Andrew Hopkinson

Concise review: evidence for CD34 as a common marker for diverse progenitors.

CD34 is a transmembrane phosphoglycoprotein, first identified on hematopoietic stem and progenitor cells. Clinically, it is associated with the selection and enrichment of hematopoietic stem cells for bone marrow transplants. Due to these historical and clinical associations, CD34 expression is almost ubiquitously related to hematopoietic cells, and it is a common misconception that CD34‐positive (CD34+) cells in nonhematopoietic samples represent hematopoietic contamination. The prevailing school of thought states that multipotent mesenchymal stromal cells (MSC) do not express CD34. However, strong evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types including muscle satellite cells, corneal keratocytes, interstitial cells, epithelial progenitors, and vascular endothelial progenitors. In many cases, the CD34+ cells represent a small proportion of the total cell population and also indicate a distinct subset of cells with enhanced progenitor activity. Herein, we explore common traits between cells that express CD34, including associated markers, morphology and differentiation potential. We endeavor to highlight key similarities between CD34+ cells, with a focus on progenitor activity. A common function of CD34 has yet to be elucidated, but by analyzing and understanding links between CD34+ cells, we hope to be able to offer an insight into the overlapping properties of cells that express CD34. Stem Cells 2014;32:1380–1389

Morphological characteristics of the limbal epithelial crypt

Aim: In 2005 we reported the discovery of a novel anatomical structure at the limbus, which we termed the limbal epithelial crypt (LEC). The purpose of this study was to further evaluate the distribution, immunophenotypical, and ultra structural characteristics of the LEC as a putative niche of stem cells. Methods: Sequential histological sections of human corneo-scleral limbal rims were examined for the presence and distribution of the LEC. Immunophenotypical characterisation of the LEC cells using a panel of antibodies of interest was undertaken. Transmission electron microscopy of the LEC was used to examine the ultra structural and morphometric features of cells within the LEC and adjacent limbus. Results: A total of 74 LECs were identified in eight corneo-scleral rims. These varied in number, size and distribution within rims. Cells within the crypt demonstrated the following phenotype: CK3−/CK19+/CD 34−/Vimentin+/p63+/Connexin 43+/MIB1 (Ki67)−. Presence of Cx43 was also demonstrated in the rete pegs adjacent to the LEC. Basal cells of the LEC were significantly smaller than basal cells found in adjacent rete pegs and also smaller than suprabasal limbal and central corneal epithelial cells (p<0.05). Morphologically they had a high nuclear:cytoplasmic ratio and were adherent to the underlying basement membrane by means of complex convolutions of cytoplasmic processes. Conclusions: LECs are sparse but a consistent finding in the human corneo-scleral limbus. The LEC contains a unique sub-population of cells expressing several characteristics that are consistent with it representing a putative stem cell niche.

Mesenchymal Stem Cells in the Human Corneal Limbal Stroma

PURPOSE Peripheral and limbal corneal stromal cells (PLCSCs), which contain keratocytes, have a complex phenotype. Knowledge of keratocyte cell properties, function, and origin is limited. Evidence available thus far has suggested both mesenchymal stromal and hematopoietic characteristics. Multipotent mesenchymal stromal cells (MSCs) are found in an increasing number of tissues and are the subject of considerable interest and investigation in the disciplines of tissue engineering, immunology, gene therapy, and oncology. METHODS Isolated PLCSCs were characterized by markers aldehyde dehydrogenase and keratocan, cultured, and analyzed against a set of criteria for the identification of MSCs developed by the International Society of Cellular Therapy (ISCT). PLCSCs were directly compared to fetal liver MSCs (flMSCs). Additional cell surface markers were also used to quantify differentiation, which was also performed on both cell types. RESULTS PLCSCs were found to be plastic adherent, displayed the correct profile and proportions of CSMs, and demonstrated trilineage potential in accordance with the ISCT guidelines. Furthermore, PLCSCs displayed a high degree of similarity to flMSCs and this likeness extended into the non-ISCT MSC cell surface markers and trilineage differentiation, which were often but not always comparable. CONCLUSIONS Herein we report a novel observation that PLCSCs conform to all the ISCT criteria and are therefore MSCs. Furthermore, this study has identified the limbal stroma as yet another MSC niche and presents a new perspective on the role of the PLCSC.

Gelatin-Based Materials in Ocular Tissue Engineering

Gelatin has been used for many years in pharmaceutical formulation, cell culture and tissue engineering on account of its excellent biocompatibility, ease of processing and availability at low cost. Over the last decade gelatin has been extensively evaluated for numerous ocular applications serving as cell-sheet carriers, bio-adhesives and bio-artificial grafts. These different applications naturally have diverse physical, chemical and biological requirements and this has prompted research into the modification of gelatin and its derivatives. The crosslinking of gelatin alone or in combination with natural or synthetic biopolymers has produced a variety of scaffolds that could be suitable for ocular applications. This review focuses on methods to crosslink gelatin-based materials and how the resulting materials have been applied in ocular tissue engineering. Critical discussion of recent innovations in tissue engineering and regenerative medicine will highlight future opportunities for gelatin-based materials in ophthalmology.

