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Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2+/ABCB5+ Limbal Epithelial Stem Cell Cultures.

ABSTRACT: Homeostasis and regeneration of corneal epithelia are sustained by limbal epithelial stem cells (LESCs); thus, an LESC deficiency is a major cause of blindness worldwide. Despite the generally promising results of cultivated LESC transplantation, it has been limited by variations in long-term success rates, the use of xenogeneic and undefined culture components, and a scarcity of donor tissues. In this study, we identified the culture conditions required to expand LESCs in vitro and established human limbus-derived highly proliferative ABCG2+/ABCB5+ double-positive LESCs. These LESCs exhibited the LESC marker profile and differentiated into corneal epithelial cells. In addition, cultured LESCs expressed high levels of the stem cell markers Sox2, Oct4, c-Myc, and Klf4, had high telomerase activity, and had stable, normal genomes. These results suggest that our novel cultivation protocol affects the phenotype and differentiation capacity of LESCs. From the limbus, which contains a heterogenous cell population, we have derived highly proliferative ABCG2+/ABCB5+ double-positive cells with the ability to differentiate into corneal epithelial cells. This study opens a new avenue for investigation of the molecular mechanism of LESC maintenance and expansion in vitro and may impact the treatment of corneal disease, particularly corneal blindness due to an LESC deficiency.

SUBMITTER: Kim EK

PROVIDER: S-EPMC5694571 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2+/ABCB5+ Limbal Epithelial Stem Cell Cultures.

Kim Eung Kweon EK Lee Ga-Hyun GH Lee Boram B Maeng Yong-Sun YS

Stem cells international 20171105

Homeostasis and regeneration of corneal epithelia are sustained by limbal epithelial stem cells (LESCs); thus, an LESC deficiency is a major cause of blindness worldwide. Despite the generally promising results of cultivated LESC transplantation, it has been limited by variations in long-term success rates, the use of xenogeneic and undefined culture components, and a scarcity of donor tissues. In this study, we identified the culture conditions required to expand LESCs in vitro and estab ...[more]

PMID: 29230251

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Project description:To use human limbal explants as an alternative source for generating conjunctival epithelium and to determine the effect of interleukin-13 (IL-13) on goblet cell number, mucin expression, and stemness. Human limbal explants prepared from 17 corneoscleral rims were cultured with or without IL-13 (IL-13+ and IL-13-, respectively) and followed up to passage 2 (primary culture [P0]-P2). Cells were characterized by alcian blue/periodic acid-Schiff (AB/PAS) staining (goblet cells); immunofluorescent staining for p63α (progenitor cells), Ki-67 (proliferation), MUC5AC (mucin, goblet cells), and keratin 7 (K7, conjunctival epithelial and goblet cells); and by quantitative real-time polymerase chain reaction for expression of the p63α (TP63), MUC5AC, MUC4 (conjunctival mucins), K3, K12 (corneal epithelial cells), and K7 genes. Clonogenic ability was determined by colony-forming efficiency (CFE) assay. Using limbal explants, we generated epithelium with conjunctival phenotype and high viability in P0, P1, and P2 cultures under IL-13+ and IL-13- conditions, i.e., epithelium with strong K7 positivity, high K7 and MUC4 expression and the presence of goblet cells (AB/PAS and MUC5AC positivity; MUC5AC expression). p63α positivity was similar in IL-13+ and IL-13- cultures and was decreased in P2 cultures; however, there was increased TP63 expression in the presence of IL-13 (especially in the P1 cultures). Similarly, IL-13 increased proliferative activity in P1 cultures and significantly promoted P0 and P1 culture CFE. IL-13 did not increase goblet cell number in the P0-P2 cultures, nor did it influence MUC5AC and MUC4 expression. By harvesting unattached cells on day 1 of P1 we obtained goblet cell rich subpopulation showing AB/PAS, MUC5AC, and K7 positivity, but with no growth potential. In conclusion, limbal explants were successfully used to develop conjunctival epithelium with the presence of putative stem and goblet cells and with the ability to preserve the stemness of P0 and P1 cultures under IL-13 influence.

Project description:Purpose:Although stem cell activity represents a crucial feature in corneal and ocular surface homeostasis, other cells populating this region and the neighboring zones might participate and influence local microenvironment. Mast cells, the long-lived and tissue-sited immune cells, have been previously reported in corneoscleral specimens. Herein, mast cells were investigated in corneoscleral tissues and related to microenvironment protein expression. Methods:Twenty-six (14 male/12 female; older than 60 years) human corneoscleral specimens were sectioned for light and fluorescent immunostaining (CD45, p63, Ck-3/7/12/19, tryptase/AA1, and chymase/CC1). Corneal, limbal, and conjunctival squares were produced for molecular and biochemical analysis. Statistical comparisons were carried out by ANOVA. Results:Toluidine blue staining identified metachromatic intact or degranulated mast cells in the area below the palisades' Vogt (Ck-3/12-positive epithelium and underneath p63 immunoreactivity). Tryptase immunoreactivity was observed close to palisades' Vogt, whereas no specific signal was detected for chymase. Tryptase/AA1 transcripts were quantified in limbal and conjunctival RNA extracts, whereas no specific amplification was detected in corneal ones. Few mediators were overexpressed in limbal extracts with respect to corneal (Neural cell adhesion molecule (NCAM), Intercellular adhesion molecule 3 (ICAM3), Brain-derived Neurotrophic factor (BDNF), and neurotrophin 3 (NT3); P < 0.00083) and conjunctival (NCAM, ICAM3, and NT3; P < 0.05) protein extracts. A trend to an increase was observed for Nerve Growth Factor (NGF) in limbal extracts (P > 0.05). Conclusions:The specific observation of tryptase phenotype and the interesting protein signature of microenvironment (adhesion molecules, growth factors, and neurotrophins), known to partake mast cell behavior, at least in other areas, would provide additional information to better understand this crucial zone in the framework of ocular surface healthiness.

Dataset Information

Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2+/ABCB5+ Limbal Epithelial Stem Cell Cultures.

Publications

Establishment of Novel Limbus-Derived, Highly Proliferative ABCG2<sup>+</sup>/ABCB5<sup>+</sup> Limbal Epithelial Stem Cell Cultures.

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