Isolation and characterization of porcine Müller cells. Myofibroblastic dedifferentiation in culture.
OBJECTIVE
To characterize phenotypic and antigenic changes in isolated Müller cells during proliferation in extended culture.


METHODS
Müller cells were isolated from porcine retina by sequential papain and DNase digestion, trituration, and density gradient centrifugation. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and delta smooth muscle actin (alpha SMA). Continuously proliferating cells established in culture were examined for changes in the expression of these antigens.


RESULTS
Primary cultures of purified Müller cells, incubated under routine culture conditions, were proliferative and lost immunodetectable CRALBP within 2 weeks. The expression of CA-II also diminished with time, but at an apparently lower rate than that of CRALBP. Loss of GFAP expression was even more gradual and was complete by passage 5. De novo expression of alpha SMA was detectable in some cells within 12 days in culture and by all cells by passage 5. During this period, vimentin expression remained qualitatively unchanged.


CONCLUSIONS
Isolated porcine Muller cells in culture undergo a phenotypic dedifferentiation to a fibroblast-like cell, which includes loss of detectable CRALBP, CA-II, and GFAP, and they acquire expression of the myoid marker alpha SMA.
Guidry
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Department of Ophthalmology, Eye Foundation Hospital, University of Alabama at Birmingham, 35294, USA.
D000818:Animals; D015153:Blotting, Western; D002256:Carbonic Anhydrases / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002455:Cell Division; D002469:Cell Separation; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D012172:Retinaldehyde / Q000378:metabolism; D013552:Swine
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D005904:Glial Fibrillary Acidic Protein; D002256:Carbonic Anhydrases; D012172:Retinaldehyde