Optimization of Amniotic Membrane (AM) Denuding for Tissue Engineering

INTRODUCTION Amniotic membrane (AM) has gained increasing popularity as a useful carrier for ex vivo-expanded cells for tissue engineering, particularly in ocular surface reconstruction. However, current methods employed for denuding AM are highly variable, and the consequent effects on the structural and molecular composition of the AM basement membrane (BM) are ambiguous. We compare the effects of the main denuding procedures, and propose a highly effective standardized alternative. METHODS AMs preserved for transplantation were denuded using published ethylenediaminetetraacetic acid (EDTA)- and dispase-based methodologies and our novel thermolysin-based procedure. Scanning and transmission electron microscopy and immunohistochemistry, for BM components (collagens IV and VII, laminin 5, and integrins alpha6 and beta4), were used to assess effectiveness of denuding epithelium, whilst maintaining the integrity of the BM. RESULTS EDTA- and dispase-based denuding techniques resulted in the disaggregation and even destruction of the BM structure and molecular composition. Employing thermolysin effectively denuded epithelium whilst maintaining BM structural and molecular integrity. CONCLUSION Current procedures for preparing AM are variable and often ineffective, resulting in nonstandard membranes. Our novel thermolysin-based technique effectively denudes the AM whilst preserving an essentially intact and consistent BM. Therefore, we propose that this novel thermolysin procedure may potentially improve overall generation of tissue-engineered constructs using AM.

Amniotic membrane transplantation for ocular surface reconstruction: indications and outcomes

Background:  The amniotic membrane is a useful tool in the management of several ocular surface diseases. Despite numerous studies, standardization of the use of the membrane and of outcome measures is lacking. Herein, the authors’ results of amniotic membrane transplantation (AMT) in ocular surface reconstruction against defined outcome measures are reported.

Limbal epithelial crypt: a model for corneal epithelial maintenance and novel limbal regional variations.

OBJECTIVE To determine the distribution of cell membrane proteins and extracellular matrix proteins around the limbal epithelial crypt (LEC) compared with adjacent limbus and corneal epithelium. METHODS Serial histological sections of human corneoscleral limbus rims were stained with antibodies of interest by standard immunohistochemistry. RESULTS Superficial cells of the limbus were desmoglein 3 positive, compared with the negative basal cells of the limbus that correspond to cells with more stemlike properties. The LEC had a much lower proportion of desmoglein 3 staining in comparison. Tenascin C staining demonstrated regional variations of the limbus depending on their association with the LEC. Limbus that was associated with or adjacent to the LEC had a greater tenascin C expression compared with normal limbus, whereas the LEC demonstrated the greatest tenascin C expression. CONCLUSIONS Based on these and similar results previously reported for connexin 43, we propose a novel model on the mechanism of corneal surface epithelium maintenance involving 3 different limbal regions: zone 1, limbus including the LEC; zone 2, limbus associated with the LEC; and zone 3, limbus distant to the LEC. CLINICAL RELEVANCE The noted limbal variations may influence the selection of the donor site for limbal grafts in the future.

The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

An overview of current techniques for ocular toxicity testing

Given the hazardous nature of many materials and substances, ocular toxicity testing is required to evaluate the dangers associated with these substances after their exposure to the eye. Historically, animal tests such as the Draize test were exclusively used to determine the level of ocular toxicity by applying a test substance to a live rabbits eye and evaluating the biological response. In recent years, legislation in many developed countries has been introduced to try to reduce animal testing and promote alternative techniques. These techniques include ex vivo tests on deceased animal tissue, computational models that use algorithms to apply existing data to new chemicals and in vitro assays based on two dimensional (2D) and three dimensional (3D) cell culture models. Here we provide a comprehensive overview of the latest advances in ocular toxicity testing techniques, and discuss the regulatory framework used to evaluate their suitability.

Comparison of choroidal and retinal endothelial cells: characteristics and response to VEGF isoforms and anti-VEGF treatments.

Neovascular eye diseases such as wet age-related macular degeneration and proliferative diabetic retinopathy are two of the most common causes of irreversible visual loss. Although mediated by vascular endothelial growth factor (VEGF), the mechanisms of these diseases are not fully understood. Molecular inhibitors of VEGF including pegaptanib, ranibizumab and bevacizumab are used as treatments for these diseases. However, there have been very few direct comparisons between these agents, and as dose and treatment regimes differ their relative efficacies are hard to determine. In vitro comparisons tend to use cells from different sites or species, which show heterogeneity in their responses. The aim of this study was to compare the characteristics of primary cultures of isolated human choroidal endothelial cells (hCEC) and retinal endothelial cells (hREC), and their proliferation responses to stimulation with VEGF 121 and 165, and to compare the anti-proliferative effects of these three drugs. hCEC and hREC were positive for the cell markers VEGFR1, VEGFR2, CD31, CD34 and von Willebrands factor (vWF), with greater expression of CD34 on the hREC compared to hCEC. Contrary to previous assumptions VEGF isoforms 121 and 165 were found to be equally potent in stimulating endothelial cell proliferation. However, hREC exhibited higher proliferation with either VEGF isoform compared to hCEC. The anti-VEGF treatments ranibizumab and bevacizumab were effective in decreasing proliferation of hCEC induced by the two VEGF isoforms, individually and in combination, with ranibizumab being moderately more effective, particularly in hREC. Pegaptanib was effective in controlling the proliferation of hCEC stimulated by VEGF 165, but was ineffective against the stimulatory effect of VEGF 121. There were found to be significant differences in microvascular endothelial cells from the retina and choroid, both in the expression of cell markers and their behaviour in response to growth factors and currently available anti-VEGF agents, highlighting the importance of targeting treatments to specific intraocular vascular beds and/or diseases.