Nancy C. Joyce

Transplantation of adult human corneal endothelium ex vivo: a morphologic study.

Purpose. To investigate the feasibility of transplanting untransformed human corneal endothelial cells as a treatment strategy and possible alternative for penetrating keratoplasty by growing donor cells in culture and then transplanting them to denuded Descemets membrane of recipient corneas. Methods. Corneas from adult donors (50–80 years old) were obtained from eye banks. To grow corneal endothelial cells, Descemets membrane with associated cells was dissected from the stroma. Endothelial cells were released by ethylenediaminetetraacetic acid treatment, grown in medium containing multiple growth factors, and identified as being of endothelial origin by morphology and by reverse-transcription polymerase chain reaction for keratin 12 and collagen type VIII. In transplantation experiments, cultured cells were seeded onto denuded Descemets membrane of a second donor cornea at 5 × 10 5 cells/mL. The recipient cornea was incubated in organ culture for as long as 2 weeks. The morphology and ultrastructure of the endothelium were evaluated 7 and 14 days after transplantation by transmission electron microscopy, and by immunolocalization of zonula occludins-1 (ZO-1). Endothelial cell density was calculated in transplants by counting ZO-1–stained cells. Results. Corneal endothelial cells cultured from adult donors consistently grew well in culture medium. Cells were identified as corneal endothelium by characteristic morphology and messenger RNA expression. Morphologic and ultrastructural studies of corneas containing transplanted endothelial cells demonstrated that with time there was an increase in endothelial cell–Descemets membrane adhesion, in the extent of cell-cell contacts and lateral interdigitation, and in formation of a single cell layer. ZO-1 staining revealed tight junction formation similar to that of corneas in vivo. Mean endothelial cell density in transplanted corneas was 1,895 cells/mm 2 (range, 1,503–2,159 cells/mm 2 ). Conclusion. Untransformed adult human corneal endothelial cells can be efficiently and consistently cultured and transplanted onto denuded Descemets membrane. Transplanted cells in organ culture exhibit morphologic characteristics and cell densities similar to corneal endothelial cells in vivo. These results provide evidence for the feasibility of developing methods for in vivo transplantation of untransformed corneal endothelial cells cultured from adult donor tissue.

Human corneal endothelial cell proliferation: potential for use in regenerative medicine.

Purpose To review and update the experience of our laboratory in culturing human corneal endothelial cells (HCEC) from young and older donors. Methods Corneas were obtained from National Disease Research Interchange, Philadelphia, PA. Data from the past 3 years were reviewed to develop criteria for selecting donor corneas to be used for endothelial cell culture. Immunocytochemical localization using mAb 9.3.E identified endothelial cells, and Ki67 staining demonstrated actively cycling cells. Cell counts demonstrated the effect of growth-promoting agents on proliferation of cells from young (<30 years old) and older (>50 years old) donors. Phase-contrast microscopy documented morphologic characteristics of cells in primary culture and the effect of growth factors on cell morphology. Results Exclusion criteria were developed to increase the chance of successful culture of HCEC. Isolation methods to remove Descemet membrane with attached endothelial cells avoided contamination with other corneal cell types. EDTA treatment combined with mechanical disruption facilitated isolation of cells. Culture medium containing FBS, EGF, NGF, and bovine pituitary extract stimulated maximal growth and facilitated normal monolayer formation. Age-related differences were detected in the density of confluent cells in primary culture and in the proliferative response to growth-promoting agents. Conclusions Untransformed HCEC can be successfully cultured from the corneas of both young and older donors by using care in the selection of donor material. Care must also be taken in the early phases of endothelial cell isolation to obtain maximal numbers of healthy cells for culture. There appear to be true age-related differences in overall proliferative capacity; however, the relative response to specific growth factors was similar in cells from young and older donors. Results of these studies provide guidelines for successful growth of untransformed HCEC for use in regenerative medicine.

Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye

Collagen VIII is localized in subendothelial and subepithelial extracellular matrices. It is a major component of Descemets membrane, a thick basement membrane under the corneal endothelium, where it forms a hexagonal lattice structure; a similar structure, albeit less extensive, may be formed in other basement membranes. We have examined the function of collagen VIII in mice by targeted inactivation of the genes encoding the two polypeptide subunits, Col8a1 and Col8a2. Analysis of these mice reveals no major structural defects in most organs, but demonstrates that type VIII collagen is required for normal anterior eye development, particularly the formation of a corneal stroma with the appropriate number of fibroblastic cell layers and Descemets membrane of appropriate thickness. Complete lack of type VIII collagen leads to dysgenesis of the anterior segment of the eye: a globoid, keratoglobus‐like protrusion of the anterior chamber with a thin corneal stroma. Descemets membrane is markedly thinned. The corneal endothelial cells are enlarged and reduced in number, and show a decreased ability to proliferate in response to different growth factors in vitro. An important function of collagen VIII may therefore be to generate a peri‐ or subcellular matrix environment that permits or stimulates cell proliferation. Hopfer, U., Fukai, N., Hopfer, H., Wolf, G., Joyce, N., Li, E., Olsen, B. R. Targeted disruption of Col8a1 and Col8a2 genes in mice leads to anterior segment abnormalities in the eye. FASEB J. 19, 1232–1244 (2005)

EGF and PGE2: Effects on corneal endothelial cell migration and monolayer spreading during wound repair in vitro

In vivo repair of the adult human corneal endothelium occurs mainly by movement of cells into the wound defect rather than by cell division. Two forms of cell movement contribute to endothelial wound repair: migration of individual cells into the defect and spreading of the confluent monolayer into the wound area. This laboratory has developed a tissue culture model using rabbit corneal endothelial cells pretreated with the mitotic inhibitor 5-fluorouracil to mimic the relatively amitotic state of human corneal endothelium in vivo. This model permits study of the effects of growth factors and other agents on individual cell migration and monolayer spreading in response to wounding. mRNA for epidermal growth factor (EGF) and its receptor has been detected in cultured corneal endothelial cells and EGF receptors have been detected on human corneal endothelial cells in situ, suggesting that this growth factor may act in an autocrine manner. Prostaglandin E2 (PGE2) is synthesized by cultured corneal endothelial cells and is present in relatively high quantity in aqueous humor in response to corneal wounding and to inflammation in the anterior chamber. Although corneal endothelial cells may be exposed to both EGF and PGE2, little is known about their effects on monolayer repair. The current study compared the effects of PGE2 alone, EGF alone, and both agents in combination on individual cell migration and monolayer spreading using the wound model system and also determined the effect of EGF on PGE2 secretion using a commercial immunoassay. A 15 min exposure of wounded cultures to exogenous PGE2 stimulated individual cell migration and suppressed monolayer spreading.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related gene response of human corneal endothelium to oxidative stress and DNA damage.

PURPOSE Nuclear oxidative DNA damage increases with age in human corneal endothelial cells (HCECs) and contributes to their decreased proliferative capacity. These studies investigated whether HCECs respond to this damage by upregulating their expression of oxidative stress and DNA damage-signaling genes in an age-dependent manner. METHODS HCECs were dissected from the corneas of young (30 years and younger) and older (50 years and older) donors. Total RNA was isolated and reverse-transcribed. Oxidative stress and DNA damage-signaling gene expression were analyzed using commercial PCR-based microarrays. Western blot analyses were conducted on selected proteins to verify the microarray results. Nuclear DNA damage foci were detected in the endothelium of ex vivo corneas by immunostaining for H2AX-Ser139. RESULTS Four of 84 genes showed a statistically significant age-related difference in the expression of oxidative stress-related genes; however, Western blot analysis demonstrated an age-related increase in only 2 (cytoglobin and GPX-1) of 11 proteins tested. No age-related differences were detected in the expression of DNA damage-signaling genes. Western blot analysis of seven DNA damage-related proteins verified this finding. Intense nuclear staining of DNA damage foci was observed in nuclei within the central endothelium of older donors. Central endothelium from young donors consistently showed a low level of positive staining. CONCLUSIONS HCECs respond to age-related increases in oxidative nuclear DNA damage by forming DNA damage repair foci; however, they do not vigorously defend against or repair this damage by upregulating the expression of multiple oxidative stress or DNA damage-signaling genes.

