Novel Enzymatic Isolation of an Entire Viable Human Limbal Epithelial Sheet

Espana, Edgar M.; Romanò, A; Kawakita, Tetsuya; Pascuale, Mario Di; Smiddy, Robert; Tseng, Scheffer C.G.

doi:10.1167/iovs.03-0089

Cited by 81 publications

(77 citation statements)

References 17 publications

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“…4B). This localization pattern for ABCG2-positive cells coincides almost identically with that for corneal epithelial stem cells demonstrated previously by several investigators [1,2,[34][35][36][37]. These findings imply that ABCG2 positive cells in the limbal epithelium are putative corneal epithelial stem cells, consistent with the observation that SP cells are present in the limbal epithelium but not in the corneal epithelium (Fig.…”

Section: Discussionsupporting

confidence: 90%

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2

WATANABE¹

2004

FEBS Letters

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Section: Discussionsupporting

confidence: 90%

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2

WATANABE¹

2004

FEBS Letters

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“…After euthanasia, the eyes were enucleated by forceps, washed profusely in phosphate-buffered saline, and incubated in DMEM containing 20 mM HEPES, 15 mg/ml dispase II (Roche Applied Science), and 100 mM sorbitol at 4°C for 18 h as previously reported (21,22). The entire corneal epithelium loosened by this treatment was subsequently removed by vigorous shaking.…”

Section: Methodsmentioning

confidence: 99%

Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-β Signaling

Kawakita

Espana

et al. 2005

Journal of Biological Chemistry

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Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-␤2 and TGF-␤ receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express ␣-smooth muscle actin, even when 10 ng/ml TGF-␤1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-␤ signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-␤. Collectively, these results indicate that down-regulation of Smad-mediated TGF-␤ signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-␤ signaling, achieved by AM stromal matrix in part via suppression of TGF-␤ gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

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“…To this end, intact limbal epithelial sheets can be successfully isolated by digestion with Dispase from several species [66,75,76]. Single cells rendered by a brief treatment with trypsin and EDTA of such isolated limbal epithelial sheets have been used by many investigators as the primary source of limbal epithelial SCs for subsequent manipulations including fluorescent-activated cell sorting (FACS) and culturing.…”

Section: How Can Limbal Epithelial Scs and Their Niche Cells Be Isolamentioning

confidence: 99%

Niche regulation of corneal epithelial stem cells at the limbus

et al. 2007

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Among all adult somatic stem cells, those of the corneal epithelium are unique in their exclusive location in a defined limbal structure termed Palisades of Vogt. As a result, surgical engraftment of limbal epithelial stem cells with or without ex vivo expansion has long been practiced to restore sights in patients inflicted with limbal stem cell deficiency. Nevertheless, compared to other stem cell examples, relatively little is known about the limbal niche, which is believed to play a pivotal role in regulating self-renewal and fate decision of limbal epithelial stem cells. This review summarizes relevant literature and formulates several key questions to guide future research into better understanding of the pathogenesis of limbal stem cell deficiency and further improvement of the tissue engineering of the corneal epithelium by focusing on the limbal niche.

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Novel Enzymatic Isolation of an Entire Viable Human Limbal Epithelial Sheet

Abstract: An intact limbal epithelial sheet can be consistently and reproducibly isolated and contains stem cell characteristics in the basal epithelium by degrading laminin 5 and part of collagen IV, and disassembling collagen VII.

Cited by 81 publications

References 17 publications

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2

Human limbal epithelium contains side population cells expressing the ATP-binding cassette transporter ABCG2

Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-β Signaling

Niche regulation of corneal epithelial stem cells at the limbus

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