The expression of native and cultured RPE grown on different matrices.
The purpose of this work was to determine the expression profiles of retinal pigment epithelial (RPE) cells grown on different matrices and to assess the degree of culture-induced artifact by comparing the profiles to native RPE. Visually confluent ARPE-19 cells were grown on plastic, Matrigel, collagen I, collagen IV, laminin, and fibronectin for 1 wk, and serum was withdrawn for 3 days. Morphologically normal, macular RPE cells were laser-capture microdissected from three human eye globes. Total RNA was extracted from 5,000 cells and reverse transcribed, and radiolabeled cDNA probes were hybridized to an array containing 4,325 known genes. Arrays were assessed by cluster analysis and significance analysis of microarrays (SAM). Real-time RT-PCR was used to validate differentially expressed genes. Despite similar morphology, ARPE-19 demonstrated different expression profiles when grown on different matrices. Cluster analysis showed that cells grown on collagen IV, laminin, and fibronectin had similar profiles that were distinct from cells grown on collagen I. Cells grown on plastic clustered closest to native RPE. This expression pattern was confirmed with supervised cluster analyses. The number of differentially expressed genes, function of differentially expressed genes, and profile of expressed and unexpressed genes suggest that the overall expression profile of cultured cells is significantly different from native RPE. RPE cells grown on collagen IV, laminin, and fibronectin have profiles more similar than cells grown on plastic, Matrigel, or collagen I. The overall mRNA phenotype, however, is different from morphologically normal, native macular RPE.
Michael Panitch Macular Degeneration Research Laboratory, Wilmer Eye Institute, Johns Hopkins Medical Institutes, Baltimore, Maryland 21287, USA.
Jane
J
Tian
Kazuki
K
Ishibashi
James T
JT
Handa
D000368:Aged; D016477:Artifacts; D016570:Bruch Membrane / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D016000:Cluster Analysis; D016326:Extracellular Matrix Proteins / Q000494:pharmacology*; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D008266:Macula Lutea / Q000166:cytology; D008297:Male; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger