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

The Stromal Ultrastructure of Normal and Pathologic Human Corneas

This thesis describes results and observations from an ultrastructural study of the stroma of various human corneo-scleral tissues. The major components of the stromal extracellular matrix are collagen fibrils and proteoglycan macromolecules. Their character and distribution in normal human cornea and sclera are first studied. The main thrust of the research then progressed to elucidating the ultrastructure in two pathologic conditions where proteoglycan anomalies were known to occur; macular corneal dystrophy and corneal oedema. Transmission electron microscopical studies, employing the proteoglycan-specific stain Cuprolinic blue, demonstrated that the arrangement of proteoglycans, with respect to the collagen fibrils, in normal human cornea differs from other mammals in that there is more 'b' band association. Meridional X-ray diffraction showed that the axial electron density of human scleral collagen was similar to rat tail tendon collagen. When used in conjunction with Cupromeronic blue-staining, it verified as non-artifactual the electron microscopical observation that proteoglycans associate with collagen near the 'd/e' staining bands in the gap zone. Transmission electron microscopy revealed that macular dystrophy corneal stromas contain numerous collagen-free lacunae. Cuprolinic blue-staining further revealed that some of these lacunae contain congregations of various sized proteoglycan filaments. Enzyme digests identified these filaments as belonging to the chondroitin/ dermatan sulphate population of proteoglycans. It was concluded that aggregation of chondroitin/dermatan sulphate proteoglycans often occurs in the macular dystrophy stroma. X-ray diffraction data supported the electron microscopical observation of normal collagen fibrils in the macular dystrophy cornea. However, meridional X -ray data, from Cuprolinic blue-stained specimens, pointed to an abnormal distribution of proteoglycans along the collagen fibrils. Equatorial X-ray diffraction results indicated heterogeneous close packing of normal diameter collagen fibrils throughout the macular dystrophy stroma, this effect was deemed responsible for the central corneal thinning in vivo; a clinical feature of macular dystrophy. By using fresh tissue in the X-ray experiments, it was shown that that cryostorage of excised corneal buttons had no effect on the fibril dimensions. A collaboration was set up to analyse serum and corneal tissue immunochemically from the macular dystrophy patients, to characterise the type of macular dystrophy under investigation. There were no specific ultrastructural differences between type I and type II macular dystrophy stromas; an overall structural heterogeneity exists which indicates that the classification system is not, as yet, complete. High-angle X-ray patterns from macular dystrophy corneas contained two “extra reflections” not obtained from other human corneas, normal or pathologic. The reflections arise from 4.61Å and 9.62Å periodic structures which are considered to be glycosaminoglycan in origin. Electron microscopy revealed the presence of “wavy” lamellae and various sized collagen free “lakes” in the stroma of the oedematous hum an cornea, with the posterior portion containing by far the largest “lakes”. The existence of stromal “lakes” was further supported by the equatorial. X-ray diffraction data. Cuprolinic blue-stained transmission electron micrographs demonstrated a D-periodic association of proteoglycans with collagen, which suggested a depletion of keratan sulphate in the oedematous stroma; this was backed-up by immunochemical evidence. Large proteoglycan filaments (possibly chondroitin/dermatan sulphate) were observed in some parts of the extracellular matrix. Scheie’s syndrome corneal stromas, which contain no α-L-iduronidase, also contained dense Cuprolinic blue-stained deposits. The possibility exists that aggregation of corneal chondroitin/dermatan sulphate is a common factor of several corneal pathologies

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

Microwave treatment of the cornea leads to localised disruption of the extracellular matrix

Microwave keratoplasty is a thermo-refractive surgical procedure that can correct myopia (short-sightedness) and pathologic corneal steepening by using microwave energy to cause localised shrinkage around an annulus of the cornea leading to its flattening and vision correction. The effects on the corneal extracellular matrix, however, have not yet been evaluated, thus the current study to assess post-procedure ultrastructural changes in an in-vivo rabbit model. To achieve this a series of small-angle x-ray scattering (SAXS) experiments were carried out across whole transects of treated and untreated rabbit corneas at 0.25 mm intervals, which indicated no significant change in collagen intra-fibrillar parameters (i.e. collagen fibril diameter or axial D-period), whereas inter-fibrillar measures (i.e. fibril spacing and the degree of spatial order) were markedly altered in microwave-treated regions of the cornea. These structural matrix alterations in microwave-treated corneas have predicted implications for corneal biomechanical strength and tissue transparency, and, we contend, potentially render microwave-treated corneas resistant to surgical stabilization using corneal cross-linking procedures currently employed to combat refractive error caused by corneal steepening

