Identification of cell surface markers glypican-4 and CD200 that differentiate human corneal endothelium from stromal fibroblasts
| Autor: | Heng-Pei Ang, Xin-Yi Seah, Zi Xian Ngoh, Zhenzhi Chng, Alan Colman, Jodhbir S. Mehta, Yuen Kuen Cheong, William Sun, Gary S L Peh |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
medicine.drug_class
Corneal Stroma Cell Culture Techniques Monoclonal antibody Immunofluorescence Polymerase Chain Reaction Glypican 4 Flow cytometry Antigen Glypicans Antigens CD medicine Humans Fibroblast Cells Cultured Cluster of differentiation medicine.diagnostic_test Chemistry Endothelium Corneal Antibodies Monoclonal Cell sorting Fibroblasts Flow Cytometry Molecular biology eye diseases medicine.anatomical_structure sense organs Biomarkers |
| Zdroj: | Investigative ophthalmologyvisual science. 54(7) |
| ISSN: | 1552-5783 |
| Popis: | Purpose 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. |
| Databáze: | OpenAIRE |
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