Generation of a TALEN-mediated, p63 knock-in in human induced pluripotent stem cells

The expression of p63 in surface ectodermal cells during development of the cornea, skin, oral mucosa and olfactory placodes is integral to the process of cellular self-renewal and the maintenance of the epithelial stem cell status. Here, we used TALEN technology to generate a p63 knock-in (KI) human induced pluripotent stem (hiPS) cell line in which p63 expression can be visualized via enhanced green fluorescent protein (EGFP) expression. The KI-hiPS cells maintained pluripotency and expressed the stem cell marker gene, ΔNp63α. They were also able to successfully differentiate into functional corneal epithelial cells as assessed by p63 expression in reconstructed corneal epithelium. This approach enables the tracing of p63-expressing cell lineages throughout epithelial development, and represents a promising application in the field of stem cell research

Topical delivery of a Rho-kinase inhibitor to the cornea via mucoadhesive film

The application of inhibitors of the Rho kinase pathway (ROCK inhibitors) to the surface of the eye in the form of eyedrops has beneficial effects which aid the recovery of diseased or injured endothelial cells that line the inner surface of the cornea. The aim of this study was to test the plausibility of delivering a selective ROCK inhibitor, Y-27,632, to the cornea using a thin polymeric film. Mucoadhesive polymeric thin films were prepared incorporating Y-27,632 and diffusional release into PBS was determined. Topical ocular delivery from the applied film was investigated using freshly excised porcine eyes and eyedrops of equivalent concentration acted as comparators; after 24 h the formulations were removed and the corneas extracted. Drug-loaded thin polymeric films, with high clarity and pliability were produced. ROCK inhibitor Y-27,632 was weakly retained within the film, with release attaining equilibrium after 1 h. This in turn facilitated its rapid ocular delivery, and an approximately three-fold greater penetration of Y-27,632 into cryoprobe-treated corneas was observed from the thin film (p < 0.01) compared to eyedrop. These findings support the further development of ROCK inhibitor delivery to the cornea via release from thin mucoadhesive films to treat vision loss cause by corneal endothelial dysfunction

Ocular surface ectoderm instigated by WNT inhibition and BMP4

We sought to elucidate how and when the ocular surface ectoderm commits to its differentiation into the corneal epithelium in eye development from human induced pluripotent stem cells (hiPSCs) under the influence of WNT signaling and the actions of BMP4. These signals are key drivers ocular surface ectodermal cell fate determination. It was discovered that secreted frizzled related protein-2 (SFRP2) and Dickkopf1 (DKK1), which are expressed in neural ectoderm, are both influential in the differentiation of hiPSCs, where they act as canonical WNT antagonists. BMP4, moreover, was found to simultaneously initiate non-neural ectodermal differentiation into a corneal epithelial lineage. Combined treatment of hiPSCs with exogenous BMP4 aligned to WNT inhibition for the initial four days of differentiation increased the ocular surface ectodermal cell population and induced a corneal epithelial phenotype. Specification of a surface ectodermal lineage and its fate is thus determined by a fine balance of BMP4 exposure and WNT inhibition in the very earliest stages of human eye development

Collagen organization in the secondary chick cornea during development

PURPOSE. The latter stages of morphogenesis in the embryonic chick cornea are instrumental in the establishment of a properly formed corneal stroma. This study was designed to provide better appreciation of collagen reorganization in the avian corneal stroma during the latter stages of embryogenesis. METHODS. High-angle synchrotron x-ray diffraction patterns were obtained from 47 developing chick corneas daily at developmental days 13 through 18 (n = 7 or 8 at each time point) and analyzed to establish collagen molecular spacing and fibril orientation. RESULTS. Collagen intermolecular x-ray reflections were of approximately constant intensity between days 13 and 15 of development, but thereafter became progressively more intense, suggesting that extra collagen is deposited in embryonic chick corneas after day 16 of development. At all times, the mean collagen intermolecular spacing measured approximately 1.43 nm. X-ray intensity was not uniform around the intermolecular x-ray reflections at earlier time points. Rather, a fourfold symmetry was evident, indicative of an orthogonal array of collagen fibrils. An index of this symmetry was essentially unchanged between developmental days 13 and 15, but thereafter diminished considerably. CONCLUSIONS. The lateral spacing of fibril-forming collagen molecules does not change as the chick cornea develops between days 13 and 18. An orthogonal array of collagen fibrils is present in the corneas of developmental day-13 to -18 chicks, but starting at developmental day 16, additional collagen is deposited in a less well-oriented manner and thus acts to obscure the overall orthogonality, with implications for the biomechanical strength and shape of the cornea