PAX6 isoforms, along with reprogramming factors, differentially regulate the induction of cornea-specific genes

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4 and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype

The biomechanics of amnion rupture: an X-ray diffraction study

Pre-term birth is the leading cause of perinatal and neonatal mortality, 40% of which are attributed to the pre-term premature rupture of amnion. Rupture of amnion is thought to be associated with a corresponding decrease in the extracellular collagen content and/or increase in collagenase activity. However, there is very little information concerning the detailed organisation of fibrillar collagen in amnion and how this might influence rupture. Here we identify a loss of lattice like arrangement in collagen organisation from areas near to the rupture site, and present a 9% increase in fibril spacing and a 50% decrease in fibrillar organisation using quantitative measurements gained by transmission electron microscopy and the novel application of synchrotron X-ray diffraction. These data provide an accurate insight into the biomechanical process of amnion rupture and highlight X-ray diffraction as a new and powerful tool in our understanding of this process

Sustained activation of the unfolded protein response induces cell death in Fuchs' endothelial corneal dystrophy

Purpose: The unfolded protein response (UPR) is believed to play a role in the pathogenesis of Fuchs' endothelial corneal dystrophy (FECD). The purpose of this study was to investigate whether unfolded proteins accumulate in the corneal endothelium in FECD and if they are involved in triggering cell death. Methods: Descemet's membranes with corneal endothelial cells (CECs) were obtained during keratoplasty, and expression of aggresomes, type 1 collagen, fibronectin, and agrin was evaluated. Endoplasmic reticulum (ER) stress of immortalized human CECs from non-FECD subjects and from FECD patients (iHCEC and iFECD, respectively) were evaluated. The effect of MG132-mediated aggresome formation on the UPR and intrinsic pathway and the effect of mitochondrial damage on UPR were also examined. The effect of CHOP knockdown on the ER stress–mediated intrinsic pathway was also evaluated. Results: Aggresome formation was higher in iFECD than in iHCEC and was colocalized with type 1 collagen, fibronectin, and agrin. GRP78, phosphorylated IRE1, PERK, and CHOP showed higher activation in iFECD than in iHCEC. MG132-mediated aggresome formation upregulated ER stress sensors, the mitochondrial membrane potential drop, cytochrome c release to the cytoplasm, and activation of caspase-9 and -3. By contrast, staurosporine-mediated mitochondrial damage did not induce ER stress. Knockdown of CHOP attenuated the ER stress-induced cleavage of caspase-9, which is caused by intrinsic pathway activation. Conclusions: Excessive synthesis of extracellular matrix proteins induced unfolded protein accumulation in FECD. Prolonged ER stress–mediated cell death, occurring via the intrinsic apoptotic signaling pathway, therefore might be associated with the pathogenesis of FECD

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

An X-ray diffraction investigation of corneal structure in lumican-deficient mice

PURPOSE. The corneas of mice homozygous for a null mutation in lumican, a keratan sulfate–containing proteoglycan, are not as clear as normal. In the present study, mutant corneas were examined by synchrotron x-ray diffraction to see what structural changes might lie behind the loss of transparency. METHODS. X-ray diffraction patterns were obtained from the corneas of 6-month-old and 2-month-old lumican-null and wild-type mice. Measured in each cornea were the average collagen fibril diameter, average collagen fibril spacing, and the level of order in the collagen array. RESULTS. The x-ray reflection arising from regularly packed collagen was well-defined on all x-ray patterns from 6-month-old wild-type corneas. Patterns from 6-month-old lumican-deficient corneas, however, contained interfibrillar reflections that were measurably more diffuse, a fact that points to a widespread alteration in the way the collagen fibrils are configured. The same distinction between mutant and wild-type corneas was also noted at 2-months of age. Average collagen fibril spacing was marginally higher in corneas of 6-month-old lumican-null mice than in corneas of normal animals. Unlike x-ray patterns from wild-type corneas, patterns from lumican-deficient corneas of both ages registered no measurable subsidiary x-ray reflection, evidence of a wider than normal range of fibril diameters. CONCLUSIONS. The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice is in disarray. There is also a considerable variation in the diameter of the hydrated collagen fibrils. These abnormalities, seen at 2 months as well as 6 months of age, probably contribute to the reduced transparency

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