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Magdalene J. Seiler, David Ferguson, Gabriel Nistor, Sabhya Rana, Irina Turovets, Joshua Almodovar, Jared Taylor, Nikolay A. Turovets, Hans S. Keirstead; Optimizing Retinal Progenitor Differentiation of hESC - Effect of RPE Co-Culture. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5897.
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© ARVO (1962-2015); The Authors (2016-present)
To optimize the differentiation of human embryonic stem cells (hESC) to retinal and photoreceptor progenitors for obtaining 3D sheets of retinal progenitors with RPE for transplantation.
hESCs were differentiated according to modifications of Nistor et al. (2010), including delayed hyaluronic acid treatment, longer time in suspension culture, and a change in concentration and treatment time with transcription factors Dkk1 and LeftyA. Additional studies focused on variations in exposure time and concentration of FGF-8, Sonic Hedgehog (shh), and Activin A. Cell cultures were analyzed by immunohistochemistry for the transcription factors Chx10, CRX, NRL and various other cell markers.
Expression of photoreceptor progenitor markers could be improved compared to the original differentiation protocol by using a higher concentration of Dkk1/LeftyA in the beginning of the culture, starting hyaluronic acid treatment after 3 weeks, and maintaining the cultures in suspension until d 28-29. FGF8 and shh treatment did not improve photoreceptor progenitor differentiation. Early Avtivin A treatment together with RPE co-culture (d 44-58 of differentiation) dramatically increased expression of the photoreceptor progenitor markers CRX and NRL in more than 90% of the cells. However, no mature photoreceptor markers such as recoverin were found at d 58 of differentiation.
Short treatment with transcription factors LeftyA and Dkk1 at high concentration (40-60 ng/ml) is sufficient to induce retinal differentiation. Early Activin A treatment, longer time in suspension culture, delayed hyaluronic acid and co-culture with RPE improves expression of photoreceptor progenitor markers. The next step is to test these tissues in retinal degenerate nude rats.
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