Keratan sulfate phenotype in the β-1,3-N-acetylglucosaminyltransferase-7-null mouse cornea

Purpose: Synthesis of keratan sulfate (KS) relies on coordinated action of multiple enzymes, including the N-acetylglucosamine–transferring enzyme, β-1,3-N-acetylglucosaminyltransferase-7 (β3GnT7). A mouse model deficient in β3GnT7 was developed to explore structural changes in KS and the extracellular matrix (ECM; i.e., the corneal stroma), elucidate the KS biosynthesis mechanism, and understand its role in corneal organization. Methods: A knockout vector for the β3GnT7-encoding gene, B3gnt7, was created to develop heterozygous- (htz) and homozygous-null (null) knockouts. Epithelial, stromal, and whole cornea thicknesses were measured from each group. Proteoglycans were stained with cupromeronic blue for visualization by electron microscopy, and Western blot analyses were conducted on the KS core protein, lumican. Corneal sections were labelled fluorescently for KS and chondroitin sulfate/dermatan sulfate (CS/DS) using monoclonal antibodies 1B4 or 2B6, respectively. Results: Wild-type (WT) and htz corneas were of similar stromal thickness, whereas null specimens measured relatively thin. Electron micrographs revealed that WT and htz samples contained comparable levels of KS- and CS/DS-PGs. Null corneas, however, lacked detectable KS and featured uncharacteristically elongated electron dense PG filaments, which were susceptible to chondroitinase ABC digestion. Western blotting revealed lumican in the null corneas was substituted with low-molecular-weight KS, relative to WT or htz tissue. KS was not immunohistochemically detectable in the null cornea, whereas CS/DS content appeared increased. Conclusions: Addition of N-acetylglucosamine via β3GnT7 to KS glycosaminoglycans is necessary for their biosynthesis. Without β3GnT7, murine corneal stromas lack KS and appear to compensate for this loss with upregulation of chondroitinase ABC-sensitive PGs

The generation of fluorometholone nanocrystal eye drops, their metabolization to dihydrofluorometholone and penetration into rabbit eyes

Fluorometholone is a widely used anti-inflammatory ophthalmic formulation, which elicits a lower ocular hypertensive response than other glucocorticoid medications. This serves to mitigate against the risk of steroid-induced glaucoma. Based on the hypothesis that an improved corneal permeability can increase the bioavailability of a drug, we sought to obtain fluorometholone in suspension with a small particle size. Accordingly, we describe the formulation of fluorometholone nanocrystal eye drops, which have a mean particle size of 201.2 ± 14.1 nm (standard deviation (s.d.)) when measured by dynamic light scattering. Scanning electron microscopy further indicates that fluorometholone nanocrystals are predominantly rectangular in shape. Fluorometholone microcrystals, on the other hand, with a mean particle size of 9.24 ± 4.51 µm (s.d.), tend to have a rod-like morphology. Powder x-ray diffraction revealed that fluorometholone microcrystal and nanocrystal formulations have the same crystal structure, with the main diffraction peaks at 2θ = 10.4 and 15.3°. The nanocrystal formulation was found to be stable, long-term, when stored at 10 °C for up to 6-months. High pressure liquid chromatography (HPLC) of the aqueous humor of rabbit eyes 15–240 mins after the in vivo application of fluorometholone eye drops to the ocular surface revealed that the molecule had been converted to 20α-dihydrofluorometholone (with no evidence of a 20β-dihydrofluorometholone fraction), and that penetration was 2–6 fold higher and longer lasting with the nanocrystal, rather than the microcrystal, formulation. In current study we show how newly generated fluorometholone nanocrystals when administered as eye drops enter the anterior chamber of the eye and become metabolized to dihydrofluorometholone

