Abstract
BackgroundHuman corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth.MethodsThree placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry.ResultsAME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures.ConclusionAME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.
Highlights
Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model
Proteomic profiles of cryopreserved AME (C-AME) versus fresh AME (F-AME) Soluble AME from 3 donor placentas were prepared with fresh amnion tissue or following cryopreservation at − 80 °C for a week
Proteomic profiling of F-AME and C-AME yielded an average of 2194 identifiable proteins and 1812 quantifiable proteins in AM3 samples; 2739 identifiable proteins and 1482 quantifiable proteins in AM8 samples; and 2907 identifiable proteins and 2889 quantifiable proteins in AM9 samples (Fig. 1b-c)
Summary
Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Due to scarring and opacities inside the corneal stroma, is a significant cause of global blindness [2, 3]. This reduces and distorts light passage, leading to decreased vision or visual loss. The development of robust strategies, like cell-based therapies, is desirable to restore stromal functions and corneal transparency for patients with corneal opacities [7,8,9,10,11]
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