The Culture of Limbal Stromal Cells and Corneal Endothelial Cells

The cornea is the transparent front part of the eye and comprises three distinct cell layers. One of these cell layers is a self-renewing epithelium long believed to harbor a resident stem cell population. The location and characteristics of corneal epithelial stem cells have now been confirmed by several research groups, and these cells are currently applied therapeutically. The corneal stroma and endothelium are largely quiescent after infancy, and until recently they were not considered to undergo self-renewal or to maintain stem cells. This view was overturned during the last two decades. At present, cell populations with characteristics of adult stem cells are routinely isolated and characterized from the limbal stroma and the corneal -endothelium. This chapter describes methods for isolation and culture of limbal stromal cells and corneal endothelial cells.

Cell Cycle Control and Replication in Corneal Endothelium

Excessive loss of endothelial cells causes loss of the barrier function of the corneal endothelium, resulting in bullous keratopathy, permanent corneal clouding, and loss of visual acuity. In vivo repair of the endothelium following cell loss occurs by cell enlargement and migration, rather than by cell division. Human corneal endothelial cells (HCEC) do not divide in vivo, because they are inhibited in G1-phase of the cell cycle; however, they retain proliferative capacity. The cell cycle is divided into multiple phases. After mitogenic stimulation, cells enter G1-phase to prepare cells for DNA duplication, which occurs in S-phase. Cells then move into G2-phase to prepare cells for division, which occurs in M-phase. Movement of cells from G1- to S-phase can be prevented by the activity of the cyclin-dependent kinase inhibitors, p27Kip1, p21Cip1, and p16INK4a. These inhibitors prevent activation of the transcription factor, E2F, which is required for S-phase entry. Several factors contribute to inhibition of the proliferation of HCEC in vivo, including formation of strong cell-cell contacts (contact inhibition), lack of autocrine or paracrine growth factor stimulation, and the suppressive effect of transforming growth factor-beta2. This inhibition appears to be mediated, in large part, by p27Kip1. Although HCEC retain the ability to divide, their capacity to proliferate decreases with increasing age. This decrease is characterized by an age-related reduction in the rate of cell cycle entry and in the relative number of dividing cells. Evidence strongly suggests that this age-related decrease is the result of an up-regulation of the expression and activity of p21Cip1 and p16INK4a, but not of p27Kip1. HCEC can be induced to divide by overcoming or bypassing G1-phase inhibition using molecular biological approaches. The most promising approach so far is ectopic expression of the transcription factor, E2F2, which increases endothelial cell proliferation in ex vivo corneas from both young ( 50 years old). Proliferative capacity and the expression of senescence characteristics are also affected by endothelial topography. Cells within the central 6.0 mm diameter of the endothelium in corneas from older donors exhibit the lowest proliferative capacity and contain the highest percentage of senescent cells. The age- and topographically related decrease in proliferative capacity observed in HCEC is not due to the presence of critically short telomeres, but appears to result from sub-lethal oxidative nuclear DNA damage. Research has led to a new hypothesis regarding the molecular basis for the age- and topographically related decrease in proliferative capacity. This hypothesis states that, with increasing age, oxidative stress increases in HCEC due to their high metabolic activity and due to chronic light exposure. This results in a gradual increase in oxidative nuclear DNA damage, which leads to a decreased ability to divide, mediated by the G1-phase inhibitors, p21Cip1, and p16INK4a. This new hypothesis provides the basis for further exploration of the molecular mechanisms under-lying the age- and topographically related decrease in proliferative capacity of HCEC. This exploration could lead to the development of methods to prevent or reverse the effects of oxidative stress on these cells, thereby increasing their ability to divide in order to repair the endothelial monolayer and prevent the devastating effect on vision of the loss of endothelial barrier function.