of tolerogenic protocols in accordance with local practice preferences is also warranted, with an emphasis on patient selection, cost ramifications, and immunological surveillance based on reliable tolerance assays.

Morphological and biomechanical analyses of the human healthy and glaucomatous aqueous outflow pathway: Imaging-to-modeling.

BACKGROUND AND OBJECTIVE: Intraocular pressure (IOP) is maintained via a dynamic balance between the production of aqueous humor and its drainage through the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and Schlemm's canal (SC) endothelium of the conventional outflow pathway. Primary open angle glaucoma (POAG) is often associated with IOP elevation that occurs due to an abnormally high outflow resistance across the outflow pathway. Outflow tissues are viscoelastic and actively interact with aqueous humor dynamics through a two-way fluid-structure interaction coupling. While glaucoma affects the morphology and stiffness of the outflow tissues, their biomechanics and hydrodynamics in glaucoma eyes remain largely unknown. This research aims to develop an image-to-model method allowing the biomechanics and hydrodynamics of the conventional aqueous outflow pathway to be studied. METHODS: We used a combination of X-ray computed tomography and scanning electron microscopy to reconstruct high-fidelity, eye-specific, 3D microstructural finite element models of the healthy and glaucoma outflow tissues in cellularized and decellularized conditions. The viscoelastic TM/JCT/SC complex finite element models with embedded viscoelastic beam elements were subjected to a physiological IOP load boundary; the stresses/strains and the flow state were calculated using fluid-structure interaction and computational fluid dynamics. RESULTS: Based on the resultant hydrodynamics parameters across the outflow pathway, the primary site of outflow resistance in healthy eyes was in the JCT and immediate vicinity of the SC inner wall, while the majority of the outflow resistance in the glaucoma eyes occurred in the TM. The TM and JCT in the glaucoma eyes showed 1.32-fold and 1.13-fold larger beam thickness and smaller trabecular space size (2.24-fold and 1.50-fold) compared to the healthy eyes. CONCLUSIONS: Characterizing the accurate morphology of the outflow tissues may significantly contribute to constructing more accurate, robust, and reliable models, that can eventually help to better understand the dynamic IOP regulation, hydrodynamics of the aqueous humor, and outflow resistance dynamic in the human eyes. This model demonstrates proof of concept for determining changes to outflow resistance in healthy and glaucomatous tissues and thus may be utilized in larger cohorts of donor tissues where disease specificity, race, age, and gender of the eye donors may be accounted for.

Genome-Wide RNA Sequencing of Human Trabecular Meshwork Cells Treated with TGF-ß1: Relevance to Pseudoexfoliation Glaucoma.

Pseudoexfoliation glaucoma (XFG) is an aggressive form of secondary open angle glaucoma, characterised by the production of exfoliation material and is estimated to affect 30 million people worldwide. Activation of the TGF-ß pathway by TGF-ß1 has been implicated in the pathogenesis of pseudoexfoliation glaucoma. To further investigate the role of TGF-ß1 in glaucomatous changes in the trabecular meshwork (TM), we used RNA-Seq to determine TGF-ß1 induced changes in the transcriptome of normal human trabecular meshwork (HTM) cells. The main purpose of this study was to perform a hypothesis-independent RNA sequencing analysis to investigate genome-wide alterations in the transcriptome of normal HTMs stimulated with TGF-ß1 and investigate possible pathophysiological mechanisms driving XFG. Our results identified multiple differentially expressed genes including several genes known to be present in exfoliation material. Significantly altered pathways, biological processes and molecular functions included extracellular matrix remodelling, Hippo and Wnt pathways, the unfolded protein response, oxidative stress, and the antioxidant system. This cellular model of pseudoexfoliation glaucoma can provide insight into disease pathogenesis and support the development of novel therapeutic interventions.

Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery.

The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.

