Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells

Tetsuya Kawakita, Edgar M. Espana, Hua He, Lung Kun Yeh, Chia Yang Liu, Scheffer C G Tseng

Research output: Contribution to journalArticle

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Abstract

PURPOSE. To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca 2+ concentration and serum in modulating their growth and differentiation. METHODS. Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4°C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca2+ concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS. Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca2+ but suppressed by 5% FBS in KSFM at passage 0. CONCLUSIONS. Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca2+ concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca2+ concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

Original languageEnglish
Pages (from-to)3507-3512
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume45
Issue number10
DOIs
Publication statusPublished - 2004 Oct
Externally publishedYes

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Serum-Free Culture Media
Keratinocytes
Epithelial Cells
Calcium
Growth
Connexin 43
Keratins
Digestion
Gentian Violet
Sorbitol
Serum
Cell Size
Immunoblotting
Plastics
Culture Media
Cultured Cells

ASJC Scopus subject areas

  • Ophthalmology

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Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells. / Kawakita, Tetsuya; Espana, Edgar M.; He, Hua; Yeh, Lung Kun; Liu, Chia Yang; Tseng, Scheffer C G.

In: Investigative Ophthalmology and Visual Science, Vol. 45, No. 10, 10.2004, p. 3507-3512.

Research output: Contribution to journalArticle

Kawakita, Tetsuya ; Espana, Edgar M. ; He, Hua ; Yeh, Lung Kun ; Liu, Chia Yang ; Tseng, Scheffer C G. / Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells. In: Investigative Ophthalmology and Visual Science. 2004 ; Vol. 45, No. 10. pp. 3507-3512.
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abstract = "PURPOSE. To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca 2+ concentration and serum in modulating their growth and differentiation. METHODS. Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4°C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca2+ concentration to 0.9 mM, with or without 5{\%} FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS. Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca2+ but suppressed by 5{\%} FBS in KSFM at passage 0. CONCLUSIONS. Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca2+ concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca2+ concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.",
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AU - Kawakita, Tetsuya

AU - Espana, Edgar M.

AU - He, Hua

AU - Yeh, Lung Kun

AU - Liu, Chia Yang

AU - Tseng, Scheffer C G

PY - 2004/10

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N2 - PURPOSE. To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca 2+ concentration and serum in modulating their growth and differentiation. METHODS. Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4°C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca2+ concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS. Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca2+ but suppressed by 5% FBS in KSFM at passage 0. CONCLUSIONS. Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca2+ concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca2+ concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

AB - PURPOSE. To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca 2+ concentration and serum in modulating their growth and differentiation. METHODS. Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4°C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca2+ concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS. Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca2+ but suppressed by 5% FBS in KSFM at passage 0. CONCLUSIONS. Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca2+ concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca2+ concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

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