Biomedical subjects

J Mario Wolosin

Publications and source records attributed to J Mario Wolosin.

6 recordsLinked to original sources

PubMed2006

Cell markers and the side population phenotype in ocular surface epithelial stem cell characterization and isolation.

The ocular surface is covered by tworapidly renewing and embryologically-related linings, the corneal and conjunctival epithelia. The long-term survival of thesetissues is ultimately dependent on their respective resident stem cells. In the corneal epithelium, the stem cells and their early precursors are exclusively circumscribed to the narrow vasscularize limbal rim that provides epithelial precursor cells to the critically transparent central cornea. Limbal damage causes an interruption of this essential cell supply and allows the invasion of the corneal surface by the conjunctival epithelium, an event that ultimately leads to corneal scarring. The limited supply of immunocompatible tissue is a major hindrance to efforts to develop effective procedures for ocular surface reconstruction. This review describes some of the current work and strategies being developed to achieve the isolation of the limbal stem cell and define its genetic, biochemical, and functional make-up. The study of isolated ocular surface stem cells will foster basic understanding of the environmentalrequisites for their survival and proliferation in a self-replicative mode, leading eventually to advances in therapeutic approaches.

ATP-Binding Cassette Transporters↗

PubMed2005

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells.

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2005

P63 expression levels in side population and low light scattering ocular surface epithelial cells.

PURPOSE: Because stem cells exhibit high self-renewal capacity, slow cycling, and high proliferative potential, and one of many markers postulated for epithelial stem cells, p63, is challenged by widespread expression within stem cell-free regions, we examined p63 expression in these stem cell-associated cohorts compared with their controls. METHODS: Rabbit limbocorneal cryosections, cytospun cell-sorted (by fluorescence-activated cell sorter) side population (SP) and low side scatter (LSSC) cells, and limbal epithelial cells over feeders were stained for p63 by indirect immunofluorescence. Clones were fixed and stained daily for 7 days. Image analysis measured p63 intensity, plotting it against colony size. RESULTS: All basal limbal cells were positive for p63, yet only 5% to 7% expressed high p63 intensities, 40% intermediate, and the majority low. Side population cells were less than 1% of total cells. The average intensity of SP staining was three times that of controls. Subpopulations displaying stemlike features exhibited highest p63 expression. Replication rates of isolated cells differed. Day 5 colonies contained 256 (16 hours/cycle) to two (96 hours/cycle) cells. Whereas all cells were positive for p63, intensity in slow-cycling cells was three to four times that in rapidly proliferating congeners. Increased cell doublings did not decrease fluorescence. CONCLUSIONS: Results suggest that p63 concentration is maximal in stem cells and decreases with differentiation. High p63 levels seem to correlate with cells of the SP and LSSC phenotypes, indicating high cell stemness. With identification of stem cells, further studies can elucidate their use in supporting ocular surface health.

Animals↗

PubMed2004

Ocular surface epithelial and stem cell development.

Phenotypic features and developmental events involved in the genesis of the limbo-corneal and conjunctival epithelia are described. Together, these two epithelia define the ocular surface. They derive from a small cohort of optic vesicle-induced PAX6+ head ectodermal cells that remain on the surface following lens vesicle formation by the main PAX6+ cell cohort. Both epithelia are stratified, and display wet, non-keratinizing phenotypes. The most significant spatial feature of the limbo-corneal epithelium is the segregation of its supporting stem and early precursor cells to the limbus, the outer vascularized rim separating the cornea from the conjunctiva. These stem cells express ABCG2, a xenobiotic transporter present in stem cells from other organs. ABCG2 transport activity excludes the DNA dye Hoechst 33342, allowing the isolation of the ocular stem cells by flow cytometry, as a unique cohort known as a side 'side population'. Limbal stem cells do not form gap junctions and exist as metabolically isolated entities. Tracking of expression changes in Cx43, the main gap junction protein expressed in both the pre-epithelial ectoderm and in the mature central corneal epithelium, indicates that a limbal stem cell phenotype starts developing very soon after lens vesicle invagination, in advance of the appearance of any recognizable anatomical sub-epithelial limbal feature. Differences in Cx43 expression also reveal the very early nature of the divergence in limbo-corneal and conjunctival lineages. The putative involvement of several early genes, including gradients of PAX6 and differences in expression patterns for members of the Id or msh gene expression regulators are reviewed.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed2003

Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry.

PURPOSE: In the epidermis, the highest clonogenicity, a feature of stem cells (SCs), is found in the smallest keratinocyte. In the limbal-corneal (LC) epithelium the SCs are exclusively localized in the basal epithelial layer of the limbal domain. The current study was conducted to determine whether this spatial SC arrangement is reflected in differences in the cell size between limbal and corneal cells. METHODS: In vivo confocal microscopy was used to scan and measure the size of the cells of the central cornea and the superior limbus in five normal subjects, from the superficial to the basal cell layer. Limbal and corneal pure epithelial sheets were isolated by dispase digestion from human tissues and dissociated into single cells by trypsin digestion. The forward (FSC; a relative measure of cell size) and side (SSC; a relative measure of cytoplasmic complexity) light-scattering properties of these cells were determined by flow cytometry. RESULTS: Confocal microscopy showed that diameters of the basal cells of the limbal and corneal zones were 10.1 +/- 0.8 and 17.1 +/- 0.8 micro m, respectively. The corresponding values for the superficial layers were 19.9 +/- 1.6 and 36.6 +/- 1.6 microm, respectively (P < 0.0001). The mean FSC and SSC of the limbal cells amounted to 65.7% +/- 8.7% and of the corneal cells, 74.4% +/- 4.6%. Furthermore, only 1.40% +/- 0.83% and 0.69% +/- 0.37% of the corneal cells had FSC and SSC equal to the lowest 15% of FSC and SCC of the limbal cells, respectively, indicating that the limbus contained a substantial proportion of very low FSC and SSC cells for which there was no corneal counterpart. CONCLUSIONS: The data collectively demonstrate that the smallest cells are located in the limbal basal epithelium. This feature may help isolate corneal SCs located in the limbus.

Adult↗

PubMed2002

Changes in connexin43 in early ocular surface development.

PURPOSE: In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43+) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. METHODS: Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. RESULTS: Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. CONCLUSIONS: Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.

Animals↗