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Y. Hirami, K. Takahashi, K. Okita, S. Yamanaka, M. Takahashi; Generation of Retinal Progenitor Cells From Induced Pluripotent Stem (iPS) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3553.
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© ARVO (1962-2015); The Authors (2016-present)
For retinal degenerative disease such as retinitis pigmentosa, transplantation of healthy retinal photoreceptor cells is needed for restore retinal function. Although embryonic stem (ES) cells have potential as donor cells for transplantation, clinical application of them has drawbacks such as immune rejection. As an alternative source of the donor cells, Takahashi and Yamanaka introduced the induced pluripotent stem (iPS) cells, the ES-like cells that were generated by reprogramming the mouse somatic cells through the retroviral activation of four definite factors (2006). The report showed the possibilities to treat patients by using their own somatic cells. Here we tried to determine whether the procedure applied to differentiate ES cells is also capable of inducing the retinal progenitor cells in mouse iPS cells.
Differentiation of mouse iPS cells was started with floating culture in 100mm low cell binding dish with serum-free medium. Floating cells that formed aggregates were plated on poly-D-lysine/laminin/fibronectin coated slide. The replated cells were then harvested for subsequent analyses of neuronal, retinal or undifferentiated marker expression using RT-PCR, immunocytochemistry and FACS.
Mouse iPS cells cultured with serum-free medium exhibited neuronal markers such as Nestin, GFAP and beta tubulin. Addition of Dkk-1 and Lefty-A to the culture medium effectively induced coexpression of Rx and Pax6, the markers of retinal progenitor cells about 15 days after differentiation was started. RT-PCR analysis of the differentiated iPS cells showed expression of forebrain to midbrain marker genes including Rx, while undifferentiated cell markers, Oct3/4 and Nanog still remained at day 10. Nanog-GFP positive undifferentiated iPS cells remained 3-4% of total cells at differential day 8 but < 0.5% at differential day 15.
We could induce Rx+/Pax6+ retinal progenitor properties from mouse iPS cells. We need to develop the methods for further differentiation into retinal photoreceptors, and to remove undifferentiated cells for prevention of tumor formation.
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