1H NMR Metabolite Monitoring during the Differentiation of Human Induced Pluripotent Stem Cells Provides New Insights into the Molecular Events That Regulate Embryonic Chondrogenesis.

The integration of cell metabolism with signalling pathways, transcription factor networks and epigenetic mediators is critical in coordinating molecular and cellular events during embryogenesis. Induced pluripotent stem cells (IPSCs) are an established model for embryogenesis, germ layer specification and cell lineage differentiation, advancing the study of human embryonic development and the translation of innovations in drug discovery, disease modelling and cell-based therapies. The metabolic regulation of IPSC pluripotency is mediated by balancing glycolysis and oxidative phosphorylation, but there is a paucity of data regarding the influence of individual metabolite changes during cell lineage differentiation. We used 1H NMR metabolite fingerprinting and footprinting to monitor metabolite levels as IPSCs are directed in a three-stage protocol through primitive streak/mesendoderm, mesoderm and chondrogenic populations. Metabolite changes were associated with central metabolism, with aerobic glycolysis predominant in IPSC, elevated oxidative phosphorylation during differentiation and fatty acid oxidation and ketone body use in chondrogenic cells. Metabolites were also implicated in the epigenetic regulation of pluripotency, cell signalling and biosynthetic pathways. Our results show that 1H NMR metabolomics is an effective tool for monitoring metabolite changes during the differentiation of pluripotent cells with implications on optimising media and environmental parameters for the study of embryogenesis and translational applications.

Genome-wide transcriptome profiling of human trabecular meshwork cells treated with TGF-ß2.

Glaucoma is a complex neurodegenerative disease resulting in progressive optic neuropathy and is a leading cause of irreversible blindness worldwide. Primary open angle glaucoma (POAG) is the predominant form affecting 65.5 million people globally. Despite the prevalence of POAG and the identification of over 120 glaucoma related genetic loci, the underlaying molecular mechanisms are still poorly understood. The transforming growth factor beta (TGF-ß) signalling pathway is implicated in the molecular pathology of POAG. To gain a better understanding of the role TGF-ß2 plays in the glaucomatous changes to the molecular pathology in the trabecular meshwork, we employed RNA-Seq to delineate the TGF-ß2 induced changes in the transcriptome of normal primary human trabecular meshwork cells (HTM). We identified a significant number of differentially expressed genes and associated pathways that contribute to the pathogenesis of POAG. The differentially expressed genes were predominantly enriched in ECM regulation, TGF-ß signalling, proliferation/apoptosis, inflammation/wound healing, MAPK signalling, oxidative stress and RHO signalling. Canonical pathway analysis confirmed the enrichment of RhoA signalling, inflammatory-related processes, ECM and cytoskeletal organisation in HTM cells in response to TGF-ß2. We also identified novel genes and pathways that were affected after TGF-ß2 treatment in the HTM, suggesting additional pathways are activated, including Nrf2, PI3K-Akt, MAPK and HIPPO signalling pathways. The identification and characterisation of TGF-ß2 dependent differentially expressed genes and pathways in HTM cells is essential to understand the patho-physiology of glaucoma and to develop new therapeutic agents.

An Optimized Method to Decellularize Human Trabecular Meshwork.

Glaucoma is linked to raised intraocular pressure (IOP). The trabecular meshwork (TM) plays a major role in regulating IOP by enabling outflow of aqueous humor from the eye through its complex 3D structure. A lack of therapies targeting the dysfunctional TM highlights the need to develop biomimetic scaffolds that provide 3D in vitro models for glaucoma research or as implantable devices to regenerate TM tissue. To artificially mimic the TM's structure, we assessed methods for its decellularization and outline an optimized protocol for cell removal and structural retention. Using bovine TM, we trialed 2 lysing agents-Trypsin (0.05% v/v) and Ammonium Hydroxide (NH4OH; 2% v/v). Twenty-four hours in Trypsin caused significant structural changes. Shorter exposure (2 h) reduced this disruption whilst decellularizing the tissue (dsDNA 26 ± 14 ng/mL (control 1970 ± 146 ng/mL)). In contrast, NH4OH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% v/v NH4OH similarly removed cells (dsDNA 52 ± 4 ng/mL (control 1965 ± 233 ng/mL)), and light microscopy and SEM presented no structural damage. X-ray computed tomography enabled a novel 3D reconstruction of decellularized human TM and observation of the tissue's intricate architecture. This study provides a new, validated method using NH4OH to decellularize delicate human TM without compromising tissue structure.

