title: Identification of cell surface markers glypican-4 and CD200 that differentiate human corneal endothelium from stromal fibroblasts.
abstract: OBJECTIVE
  There is a lack of definitive cell surface markers to differentiate cultured human corneal endothelial cells (HCECs) from stromal fibroblasts, which could contaminate HCEC cultures. The aim of our study is to discover cell surface antigens on HCECs that can be used to identify and purify HCECs from stromal fibroblasts.
  
  
  METHODS
  RNA sequencing (RNA-seq) was used to find differentially overexpressed genes in HCECs and commercial antibodies against these overexpressed antigens were screened by immunofluorescence assay. Similarly, 242 commercial antibodies against cell-surface antigens also were screened. Selected antibodies were used to sort HCECs from stromal fibroblasts by fluorescence-activated cell sorting (FACS).
  
  
  RESULTS
  Two monoclonal antibodies, anti-GPC4 and anti-CD200, were identified to stain HCECs specifically. FACS was used successfully to sort HCECs away from stromal fibroblasts. Recovery efficiency of HCECs after sorting using anti-GPC4 antibody was higher compared to anti-CD200 antibody, but purity of HCECs culture using either antibody was comparable.
  
  
  CONCLUSIONS
  Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts.
authors.affiliation: Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR), Singapore.
authors.forename: Yuen Kuen
authors.initials: YK
authors.lastname: Cheong
authors.forename: Zi Xian
authors.initials: ZX
authors.lastname: Ngoh
authors.forename: Gary Swee Lim
authors.initials: GS
authors.lastname: Peh
authors.forename: Heng-Pei
authors.initials: HP
authors.lastname: Ang
authors.forename: Xin-Yi
authors.initials: XY
authors.lastname: Seah
authors.forename: Zhenzhi
authors.initials: Z
authors.lastname: Chng
authors.forename: Alan
authors.initials: A
authors.lastname: Colman
authors.forename: Jodhbir S
authors.initials: JS
authors.lastname: Mehta
authors.forename: William
authors.initials: W
authors.lastname: Sun
mesh_terms: D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000276:immunology / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology* / Q000378:metabolism; D004728:Endothelium, Corneal / Q000276:immunology / Q000378:metabolism*; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D053673:Glypicans / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015415:Biomarkers; D053673:Glypicans; C042739:antigens, CD200
keywords: antibodies; biomarker; corneal endothelial cells; stromal fibroblast