title: Induction of differentiation by pyruvate and DMEM in the human retinal pigment epithelium cell line ARPE-19.
abstract: OBJECTIVE
  Cultured retinal pigment epithelium (RPE) may become a therapeutic option for transplantation in retinal disease. However maintaining a native RPE phenotype in vitro has proven challenging. The human RPE cell-line ARPE-19 is used widely as an alternative to primary RPE. It is grown in DMEM/F12 medium as standard, but its phenotype is dependent on culture conditions, and many differentiation markers are usually absent. The purpose of this study was to examine how this sensitive phenotype of ARPE-19 can be modulated by growth media with or without the metabolite pyruvate to elucidate better RPE growth conditions.
  
  
  METHODS
  ARPE-19 cells at passages p22 to p28 were cultured on filters for up to 3 months in DMEM/F12 or DMEM media with or without pyruvate and 1% fetal calf serum. Assessment of differentiation was performed using pigmentation, immunocytochemistry, protein/mRNA expression, transepithelial resistance, VEGF secretion, and ultrastructure.
  
  
  RESULTS
  Pyruvate, in combination with DMEM, induced dark pigmentation and promoted differentiation markers such as CRALBP and MerTK. Importantly, RPE65 protein was detected by Western blotting and was enhanced by pyruvate, high glucose, and DMEM. ARPE-19 cells maintained in this medium could also phagocytose human photoreceptor outer segments (POS). VEGF secretion was greater in DMEM cultures and was affected by glucose but not by pyruvate. Pigmentation never occurred in DMEM/F12.
  
  
  CONCLUSIONS
  This study demonstrated important differentiation markers, including pigmentation and Western blots of RPE65 protein, and showed human POS phagocytosis in ARPE-19 cultures using a simple differentiation protocol. The results favor the use of high-glucose DMEM with pyruvate for future RPE differentiation studies.
authors: Department of Ocular Biology and Therapeutics, University College London (UCL), Institute of Ophthalmology, London, United Kingdom. a.ahmado@ucl.ac.uk
authors: Ahmad
authors: A
authors: Ahmado
authors: Amanda-Jayne
authors: AJ
authors: Carr
authors: Anthony A
authors: AA
authors: Vugler
authors: Ma'ayan
authors: M
authors: Semo
authors: Carlos
authors: C
authors: Gias
authors: Jean M
authors: JM
authors: Lawrence
authors: Li Li
authors: LL
authors: Chen
authors: Fred K
authors: FK
authors: Chen
authors: Patric
authors: P
authors: Turowski
authors: Lyndon
authors: L
authors: da Cruz
authors: Peter J
authors: PJ
authors: Coffey
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005136:Eye Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D010587:Phagocytosis; D010641:Phenotype; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019289:Pyruvic Acid / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D000076202:c-Mer Tyrosine Kinase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
chemical_list: D015415:Biomarkers; D002352:Carrier Proteins; D003470:Culture Media; D005136:Eye Proteins; D011518:Proto-Oncogene Proteins; D019289:Pyruvic Acid; D020794:Receptor Protein-Tyrosine Kinases; D042461:Vascular Endothelial Growth Factor A; D000076202:c-Mer Tyrosine Kinase; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C020932:11-cis-retinal-binding protein; C467484:VEGFA protein, human; C097072:MERTK protein, human