Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane

Wei Li, Hua He, Ching Liang Kuo, Yingying Gao, Tetsuya Kawakita, Scheffer C G Tseng

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

PURPOSE. To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS. Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin α6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS. All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS. After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.

Original languageEnglish
Pages (from-to)2381-2389
Number of pages9
JournalInvestigative Ophthalmology and Visual Science
Volume47
Issue number6
DOIs
Publication statusPublished - 2006 Jun
Externally publishedYes

Fingerprint

Amnion
Basement Membrane
Epithelial Cells
Matrix Metalloproteinase 2
Matrix Metalloproteinase 9
Matrix Metalloproteinase 1
Tissue Inhibitor of Metalloproteinase-1
Keratin-12
Keratin-3
Collagen Type VII
Phenotype
Matrix Metalloproteinase Inhibitors
Differentiation Antigens
Gelatin
Conditioned Culture Medium
Integrins
Wound Healing
Regeneration
Cell Differentiation
Collagen

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. / Li, Wei; He, Hua; Kuo, Ching Liang; Gao, Yingying; Kawakita, Tetsuya; Tseng, Scheffer C G.

In: Investigative Ophthalmology and Visual Science, Vol. 47, No. 6, 06.2006, p. 2381-2389.

Research output: Contribution to journalArticle

Li, Wei ; He, Hua ; Kuo, Ching Liang ; Gao, Yingying ; Kawakita, Tetsuya ; Tseng, Scheffer C G. / Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. In: Investigative Ophthalmology and Visual Science. 2006 ; Vol. 47, No. 6. pp. 2381-2389.
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abstract = "PURPOSE. To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS. Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin α6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS. All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS. After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.",
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T1 - Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane

AU - Li, Wei

AU - He, Hua

AU - Kuo, Ching Liang

AU - Gao, Yingying

AU - Kawakita, Tetsuya

AU - Tseng, Scheffer C G

PY - 2006/6

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N2 - PURPOSE. To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS. Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin α6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS. All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS. After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.

AB - PURPOSE. To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS. Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin α6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS. All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS. After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.

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