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Y. Hirami, F. Osakada, K. Takahashi, S. Yamanaka, Y. Kurimoto, M. Takahashi; Generation of Retinal Progenitor Cells From Human Induced Pluripotent Stem Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3063.
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Previously, we generated putative photoreceptors and RPE cells from primate ES cells using step-wise induction with defined factors. Here we tested whether human induced pluripotent stem (hiPS) cells can be differentiated into retinal progenitors, RPE and photoreceptors with the same procedure used for ES cell differentiation.
Human iPS cells were dissociated into small clumps of 5-10 cells and seeded in Petri dishes as suspension cultures with serum-free medium containing Dkk-1 and Lefty A. Under these conditions, hiPS cells formed embryoid body-like aggregates. On day 20, aggregates were plated onto glass slides coated with poly-D-lysine, laminin, and fibronectin. For photoreceptor differentiation, hiPS cells were incubated under SFEB/DL conditions for 90 days, and subsequently in the medium containing retinoic acid and taurine for at least 30 days.
Human iPS cells were cultured under the SFEB conditions and on day 35, we observed marked expression of the neural differentiation marker nestin and betaIII-tubulin. We observed hiPS colonies that positive for the retinal progenitor markers Rx, Pax6 and Mitf. On day 40, pigmented cells were evident under the light microscopy in the SFEB/DL cultures. Over time, these cells adopted a polygonal morphology, a characteristic of RPE cells. On day 80, we observed hiPS colonies that positive for Crx, a photoreceptor precursor maker. To determine the potential for hiPS cells to express photoreceptor properties, we treated these cells with SFEB/DL, and subsequently with retinoic acid and taurine, beginning at differentiation day 90. On day 120, we observed hiPS cells co-expressed the photoreceptor marker recoverin and the rod photoreceptor marker rhodopsin.
We induced retinal progenitor cells from hiPS cells by serum-free embryoid body-like culture added with Wnt and Nodal inhibitors. Prolonged culture period generated mature RPE cells and addition of retinoic acid and taurine in culture condition promoted differentiation of photoreceptors. Our result showed that iPS cells differentiate into retinal cells by the same method as used in ES cells and have the possibility as a tool for cell transplantation therapy.
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