G M Seigel1, A L Mutchler, E L Imperato. 1. Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue Rochester, NY 14642, USA. GAIL_SEIGEL@urmc.rochester.edu
Abstract
PURPOSE: To determine glial characteristics of the retinal precursor cell line R28, which has previously been shown to express proteins immunoreactive with photoreceptor markers IRBP, S-Ag, recoverin, the ganglion cell marker 2G12, as well as the Muller cell marker RetG1. METHODS: R28, an immortalized retinal precursor cell line derived from P6 rat retinal tissue, was analyzed to determine expression of glial cell markers. R28 cells were analyzed both immunocytochemically and by western immunoblot for GFAP, S-100, and vimentin. These results were compared with the primary postnatal day 6 retina. Double fluorescence immunolabelling was used to identify R28 cells which simultaneously expressed vimentin and the photoreceptor marker IRBP (interphotoreceptor retinoid-binding protein). RESULTS: GFAP, S-100 and vimentin immunoreactive proteins were detected in R28 cells. Western blot analysis showed the GFAP immunoreactive band to migrate at a slightly higher apparent molecular weight for R28 than for P6 retina, and demonstrated a less fibrillary staining pattern than P6 retina, but appeared to be present to some degree in all R28 cells. Variations in molecular weight were seen for S-100, although the nuclear staining pattern was the same for both the R28 cell line and P6 retina. S-100 immunoreactivity was seen in approximately 50% of the R28 cell population. Vimentin was expressed by virtually all R28 cells, and to a greater degree than that seen in P6 retina (both in cell number and intensity). Double labelling studies revealed R28 cells which expressed both vimentin and IRBP simultaneously. CONCLUSIONS: There is a very strong glial component to the R28 retinal precursor cell line, as evidenced by the expression of proteins immunoreactive to GFAP, S-100 and vimentin. However, even the most strongly immunoreactive marker vimentin was compatible with co-expression of the photoreceptor marker IRBP as evidence of the "multi-phenotypic" nature of the precursor-like R28 cells. Ongoing studies will assess the differentiation potential of R28 cells and applicability in future studies of retinal cell differentiation and gene expression.
PURPOSE: To determine glial characteristics of the retinal precursor cell line R28, which has previously been shown to express proteins immunoreactive with photoreceptor markers IRBP, S-Ag, recoverin, the ganglion cell marker 2G12, as well as the Muller cell marker RetG1. METHODS: R28, an immortalized retinal precursor cell line derived from P6 rat retinal tissue, was analyzed to determine expression of glial cell markers. R28 cells were analyzed both immunocytochemically and by western immunoblot for GFAP, S-100, and vimentin. These results were compared with the primary postnatal day 6 retina. Double fluorescence immunolabelling was used to identify R28 cells which simultaneously expressed vimentin and the photoreceptor marker IRBP (interphotoreceptor retinoid-binding protein). RESULTS:GFAP, S-100 and vimentin immunoreactive proteins were detected in R28 cells. Western blot analysis showed the GFAP immunoreactive band to migrate at a slightly higher apparent molecular weight for R28 than for P6 retina, and demonstrated a less fibrillary staining pattern than P6 retina, but appeared to be present to some degree in all R28 cells. Variations in molecular weight were seen for S-100, although the nuclear staining pattern was the same for both the R28 cell line and P6 retina. S-100 immunoreactivity was seen in approximately 50% of the R28 cell population. Vimentin was expressed by virtually all R28 cells, and to a greater degree than that seen in P6 retina (both in cell number and intensity). Double labelling studies revealed R28 cells which expressed both vimentin and IRBP simultaneously. CONCLUSIONS: There is a very strong glial component to the R28 retinal precursor cell line, as evidenced by the expression of proteins immunoreactive to GFAP, S-100 and vimentin. However, even the most strongly immunoreactive marker vimentin was compatible with co-expression of the photoreceptor marker IRBP as evidence of the "multi-phenotypic" nature of the precursor-like R28 cells. Ongoing studies will assess the differentiation potential of R28 cells and applicability in future studies of retinal cell differentiation and gene expression.
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