Effects of ciliary neurotrophic factor on differentiation of late retinal progenitor cells.
Ciliary neurotrophic factor (CNTF) has been shown to be a potent regulator of retinal cell differentiation. The present study was undertaken to investigate the effects of CNTF on in vitro differentiation of expanded late retinal progenitor cells. Retinal progenitor cells used in these studies were isolated from the neural retina of postnatal day-1 green fluorescent protein (GFP) transgenic mice. The resulting GFP-positive neurospheres were dissociated into a single-cell suspension and grown on poly-D-lysine/laminin-coated tissue culture flasks or slides to generate adherent retinal progenitor cells. These adherent cells were treated with 20 ng/ml of CNTF for up to 14 days, and expression of specific retinal cell markers was determined by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and immunoblot analysis. In vitro studies showed that CNTF treatment of late retinal progenitor cells resulted in changes in cellular morphology. Immunocytochemical studies showed an increase in the proportion of cells expressing markers of bipolar cells but not rod differentiation. In addition, an increase in the proportion of cells expressing glial cell markers was observed. RT-PCR analysis showed downregulation in Hes1, Nestin, Notch1, and Pax6 transcripts along with a concomitant increase in protein kinase C (PKC) alpha and glial fibrillary acidic protein (GFAP) transcripts. These findings were confirmed by immunoblot analysis, where downregulation in Nestin expression and simultaneous upregulation in PKC alpha and GFAP were observed. The data indicate that CNTF treatment of multipotential late retinal progenitors increases the proportion of cells that express markers of bipolar neurons and glia.
Zahir
Tasneem
T
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA.
Klassen
Henry
H
Young
Michael J
MJ
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology*; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D039902:Multipotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D009420:Nervous System / Q000166:cytology; D064231:Nestin; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051603:Recoverin; D012160:Retina / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D015415:Biomarkers; D002135:Calcium-Binding Proteins; D020934:Ciliary Neurotrophic Factor; D005136:Eye Proteins; D005904:Glial Fibrillary Acidic Protein; D007381:Intermediate Filament Proteins; D019394:Ki-67 Antigen; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; C577485:Nes protein, mouse; D064231:Nestin; C496732:Rcvrn protein, mouse; D051603:Recoverin; D049452:Green Fluorescent Proteins; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha