Salim Ismail

Spectrum of bacterial colonization associated with urothelial cells from patients with chronic lower urinary tract symptoms.

ABSTRACT Chronic lower urinary tract symptoms (LUTS), such as urgency and incontinence, are common, especially among the elderly, but their etiology is often obscure. Recent studies of acute urinary tract infections implicated invasion by Escherichia coli into the cytoplasm of urothelial cells, with persistence of long-term bacterial reservoirs, but the role of infection in chronic LUTS is unknown. We conducted a large prospective study with eligible patients with LUTS and controls over a 3-year period, comparing routine urine cultures of planktonic bacteria with cultures of shed urothelial cells concentrated in centrifuged urinary sediments. This comparison revealed large numbers of bacteria undetected by routine cultures. Next, we typed the bacterial species cultured from patient and control sediments under both aerobic and anaerobic conditions, and we found that the two groups had complex but significantly distinct profiles of bacteria associated with their shed bladder epithelial cells. Strikingly, E. coli, the organism most responsible for acute urinary tract infections, was not the only or even the main offending pathogen in this more-chronic condition. Antibiotic protection assays with shed patient cells and in vitro infection studies using patient-derived strains in cell culture suggested that LUTS-associated bacteria are within or extremely closely associated with shed epithelial cells, which explains how routine cultures might fail to detect them. These data have strong implications for the need to rethink our common diagnoses and treatments of chronic urinary tract symptoms.

Limbal stem cells: Central concepts of corneal epithelial homeostasis.

A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent studies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface.

Sphere‐forming cells from peripheral cornea demonstrate polarity and directed cell migration

Sphere‐forming cells from peripheral cornea represent a potential source of progenitor cells for treatment of corneal degenerative diseases. Control of cellular repopulation on transplantable substrates is important to prevent uncontrolled growth in unfavourable directions. The coordination of cellular outgrowth may be in response to environmental cues and/or cellular signals from other spheres. To investigate this, cell migration patterns were observed following placement of spheres on an adhesive surface. Human peripheral corneal cells were maintained using a sphere‐forming assay and their behaviour on collagen substrate recorded by time‐lapse imaging. Immunocytochemistry and proliferation assays were used to detect protein expression and cell division. Proliferation assays showed that spheres formed by a combination of cell division and aggregation. Cell division continued within spheres for up to 4 months and was up‐regulated when exposed to differentiation medium and collagen substrate. The spheres expressed both epithelial and stromal cell markers. When exposed to collagen; (1) 25% of the spheres showed spontaneous polarised outgrowth. (2) One sphere initially showed polarised outgrowth followed by collective migration with discrete morphological changes to form leading and trailing compartments. (3) A sphere which did not show polarised outgrowth was also capable of collective migration using cell protrusion and retraction. (4) Active recruitment of cells into spheres was observed. (5) Placement of spheres in close proximity led to production of a cell exclusion area adjacent to spheres. Thus peripheral corneal cell spheres are dynamic entities capable of developing polarity and modifying migration in response to their environment.

Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface

BackgroundThe limbus forms the outer rim of the cornea at the corneoscleral junction and harbours a population of stem cells for corneal maintenance. Injuries to the limbus, through disease or accidents such as chemical injuries or burns, may lead to significant visual impairment due to depletion of the native stem cells of the tissue.MethodsSphere-forming cells were isolated from peripheral cornea for potential use as transplantable elements for limbal stem cell repopulation and limbal reconstruction. Immunocytochemistry, live cell imaging and quantitative PCR were used to characterize spheres and elucidate activity post implantation into human cadaveric corneal tissue.ResultsSpheres stained positively for stem cell markers ∆NP63α, ABCG2 and ABCB5 as well as the basal limbal marker and putative niche marker, notch 1. In addition, spheres also stained positively for markers of corneal cells, vimentin, keratin 3, keratocan and laminin, indicating a heterogeneous mix of stromal and epithelial-origin cells. Upon implantation into decellularized corneoscleral tissue, 3D, polarized and radially orientated cell migration with cell proliferation was observed. Cells migrated out from the spheres and repopulated the entire corneal surface over 14 days. Post-implantation analysis revealed qualitative evidence of stem, stromal and epithelial cell markers while quantitative PCR showed a quantitative reduction in keratocan and laminin expression indicative of an enhanced progenitor cell response. Proliferation, quantified by PCNA expression, significantly increased at 4 days subsequently followed by a decrease at day 7 post implantation.ConclusionThese observations suggest great promise for the potential of peripheral corneal spheres as transplantable units for corneal repair, targeting ocular surface regeneration and stem cell repopulation.

Sphere-forming cells from peripheral cornea demonstrate a wound-healing response to injury.

The cornea is the initial refractive interface of the eye. Its transparency is critical for clear vision and is maintained by stem cells which also act to repair injury inflicted by external insults, such as chemical and thermal burns. Damage to the epithelium compromises its clarity and can reduce or eliminate the stem cell population, diminishing the ability for self‐repair. This condition has been termed “limbal stem cell deficiency”; severe cases can lead to corneal blindness. Sphere‐forming cells isolated from peripheral cornea are a potential source of stem and progenitor cells for corneal repair. When provided with appropriate substrate, these spheres have the ability to adhere and for cells to migrate outwards akin to that of their natural environment. Direct compression injury and remote scratch injury experiments were conducted on the sphere cells to gauge their wound healing capacity. Measures of proliferation, differentiation, and migration were assessed by immunohistochemical detection of EdU incorporation, α‐smooth muscle actin expression and confocal image analysis, respectively. Both modes of injury were observed to draw responses from the spheres indicating wound healing processes. Direct wounding induced a rapid, but transient increase in expression of α‐SMA, a marker of corneal myofibroblasts, followed by a proliferative and increasing migratory response. The spheres were observed to respond to remote injury as entire units, with no directional response seen for targeted repair over the scratch injury area. These results give strength to the future use of these peripheral corneal spheres as transplantable units for the regeneration of corneal tissue.

Histopathology (from Keratoconus Pathology to Pathogenesis)

Histopathology has long been used to confirm the diagnosis of keratoconus and to determine the extent of disease progression in tissue post-corneal transplant. We examine here how modern advances in histopathology have informed the pathology of the condition and describe how the technique of immunohistochemistry, in particular, has enabled researchers to describe pathology down to the protein and molecular level. This chapter examines how this information has been used to decipher clues towards understanding the pathogenesis of keratoconus.

Autologous Corneal Repair Using in-vitro Adult Stem Cell Expansion

The human cornea requires a smooth, transparent, robust and renewable surface to maintain its key optical and protective functions. The corneal epithelium is maintained by a local population of “stem cells”, which although not truly pluripotent, are capable of self-regeneration by asymmetric division. These cells are located at the limbus - the region where the conjunctiva-covered sclera meets the cornea. The widely accepted “XYZ” hypothesis of corneal epithelial maintenance postulates that limbal stem cells give rise to transient amplifying cells, which migrate and mature in both a centripetal and anterograde fashion towards the corneal surface. Many diseases and injuries to the limbus can cause the loss of this vital reservoir of cells and, due to subsequent surface irregularity and loss of corneal transparency, result in severe visual impairment, including blindness. Indeed, in some regions of the developing world corneal blindness may be more prevalent than cataract blindness. Such corneal blindness was previously irreversible, but over the past 30 years it has been possible to improve or restore vision using limbal “stem cell” transplants. Pioneering techniques required large tissue grafts that could compromise the donor eye, but recent developments allow the harvesting, ex-vivo expansion and transplantation of limbal stem cells from relatively small biopsies. In this review we outline limbal stem cell physiology in health and disease, describe our surgical approach to limbal stem cell deficiency, illustrate results of this intervention in our practice, and consider current and future advances in this arena.