A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

Beau J. Fenner, Nur Zahirah B.M. Yusoff, Matthias Fuest, Lei Zhou, Francisco Bandeira, Howard Y. Cajucom-Uy, H. K. Tan, Jodhbir S. Mehta, Gary H.F. Yam

Research output: Journal article publicationJournal articleAcademic researchpeer-review

10 Citations (Scopus)

Abstract

Background: 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. Methods: Three 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. Results: AME 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. Conclusion: AME 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.

Original languageEnglish
Article number30
JournalEye and Vision
Volume6
Issue number1
DOIs
Publication statusPublished - Dec 2019
Externally publishedYes

Keywords

  • Amnion extract
  • Corneal stromal keratocytes
  • Marker expression
  • Proteomics

ASJC Scopus subject areas

  • Ophthalmology
  • Health Professions (miscellaneous)

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