Abcg2-Dependent Dye Exclusion Activity And Clonal Potential In Epithelial Cells Continuously Growing For 1 Month From Limbal Explants

Autor: J. Mario Wolosin, Mohaned Ahmed, Ozlem Barut Selver, Alexander Barash
Rok vydání: 2011
Předmět:
Adult
ATP Binding Cassette Transporter
Subfamily B
Time Factors
animal structures
Somatic cell
Population
Cell Separation
Biology
In Vitro Techniques
Limbus Corneae
3T3 cells
Colony-Forming Units Assay
Mice
Side population
medicine
Cadaver
ATP Binding Cassette Transporter
Subfamily G
Member 2
Animals
Humans
Progenitor cell
education
skin and connective tissue diseases
Aged
Cell Proliferation
Fluorescent Dyes
education.field_of_study
Staining and Labeling
Polyethylene Terephthalates
Regeneration (biology)
Biological Transport
Epithelial Cells
Membranes
Artificial
Articles
3T3 Cells
Carbocyanines
Middle Aged
eye diseases
Cell biology
Neoplasm Proteins
medicine.anatomical_structure
Immunology
embryonic structures
ATP-Binding Cassette Transporters
Benzimidazoles
Rabbits
sense organs
Stem cell
Explant culture
Popis: The limbal zone contains the majority of the stem cells (SCs) for the limbal–corneal epithelial lineage.1 Consequently, the limbus plays a critical role in the survival of this vision-essential lineage. Epithelium and/or limbal zone loss, due to disease, trauma, or congenital deficiencies, results in visual decrease or vision loss,2,3 and reintroduction of epithelial cells derived from a contralateral eye pre-expanded in vitro can reestablish a fully functional lineage,4–8 provided the engrafted cell population incorporates cells retaining the SC/progenitor phenotype.9 One indicator of the stem/progenitor cell phenotype is the expression of the multidrug resistance protein ABCG2/BCRP1, a xenobiotic transporter expressed at high levels within subpopulations of adult SCs of multiple lineages.10,11 ABCG2 is a very efficient efflux transporter of the supravital DNA binding dye Hoechst 33342; thus, cells with the highest levels of functional ABCG2 exclude the dye. Due to concentration-dependent bathochromic features of ABCG2, the dye exclusion results in a characteristic dual-wavelength emission flow cytometry pattern known as a side population (SP). This feature has allowed the isolation of viable stem and progenitor cells by flow cytometry from multiple lineages and organs, including cells from the limbal and conjunctival epithelia.12–20 We demonstrated that the SP cells identify with cells that before isolation have been in the slow cycling state that is unique to tissue SCs.17,21 SP cells obtained from fresh adult tissue show a very low propensity to start proliferating in clonal conditions. This failure to proliferate may reflect the immediate transition of these quiescent cells from the nurturing in vivo environment to an in vitro environment lacking the conditions for proper activation of proliferation.16,17,19,21 One common approach to generate therapeutic epithelial cell populations for ocular surface reconstruction is based on the explant method, where cells outgrow into a suitable biological or synthetic substratum from a small tissue segment.22–26 Retention of SC- associated features in these explants has undergone extensive characterization. In outgrowth over an amniotic membrane, ABCG2 is well expressed in the proximity of the outgrowth and decreases in intensity toward the margin of the culture, whereas keratin 3, a marker of differentiation, displays the opposite distribution.26 These characterizations notwithstanding, many questions about the origin of the outgrowth population and the explant's biological dynamics have not been fully addressed. In particular, given the slow cycling/quiescent features of somatic adult tissue SCs they need to become activated to contribute to the outgrowth population. It is not clear what the contribution of bona fide SCs to the outgrowth is, vis-a-vis the contribution of other proliferative limbal epithelial cells. In particular, the unique niche localization of SCs also raises questions about the permanence or survival of the SC phenotype within the explant proper, which is usually not included with the cell populations used for corneal regeneration. To start addressing these questions we have now investigated the ability of limbal explants to generate outgrowths over prolonged time periods, and the relative density of cells exhibiting a high level of ABCG2 transport. In these studies we have replaced Hoechst 33342, a DNA binding dye that displays substantial toxicity, with JC1, a new ABCG2 substratum that binds to mitochondria instead of nuclei and shows minimal toxicity. The results show that (1) limbal explants could continuously give rise to outgrowths for at least 1 month; (2) JC1low cells are as much as 10- to 15-fold more frequent in these outgrowths than in the cell population isolated from fresh tissue (FT); (3) the percentage of JC1low cells is higher in the second outgrowth round than that in the first one; and (4) unlike the cells isolated from FT, the JC1low cells isolated from the outgrowths display a colony formation efficiency (CFE) that is 14-fold higher than that of the JC1main cells and incorporate the majority of the colony-forming cells within the outgrowths. The potential implications of these findings for the application of limbal explant methodology to ocular surface reconstruction are discussed.
Databáze: OpenAIRE
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