Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane.
OBJECTIVE
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 alpha6, 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.
Li
Wei
W
Ocular Surface Center and TissueTech, Inc., Miami, Florida 33173, USA.
He
Hua
H
Kuo
Ching-Liang
CL
Gao
Yingying
Y
Kawakita
Tetsuya
T
Tseng
Scheffer C G
SC
D000650:Amnion; D001485:Basement Membrane / Q000502:physiology*; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000166:cytology*; D022002:Hemidesmosomes / Q000378:metabolism; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis*; D013234:Stem Cells / Q000166:cytology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism
D008565:Membrane Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9