The Culture And Identification Of Retinal Photoreceptor Precursors From Newborn Mouse Eye

Photoreceptor loss causes irreversible blindness in many retinal diseases. Transplant stem cells and integrate them into retina,induce them to differentiate into new photoreceptors to repair such damage is one of the most feasible means,and it is investigative hotspot at present.In recent years,many researchers had obtained the delightful result and achievement on retina cell transplant,according to the various basic research on the embryo stem cell(ESC)and the adult stem cell including the mesenchyma stem cell(MSC),the nerve stem cell(NSC)and the retina stem cell/progenitor cell(RPC).So far,all style of derived stem cells transplanted into adult retina have shown little evidence of being able to integrate into the outer nuclear layer and differentiate into new photoreceptor,form functional synaptic connections with other neuron in the recipient retina or restore visual function.This might be because the mature mammalian retina lacks the ability to accept and incorporate stem cells or to promote photoreceptor differentiation.In the process of retinal development, retinal progenitor continuously proliferates,and differentiates into all types of mature retinal cells according to appropriate space-time gradation.The retinal progenitor cells at later ontogenetic stages are named retinal photoreceptor precursors,if they are regarded as donor cells,there might have higher probability of success upon transplantation.Objective:To confirm that there exist photoreceptor precursors in newborn mice's retina,which have characteristic of neuronal stem cells,and can offer investigative basic for transplanting retinal photoreceptor precursors according to isolating and culturing the retinal photoreceptor precursors of newborn mice in vitro, identifying their character.Methods:The retinal photoreceptor precursors were obtained from the retina of newborn mice in littermates(postnatal day 1-7),and cultured in vitro.Their growth status was observed under phase-contrast microscope.After expansion,the specific antibodies(BrdU,Nestin,Nrl and Opsin)were used to indentify their character by cell immunohistochemistry and immunofluorescence,to confirm that there exist photoreceptor precursors in newborn mice's retina,which were immature precursors at post-mitotic period,and about to differentiate into mature photoreceptor.Results:The retinal cells from newborn mice can grow and proliferate by attached condition.After the first generation,mark them by antibodies of BrdU, Nestin,Nrl and Opsin,the result of cell immunohistochemistry and immunofluorescence indicates:(1)The most cells expressed cell division marker BrdU,which demonstrated that the growing cells were composed of proliferating cells mostly.(2)The growing cells could express specific neural stem cell marker Nestin,which demonstrated that the growing cells had characteristic of neuronal stem cells.(3)The growing cells could express Nrl(the specific transcription factor of post-mitotic photoreceptor,which expresses in mature rod photoreceptor continuously),and the cells from postnatal day 3-4 newborn mice expressed Nrl greatly.(4)The most growing cells didn't expressed Opsin(expressed by mature retinal photoreceptor),which indicated the cells at this period were immature retinal photoreceptor precursors.Conclusion:(1)The peak of retinal photoreceptor genesis of mouse is at the early postnatal period,and is greatest in postnatal day 3-4 newborn mice.(2)The retinal cells at this period are immature photoreceptor precursors at later ontogenetic stages,and about to differentiate into mature photoreceptor.(3)These retinal photoreceptor precursors have capability of proliferation and potential of differentiation,at this period,they begin to express the transcript ion factor Nrl, which can promote them differentiate into mature photoreceptors.