Short and long-term effect of dexamethasone on the transcriptome profile of primary human trabecular meshwork cells in vitro.

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma.

Biofabrication of Artificial Stem Cell Niches in the Anterior Ocular Segment.

The anterior segment of the eye is a complex set of structures that collectively act to maintain the integrity of the globe and direct light towards the posteriorly located retina. The eye is exposed to numerous physical and environmental insults such as infection, UV radiation, physical or chemical injuries. Loss of transparency to the cornea or lens (cataract) and dysfunctional regulation of intra ocular pressure (glaucoma) are leading causes of worldwide blindness. Whilst traditional therapeutic approaches can improve vision, their effect often fails to control the multiple pathological events that lead to long-term vision loss. Regenerative medicine approaches in the eye have already had success with ocular stem cell therapy and ex vivo production of cornea and conjunctival tissue for transplant recovering patients' vision. However, advancements are required to increase the efficacy of these as well as develop other ocular cell therapies. One of the most important challenges that determines the success of regenerative approaches is the preservation of the stem cell properties during expansion culture in vitro. To achieve this, the environment must provide the physical, chemical and biological factors that ensure the maintenance of their undifferentiated state, as well as their proliferative capacity. This is likely to be accomplished by replicating the natural stem cell niche in vitro. Due to the complex nature of the cell microenvironment, the creation of such artificial niches requires the use of bioengineering techniques which can replicate the physico-chemical properties and the dynamic cell-extracellular matrix interactions that maintain the stem cell phenotype. This review discusses the progress made in the replication of stem cell niches from the anterior ocular segment by using bioengineering approaches and their therapeutic implications.

Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control?

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM's structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

Exploiting biomaterial approaches to manufacture an artificial trabecular meshwork: A progress report.

Glaucoma is the second leading cause of irreversible blindness worldwide. Glaucoma is a progressive optic neuropathy in which permanent loss of peripheral vision results from neurodegeneration in the optic nerve head. The trabecular meshwork is responsible for regulating intraocular pressure, which to date, is the only modifiable risk factor associated with the development of glaucoma. Lowering intraocular pressure reduces glaucoma progression and current surgical approaches for glaucoma attempt to reduce outflow resistance through the trabecular meshwork. Many surgical approaches use minimally invasive glaucoma surgeries (MIGS) to control glaucoma. In this progress report, biomaterials currently employed to treat glaucoma, such as MIGS, and the issues associated with them are described. The report also discusses innovative biofabrication approaches that aim to revolutionise glaucoma treatment through tissue engineering and regenerative medicine (TERM). At present, there are very few applications targeted towards TM engineering in vivo, with a great proportion of these biomaterial structures being developed for in vitro model use. This is a consequence of the many anatomical and physiological attributes that must be considered when designing a TERM device for microscopic tissues, such as the trabecular meshwork. Ongoing advancements in TERM research from multi-disciplinary teams should lead to the development of a state-of-the-art device to restore trabecular meshwork function and provide a bio-engineering solution to improve patient outcomes.

Plasma polymer surface modified expanded polytetrafluoroethylene promotes epithelial monolayer formation in vitro and can be transplanted into the dystrophic rat subretinal space.

