Purchase this article with an account.
Panah Liravi, Naresh Polisetti, Naoki Okumura, Noriko Koizumi, Shigeru Kinoshita, Friedrich E Kruse, Ursula Schlotzer-Schrehardt; Laminin-511 and -521-based matrices for efficient ex vivo-expansion of human limbal epithelial progenitor cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1380.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Optimization and standardization of culture conditions for human limbal epithelial stem/progenitor cells (LEPC) considering cell-matrix interactions are required to enhance LEPC ex vivo-expansion and transplantation efficiency. Here, we investigated the efficacy of laminin (LN) isoforms specifically expressed in the limbal niche as culture matrices for epithelial tissue engineering approaches.
Expression patterns of specific LN chains in the limbal niche were analyzed on the protein and mRNA level using laser capture microdissected (LCM) LEPC clusters, cryosections of donor eyes, and cultured LEPC. The effect of human recombinant LN isoforms and short LN-511-E8 fragments on LEPC adhesion, migration, proliferation, and differentiation was evaluated. Neutralizing antibodies were used to determine involvement of α3β1 and α6β1 integrins in cell adhesion. Limbal epithelial cell sheets cultured on fibrin gels prepared with or without LN-511-E8 were analyzed by light and electron microscopy and immunohistochemistry.
Expression levels of LN-α2, -α4, -α5, -β2, -β3 and -γ2 chains were significantly increased in LCM-dissected LEPC clusters compared to basal corneal epithelial cells, whereas other LN chains were not differentially expressed. Confocal microscopy showed increased expression of LN-α2, -α5, -β1, -β2, -γ1, -γ2 and -γ3 in the limbal compared to the corneal basement membrane. Comparative analysis of cultured LEPC and their associated stromal niche cells revealed that LN-α5 was primarily expressed in LEPC, whereas LN-α2 was highly expressed in niche cells. The LN-α5 containing isoforms LN-521, LN-511, and LN-511-E8 significantly enhanced in vitro LEPC adhesion, migration, proliferation, and preservation of an undifferentiated phenotype. Functional blocking of α3β1 and α6β1 integrins suppressed adhesion of LEPC to LN-511/521-coated surfaces. Cultivation of LEPC on fibrin gels containing LN-511-E8 resulted in stratified epithelial constructs expressing progenitor cell markers in their basal layers.
LN-α5 containing isoforms LN-511 and LN-521 promote LEPC adhesion, migration, and proliferation and enable efficient ex vivo-expansion and maintenance of LEPC during cultivation. LN-511-E8 fragments display a similar efficacy to full-length laminin and thus represent a defined xeno-free substrate for epithelial tissue engineering and clinical application.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
This PDF is available to Subscribers Only