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

作　　者：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 

机构地区：[1]Tissue Engineering and Stem Cell Group,Singapore Eye Research Institute,20 College Road,The Academia,Discovery Tower Level 6,Singapore 169856,Singapore [2]Singapore National Eye Centre,Singapore,Singapore [3]Department of Ophthalmology,RWTH Aachen University,Aachen,Germany [4]Eye-Academic Clinical Programme,Duke-NUS Graduate Medical School,Singapore,Singapore [5]Proteomics Platform,Singapore Eye Research Institute,Singapore,Singapore [6]Federal University of São Paulo,Sao Paulo,Brazil [7]Singapore Eye Bank,Singapore,Singapore [8]Department of Obstetrics and Gynaecology,Singapore General Hospital,Singapore,Singapore

出　　处：《Eye and Vision》2019年第1期247-260,共14页眼视光学杂志（英文）

基　　金：Singapore National Eye Centre HREF 0618-2;Singapore National Research Foundation under Clinician Scientist Award-Senior Investigator Category(JRNMRR163801);National Medical Research Council,Ministry of Health,Singapore.

摘　　要：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 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.

关 键 词：Amnion extract PROTEOMICS Corneal stromal keratocytes Marker expression 

分 类 号：R73[医药卫生—肿瘤]