The aim of this study was to evaluate whether the surface modification of expanded polytetrafluoroethylene (ePTFE) using an n-heptylamine (HA) plasma polymer would allow for functional epithelial monolayer formation suitable for subretinal transplant into a non-dystrophic rat model. Freshly isolated iris pigment epithelial (IPE) cells from two rat strains (Long Evans [LE] and Dark Agouti [DA]) were seeded onto HA, fibronectin-coated n-heptylamine modified (F-HA) and unmodified ePFTE and fibronectin-coated tissue culture (F-TCPS) substrates. Both F-HA ePTFE and F-TCPS substrates enabled functional monolayer formation with both strains of rat. Without fibronectin coating, only LE IPE formed a monolayer on HA-treated ePTFE. Functional assessment of both IPE strains on F-HA ePTFE demonstrated uptake of POS that increased significantly with time that was greater than control F-TCPS. Surgical optimization using Healon GV and mixtures of Healon GV: phosphate buffered saline (PBS) to induce retinal detachment demonstrated that only Healon GV:PBS allowed F-HA ePTFE substrates to be successfully transplanted into the subretinal space of Royal College of Surgeons rats, where they remained flat beneath the neural retina for up to 4 weeks. No apparent substrate-induced inflammatory response was observed by fundus microscopy or immunohistochemical analysis, indicating the potential of this substrate for future clinical applications.

Thrombospondin-2 is up-regulated by TGFß2 and increases fibronectin expression in human trabecular meshwork cells.

Elevated intraocular pressure (IOP) is a major risk factor for the development of primary open-angle glaucoma (POAG). This is from an increased aqueous humour (AH) outflow resistance through the trabecular meshwork (TM). The pathogenic mechanisms leading to the increase in TM outflow resistance are poorly understood but are thought to be from a dysregulation of the TM extracellular matrix (ECM) environment. ECM modification and turnover are crucial in regulating the resistance to aqueous outflow. ECM turnover is influenced by a complex interplay of growth factors such as transforming growth factors (TGFß) family and matrix metalloproteinases (MMPs). Elevated TGFß2 levels result in an increase in ECM deposition such as fibronectin leading to increased resistance. Fibronectin is a major component of TM ECM and plays a key role in its maintenance. Thrombospondins (TSP)-1 and -2 are important regulators of the ECM environment. TSP-1 has been implicated in the pathogenesis of POAG through activation of TGFß2 within the TM. TSP-2 does not contain the catalytic domain to activate latent TGFß, but is able to mediate the activities of MMP 2 and 9, thereby influencing ECM turnover. TSP-2 knock out mice show lower IOP levels compared to their wild type counterparts, suggesting the involvement of TSP-2 in the pathogenesis of POAG but its role in the pathogenesis of POAG remains unclear. The purpose of this study was to investigate the role of TSP-2 in trabecular meshwork ECM regulation and hence the pathogenesis of POAG. TSP-1 and TSP-2 expressions in immortalised glaucomatous TM cells (GTM3) and primary human non-glaucomatous (NTM) and glaucomatous cells (GTM) were determined by immunocytochemistry, immuno-blot analysis and qPCR following treatment with TGFß2 and Dexamethasone. The level of ECM protein fibronectin was determined in TM cells using immuno-blot analysis following treatment with TSP-1 or -2. TM cells secrete TSP-1 and -2 under basal conditions at the protein level and TSP-2 mRNA and protein levels were increased in response to TGFß2 three days post treatment. Exogenous treatment with TSP-2 up-regulated the expression of fibronectin protein in GTM3 cells, primary NTM and GTM cells. TSP-1 did not affect fibronectin protein levels in GTM3 cells. This suggests that the role of TSP-2 might be distinct from that of TSP-1 in the regulation of the TM cell ECM environment. TSP-2 may be involved in the pathogenesis of POAG and contribute to increased IOP levels by increasing the deposition of fibronectin within the ECM in response to TGFß2.

Differential Distribution of Laminin N-Terminus α31 Across the Ocular Surface: Implications for Corneal Wound Repair.

