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M. Kayama, M. S. Kurokawa, Y. Ueda, H. Ueno, Y. Kumagai, C. Masuda, E. Takada, S. Ueno, M. Tadokoro, N. Suzuki; Induction of Differentiation Into Retinal Ganglion Cells of Mouse Es Cells by Pax6 Gene Transfection. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4072.
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Glaucoma causes the apoptotic death of retinal ganglion cells (RGC), leading to visual field defects. RGC which originate from forebrain do not have regenerative abilities. RGC replacement treatment may become alternative modality for improving visual acuity. Embryonic stem (ES) cells have the ability to differentiate into multiple cell types and may become the source of RGC. pax6 gene is a homeobox transcription factor acknowledged to have a crucial role in early eye development including generation of RGC. Here, we have attempted to establish cloned RGC from ES cells transfected with pax6 gene.
Undifferentiated mouse ES cells were transfected with pax6 cDNA by using electroporation, followed by selection with G418. We conducted limiting dilution culture of the pax6 transfected cells. We expanded the cloned pax6 transfected cell lines which expressed nestin and musashi1 for further characterization in media including fibronectin. The cells were characterized by using RT-PCR, immunostaining, electron microscopy and Ca imaging.
We obtained three cloned pax6 transfected cell lines with bipolar configuration, all of which were positive for Islet1, Brn3, Thy1, and melanopsin. When transplanted into mouse renal capsule, they differentiated into neurons with bipolar appearance, expressing betaIIItubulin and neurofilament middle chain and they were free from teratoma development. Electron microscopic examination showed neurotubules and neurofilament in the axon like processes of the cloned pax6 transfected cell lines. A high KCl stimulated caused increase of free Ca influx on Ca2+ imaging.
pax6 gene transfection into mouse ES cells leads to emergence of the cloned cell lines with bipolar configuration that express RGC markers. These cells may be applicable for the new therapeutic strategy to improve visual acuity.
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