Novel enzymatic isolation of an entire viable human limbal epithelial sheet

Edgar M. Espana, Andre C. Romano, Tetsuya Kawakita, Mario Di Pascuale, Robert Smiddy, Scheffer C G Tseng

Research output: Contribution to journalArticle

69 Citations (Scopus)

Abstract

OBJECTIVE. To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS. Human pigmented limbus was incubated at 4°C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS. Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% ± 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS. 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.

Original languageEnglish
Pages (from-to)4275-4281
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number10
DOIs
Publication statusPublished - 2003 Oct 1
Externally publishedYes

Fingerprint

Collagen
Keratin-3
Negative Staining
Staining and Labeling
Basement Membrane
Epithelium
Western Blotting
Epithelial Cells
Connexin 43
Sorbitol
Cell Extracts
Plastics
Cell Survival
Stem Cells
kalinin

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Espana, E. M., Romano, A. C., Kawakita, T., Di Pascuale, M., Smiddy, R., & Tseng, S. C. G. (2003). Novel enzymatic isolation of an entire viable human limbal epithelial sheet. Investigative Ophthalmology and Visual Science, 44(10), 4275-4281. https://doi.org/10.1167/iovs.03-0089

Novel enzymatic isolation of an entire viable human limbal epithelial sheet. / Espana, Edgar M.; Romano, Andre C.; Kawakita, Tetsuya; Di Pascuale, Mario; Smiddy, Robert; Tseng, Scheffer C G.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 10, 01.10.2003, p. 4275-4281.

Research output: Contribution to journalArticle

Espana, EM, Romano, AC, Kawakita, T, Di Pascuale, M, Smiddy, R & Tseng, SCG 2003, 'Novel enzymatic isolation of an entire viable human limbal epithelial sheet', Investigative Ophthalmology and Visual Science, vol. 44, no. 10, pp. 4275-4281. https://doi.org/10.1167/iovs.03-0089
Espana, Edgar M. ; Romano, Andre C. ; Kawakita, Tetsuya ; Di Pascuale, Mario ; Smiddy, Robert ; Tseng, Scheffer C G. / Novel enzymatic isolation of an entire viable human limbal epithelial sheet. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 10. pp. 4275-4281.
@article{dd07ea2aad6f4aa9a6ca337004c80eca,
title = "Novel enzymatic isolation of an entire viable human limbal epithelial sheet",
abstract = "OBJECTIVE. To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS. Human pigmented limbus was incubated at 4°C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS. Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7{\%} ± 9.1{\%}. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS. 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.",
author = "Espana, {Edgar M.} and Romano, {Andre C.} and Tetsuya Kawakita and {Di Pascuale}, Mario and Robert Smiddy and Tseng, {Scheffer C G}",
year = "2003",
month = "10",
day = "1",
doi = "10.1167/iovs.03-0089",
language = "English",
volume = "44",
pages = "4275--4281",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "10",

}

TY - JOUR

T1 - Novel enzymatic isolation of an entire viable human limbal epithelial sheet

AU - Espana, Edgar M.

AU - Romano, Andre C.

AU - Kawakita, Tetsuya

AU - Di Pascuale, Mario

AU - Smiddy, Robert

AU - Tseng, Scheffer C G

PY - 2003/10/1

Y1 - 2003/10/1

N2 - OBJECTIVE. To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS. Human pigmented limbus was incubated at 4°C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS. Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% ± 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS. 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.

AB - OBJECTIVE. To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS. Human pigmented limbus was incubated at 4°C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS. Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% ± 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS. 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.

UR - http://www.scopus.com/inward/record.url?scp=0141653008&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0141653008&partnerID=8YFLogxK

U2 - 10.1167/iovs.03-0089

DO - 10.1167/iovs.03-0089

M3 - Article

C2 - 14507871

AN - SCOPUS:0141653008

VL - 44

SP - 4275

EP - 4281

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 10

ER -