Proteomic analysis of epithelium-denuded human amniotic membrane as a limbal stem cell niche.

A new strategy of treating ocular surface reconstruction is to transplant a bioengineered graft by expanding limbal stem cells (SCs) ex vivo on the amniotic membrane (AM). The reasons for the exceptional success on the AM are not fully understood but are believed to be related to its unique composition. We investigated the proteome of the epithelium-denuded AM to increase our understanding of the mechanisms by which AM may confer its beneficial effects. We compared the epithelially denuded-human AM with matrigel and collagen on the expansion of limbal SCs by evaluating the expression of specific markers. The protein pattern of the epithelium-denuded AM was analyzed using two dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) identification of proteins. Epithelial outgrowth of limbal explants on AM expressed more p63 and K19 (SC markers) and less K3 and connexin 43 (corneal differentiation markers) in comparison with other extracellular matrices (ECMs). Moreover, in all groups, the cells expressed ABCG2, K19, K12, p63, and Pax6 as shown by reverse transcription polymerase chain reaction (RT-PCR). Out of about 600 protein spots analyzed on six 2-DE gels, 515 spots could be detected in all replicates. A high average correlation coefficiency (CC) of 0.926 implied good intra-sample reproducibility. Forty major proteins of AM were identified using MALDI TOF/TOF MS of which different isoforms of lumican and osteoglycin were responsible for around 23% of the total proteome on gels. Our results showed that epithelium-denuded AM provides a superior niche for limbal SC proliferation and phenotype maintenance in vitro and the denuded human AM is a protein enriched ECM. This will prove critical to the future understanding of the biological and therapeutic mechanisms involved in AM transplantation and regeneration. The identification of highly abundant proteins in denuded-AM, such as lumican, osteoglycin/memican, collagen alpha type IV, and fibrinogen, further explains its unique properties and will assist in the efforts to generate bioengineered and artificial AM constructs.