A study of corneal thickness, shape and collagen organisation in keratoconus using videokeratography and X-ray scattering techniques

In keratoconus, the cornea becomes progressively ectactic resulting in severe visual impairment. Here, we use a combination of videokeratography and synchrotron X-ray diffraction to investigate the relationship between corneal shape and thickness, and the distribution and predominant orientation of stromal fibrillar collagen in five keratoconus corneas. In all but the least advanced case, the thinning and ectasia measured in vivo using corneal videokeratography was accompanied by corresponding changes in the relative distribution and orientation of stromal collagen in the excised corneal buttons. Although the most severe case of keratoconus possessed the most pronounced stromal collagen alterations, and only a minor disruption to stromal collagen arrangement was seen in the least advanced case, a variability in the extent of stromal collagen alteration was seen between these clinical extremes. The observed abnormalities in collagen distribution and orientation are consistent with a mechanism of keratoconus progression that involves inter-fibrillar or inter-lamellar slippage causing a redistribution of tissue within the cornea

CD200 facilitates the isolation of corneal epithelial cells derived from human pluripotent stem cells

The in vitro induction of corneal epithelial cells (CECs) from human induced pluripotent stem cells (iPSCs) represents a new strategy for obtaining CE stem/progenitor cells for the surgical reconstruction of a diseased or injured ocular surface. The clinical promise of this strategy is considerable, but if the approaches’ potential is to be realised, robust methods for the purification of iPSC-derived CE lineage cells need to be developed to avoid contamination with other cells that may carry the risk of unwanted side effects, such as tumorigenesis. Experiments conducted here revealed that during CEC isolation, CD200-negative selection using a cell sorter considerably reduced the contamination of the cell population with various non-CECs compared with what could be achieved using TRA-1-60, a conventional negative marker for CECs. Furthermore, CD200-negative sorting did not affect the yield of CECs nor that of their stem/progenitor cells. Single-cell gene expression analysis for CEC sheets obtained using CD200-negative sorting showed that all analysed cells were CE-lineage cells, expressing PAX6, delta-N p63, and E-cadherin. Non-CECs, on the other hand, expressed non-CEC genes such as FGFR1 and RPE65. CD200, thus, represents a robust negative marker for purification of induced CE lineage cells, which is expressed by undifferentiated iPSCs and non-CECs, including iPSC-derived neural and retinal cells

Developmental changes in patterns of distribution of fibronectin and tenascin-C in the chicken cornea: evidence for distinct and independent functions during corneal development and morphogenesis

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior–posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman’s layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles

The microanatomy of Bowman’s layer in the cornea of the pig: changes in collagen fibril architecture at the corneoscleral limbus

In most animals, Bowman’s layer is a feature of the cornea of the eye, and lies between the sur-face epithelium and the stromal extracellular matrix that makes up the bulk of the cornea. It is com-prised of a condensation of disorganised collagen fibrils. However, it has been conjectured that not all species possess Bowman’s layer, and pigs are a species that has classically been stated to lack this anatomical structure, although there is disa-greement in the published literature. Here, we studied the porcine cornea using transmission and scanning electron microscopy (TEM and SEM) to ascertain whether Bowman’s layer existed. TEM identified a thin band of disorganised collagen fi-brils between the epithelial basement membrane and corneal stroma. SEM images of the central and peripheral corneal surfaces, following removal of the corneal epithelium by cell maceration, re-vealed a disorganised meshwork of collagen fibrils, with a highly aligned annulus of collagen at the limbus. In between the peripheral cornea and lim-bus, a “transition zone” is observed where collagenfibrils start to align. Quantification of fibril alignment demonstrates a significant increase in collagen alignment from 0.08 ± 0.04 to 0.33 ± 0.07 (p < 0.001; n = 60; 0 = no alignment, 1 = full alignment) with increasing distance from the corneal centre. These data together lead us to conclude that the porcine cornea does include Bowman’s layer, though it is thin (contributing roughly 0.2% of cor-neal thickness), and thus, reaffirms the porcine cornea’s similarity to its human counterpart and usefulness as a model system