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

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 Fand CAME , while 286 proteins were reduced (less than 0.5-fold) in CAME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between FAME and CAME 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 FAME or CAME showed similar morphology and viability, while cell proliferation was mildly suppressed with CAME (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.

Comprehensive quantitative characterization of the human term amnion proteome

“…With our approach, we identified 117 core ECM proteins and 168 matrisome-associated proteins ( Table S5 ). This is a significantly higher number than previous similar reports in which 47 core ECM proteins and 63 matrisome-associated proteins were identified [36] . We hypothesize that the higher number of ECM proteins can likely be attributed to the applied extraction method, that is not only extracting soluble proteins, and to the used instrument.…”

“…A study focusing on epithelium-denuded amniotic membrane [40] identified 43 highly abundant proteins by Matrix Assisted Laser Desorption/Ionization (MALDI-TOF). More recent work [36] compared the soluble proteins of fresh and cryopreserved human amniotic membranes by isobaric tagging for relative quantification (iTRAQ) and identified 1949 soluble proteins that were unaltered by cryopreservation.…”

“…The protein composition of the human amnion has been characterized using methods such as western blot and protein microarrays [35] , [36] . Although highly specific, these methods are limited by their low-medium throughput and the low yield of identifications.…”

“…Furthermore, these methods make quantification of protein abundance difficult. First proteomic studies on the amnion were based on 2-dimensional gel electrophoresis followed by peptide mass fingerprint (PMF) analysis [38] , [39] , [40] , or used isobaric tagging for relative quantification (iTRAQ) and focused solely on the soluble fraction of the amnion proteome [36] . In the past decade, mass spectrometry-based proteomics emerged as the method of choice to obtain an unbiased and untargeted proteome of various tissues [41] and was the method employed when drafting the human proteome in 2014 [42] .…”

Cell-Free Biological Approach for Corneal Stromal Wound Healing

“…One option is the soluble amnion membrane extract (AME). In vitro stromal keratocyte culture studies have shown that the human amnion matrix and its soluble protein extract reduced TGFβ1 and β2-related signaling (Tseng et al, 1999;Espana et al, 2003;Kawakita et al, 2005;Yam et al, 2015;Fenner et al, 2019). Cultured keratocytes had a minimal transition to fibroblastic phenotypes during early passages, even in the presence of serum.…”