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

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

Investigating Town Design and Social Organization at Port Tobacco, Maryland, Through the Use of Archaeology and Geophysics

This thesis examines the connection between town planning and social organization at the small town site of Port Tobacco in south-central Charles County, Maryland from the beginning of the 18th century through to the end of the 19th century. By employing a methodology of both geophysical techniques and archaeological excavations, I was able to locate and map numerous structures and features associated with town planning and examine how these spaces were used. This data was used to show how social order, power, and wealth transformed the town layout from a linear settlement along the river into a grid-like pattern. Specifically, I was able to show that these changes in town layout were dominated by the power of the local elite landowners and tobacco merchants

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

Chronic Scleroedema

Two patients with chronic scleroedema are presented. One patient had severe joint contractures. The literature on scleroedema is reviewed and the relationship of scleroedema to the stiff skin syndrome is discussed.S. Afr. Med. J., 48, 164, (1974)

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

Bioactive films produced from self-assembling peptide amphiphiles as versatile substrates for tuning cell adhesion and tissue architecture in serum-free conditions

The development of versatile bioactive surfaces able to emulate in vivo conditions is of enormous importance to the future of cell and tissue therapy. Tuning cell behaviour on two-dimensional surfaces so that the cells perform as if they were in a natural three-dimensional tissue represents a significant challenge, but one that must be met if the early promise of cell and tissue therapy is to be fully realised. Due to the inherent complexities involved in the manufacture of biomimetic three-dimensional substrates, the scaling up of engineered tissue-based therapies may be simpler if based upon proven two-dimensional culture systems. In this work, we developed new coating materials composed of the self-assembling peptide amphiphiles (PAs) C16G3RGD (RGD) and C16G3RGDS (RGDS) shown to control cell adhesion and tissue architecture while avoiding the use of serum. When mixed with the C16ETTES diluent PA at 13 : 87 (mol mol-1) ratio at 1.25 times 10-3 M, the bioactive {PAs} were shown to support optimal adhesion, maximal proliferation, and prolonged viability of human corneal stromal fibroblasts ({hCSFs)}, while improving the cell phenotype. These {PAs} also provided stable adhesive coatings on highly-hydrophobic surfaces composed of striated polytetrafluoroethylene ({PTFE)}, significantly enhancing proliferation of aligned cells and increasing the complexity of the produced tissue. The thickness and structure of this highly-organised tissue were similar to those observed in vivo, comprising aligned newly-deposited extracellular matrix. As such, the developed coatings can constitute a versatile biomaterial for applications in cell biology, tissue engineering, and regenerative medicine requiring serum-free conditions