Purification of mammalian cone photoreceptors by lectin panning and the enhancement of their survival in glia-conditioned medium.
OBJECTIVE
In retinal diseases characterized by photoreceptor degeneration, the main cause of clinically significant vision loss is cone, rather than rod, loss. In the present study, a technique was designed to purify cones to make it possible to screen for neuroprotective molecules.


METHODS
A suspension of porcine retinal cells was incubated on coverslips coated with the peanut agglutinin (PNA) lectin, which selectively binds to cones. Cones were identified and quantified by using an antibody specific for cone arrestin. Their identity and viability were also assessed by single-cell RT-PCR and patch-clamp recording.


RESULTS
This panning method provided a population of cones that was 80% to 92% pure, depending on the counting strategy used. The panned cells contained both short (S)- and medium/long (M/L)-wavelength opsin cones. The panned retinal cells exhibited the physiological signature of cone photoreceptors and single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) showed that they expressed the cone arrestin mRNA. Most (69%) cone photoreceptors produced neurites and survived for up to 7 days when cultured in a glia-conditioned medium, whereas very few (4%) survived after 7 days in the control medium.


CONCLUSIONS
This PNA-lectin-panning method can provide highly pure and viable mammalian cones, the survival of which can be prolonged by glia-conditioned medium. Because PNA lectin binds to cone photoreceptors from various species in both normal and pathologic conditions, this technique should enable the screening of neuroprotective molecules like those released by glial cells and enable the physiological, genomic, and proteomic characterization of cones.
Balse
Elise
E
Institute National de la Santé et de la Recherche Médicale (INSERM) U-592, Université Pierre et Marie Curie, Paris, France.
Tessier
Luc-Henri
LH
Fuchs
Céline
C
Forster
Valérie
V
Sahel
José A
JA
Picaud
Serge
S
D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D019084:Fluorescent Antibody Technique, Indirect; D009457:Neuroglia / Q000502:physiology*; D018408:Patch-Clamp Techniques; D019887:Peanut Agglutinin / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000166:cytology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism; D013552:Swine
D019393:Arrestin; D017077:Culture Media, Conditioned; D019887:Peanut Agglutinin; D012333:RNA, Messenger; D017299:Rod Opsins