Purpose: Laminin N-terminus (LaNt) α31 is a relatively unstudied protein derived from the laminin α3 gene but structurally similar to netrins. LaNt α31 has, to date, been investigated only in two-dimensional (2D) keratinocyte culture where it influences cell migration and adhesion, processes integral to wound repair. Here we investigated LaNt α31 distribution in ocular surface epithelium, during limbal stem cell activation, and corneal wound healing. Methods: Human, mouse, and pig eyes, ex vivo limbal explant cultures, and alkali burn wounds were processed for immunohistochemistry with antibodies against LaNt α31 along with progenitor cell-associated proteins. LaNt α31 expression was induced via adenoviral transduction into primary epithelial cells isolated from limbal explants, and cell spreading and migration were analyzed using live imaging. Results: LaNt α31 localized to the basal layer of the conjunctival, limbal, and corneal epithelial cells. However, staining was nonuniform with apparent subpopulation enrichment, and some suprabasal reactivity was also noted. This LaNt α31 distribution largely matched that of keratin 15, epidermal growth factor receptor, and transformation-related protein 63α (p63α), and displayed similar increases in expression in activated limbal explants. During active alkali burn wound repair, LaNt α31 displayed increased expression in limbal regions and loss of basal restriction within the cornea. Distribution returned to predominately basal cell restricted once the wounded epithelium matured. Cultured corneal epithelial cells expressing LaNt α31 displayed increased 2D area and reduced migration, suggesting a functional link between this protein and key wound repair activities. Conclusions: These data place LaNt α31 in position to influence laminin-dependent processes including wound repair and stem cell activation.

The formation of a functional retinal pigment epithelium occurs on porous polytetrafluoroethylene substrates independently of the surface chemistry.

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells may have the potential to preserve or restore vision in patients affected by blinding diseases such as age-related macular degeneration (AMD). One of the critical steps in achieving this is the ability to grow a functioning retinal pigment epithelium, which may need a substrate on which to grow and to aid transplantation. Tailoring the physical and chemical properties of the substrate should help the engineered tissue to function in the long term. The purpose of the study was to determine whether a functioning monolayer of RPE cells could be produced on expanded polytetrafluoroethylene substrates modified by either an ammonia plasma treatment or an n-Heptylamine coating, and whether the difference in surface chemistries altered the extracellular matrix the cells produced. Primary human RPE cells were able to form a functional, cobblestone monolayer on both substrates, but the formation of an extracellular matrix to exhibit a network structure took months, whereas on non-porous substrates with the same surface chemistry, a similar appearance was observed after a few weeks. This study suggests that the surface chemistry of these materials may not be the most critical factor in the development of growth of a functional monolayer of RPE cells as long as the cells can attach and proliferate on the surface. This has important implications in the design of strategies to optimise the clinical outcomes of subretinal transplant procedures.

Yield and Viability of Human Limbal Stem Cells From Fresh and Stored Tissue.

PURPOSE: We compared cell number, putative stem cell markers, and clonogenic ability in fresh uncultured human limbal epithelial cells to that obtained from stored organ-cultured tissue. METHODS: Cell suspensions were formed from fresh and organ culture-stored human limbal epithelium. Expression of putative stem cell markers ΔNp63 and TrkA was performed using immunofluorescent staining before culture. Colony-forming efficiency (CFE) assays were performed at first passage. The effects of tissue storage, age, and postmortem/culture times were analyzed in a general linear model. RESULTS: Limbal tissue from 94 donors (34 fresh and 60 stored) was compared. Three times more cells were obtained per eye from fresh (35.34 × 104; SD, 17.39) than stored (11.24 × 104; SD, 11.57; P < 0.01) tissue. A higher proportion of cells from fresh tissue were viable (91.9%; SD, 5.7 vs. 85%; SD, 10.8) P < 0.01. Higher total cell expression of ΔNp63 (20.19 × 104; SD, 15.5 vs. 3.28 104; SD, 4.33) and TrkA (59.24 × 104; SD, 13.21 vs. 7.65 × 104; SD, 1.05) was observed in fresh than stored tissue (P < 0.01). Colony-forming efficiency was higher for fresh (1.42; SD, 0.12) than stored (0.43; SD, 0.15; P < 0.01) cells. For stored tissue only, there was a significant inverse relationship between donor age and total number of cells isolated (R2 = 0.27, P < 0.001). CONCLUSIONS: Storage of corneoscleral discs in organ culture medium leads to significant reduction in limbal epithelial cell number, expression of ΔNp63 and TrkA, and viability compared to fresh tissue. There is a smaller basal stem cell population in stored compared to fresh tissue.

Concise Review: An Update on the Culture of Human Corneal Endothelial Cells for Transplantation.

The cornea forms the front window of the eye, enabling the transmission of light to the retina through a crystalline lens. Many disorders of the cornea lead to partial or total blindness, and therefore corneal transplantation becomes mandatory. Recently, selective corneal layer (as opposed to full thickness) transplantation has become popular because this leads to earlier rehabilitation and visual outcomes. Corneal endothelial disorders are a common cause of corneal disease and transplantation. Corneal endothelial transplantation is successful but limited worldwide because of lower donor corneal supply. Alternatives to corneal tissue for endothelial transplantation therefore require immediate attention. The field of human corneal endothelial culture for transplantation is rapidly emerging as a possible viable option. This manuscript provides an update regarding these developments. Significance: The cornea is the front clear window of the eye. It needs to be kept transparent for normal vision. It is formed of various layers of which the posterior layer (the endothelium) is responsible for the transparency of the cornea because it allows the transport of ions and solutes to and from the other layers of the cornea. Corneal blindness that results from the corneal endothelial dysfunction can be treated using healthy donor tissues. There is a huge demand for human donor corneas but limited supply, and therefore there is a need to identify alternatives that would reduce this demand. Research is underway to understand the isolation techniques for corneal endothelial cells, culturing these cells in the laboratory, and finding possible options to transplant these cells in the patients. This review article is an update on the recent developments in this field.

Human Conjunctival Stem Cells are Predominantly Located in the Medial Canthal and Inferior Forniceal Areas.

PURPOSE: The conjunctiva plays a key role in ocular surface defence and maintenance of the tear film. Ex vivo expansion of conjunctival epithelial cells offers potential to reconstruct the ocular surface in cases of severe cicatrising disease, but requires initial biopsies rich in stem cells to ensure long-term success. The distribution of human conjunctival stem cells, however, has not been clearly elucidated. METHODS: Whole human cadaveric conjunctiva was retrieved and divided into specific areas for comparison. From each donor, all areas from one specimen were cultured for colony-forming efficiency assays and immunocytochemical studies; all areas from the other specimen were fixed and paraffin embedded for immunohistochemical studies. Expression of CK19, p63, and stem cell markers ABCG2, ΔNp63, and Hsp70 were analyzed. Results were correlated to donor age and postmortem retrieval time. RESULTS: Conjunctiva was retrieved from 13 donors (26 specimens). Colony-forming efficiency and expression of stem cell markers ABCG2, ΔNp63, and Hsp70 in cultures and ABCG2 in fixed tissue were all consistently demonstrated throughout the tissue but with highest levels in the medial canthal and inferior forniceal areas (P < 0.01 for each). Both increasing donor age and longer postmortem retrieval times were associated with significantly lower colony-forming efficiency, stem cell marker expression in cell cultures and ABCG2 expression in fixed tissue. CONCLUSIONS: Biopsies from the medial canthus and inferior forniceal areas, from younger donors, and with short postmortem retrieval times offer the greatest potential to developing conjunctival stem cell-rich epithelial constructs for transplantation.