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Kimberly A Wong, Michael Trembley, Ichiro Hiratani, David M Gilbert, Andrea Sophia Viczian; Generating cone photoreceptor-like cells by inhibiting BMP and Activin/TGFβ signaling in early primitive ectoderm-like cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1131.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptor cells derived from mouse embryonic stem (mES) cells are an attractive source for studying photoreceptor-replacement therapies; however, current studies have been unsuccessful in generating a sufficient number of cone photoreceptors, due to a lack of understanding about how cones are specified. In Xenopus, we have found that inhibition of bone morphogenetic protein (BMP) and Activin/TGFβ signaling pathways in primitive ectoderm is sufficient to bias cells to retinal progenitor cells. We hypothesize that a similar treatment with BMP and Activin/TGFβ inhibitors will direct mES-derived primitive ectoderm to become retinal progenitors, including cone photoreceptors.
Mouse D3 ES cells were treated with conditioned media in suspension and transformed into primitive ectoderm-like (EPL) spheroids. Cultures were then supplemented with Noggin, or the chemical inhibitors dorsomorphin and SB431542, to inhibit BMP and Activin/TGFβ signaling, respectively. After differentiation, we assayed for the presence of various neural and retinal cell types by immunofluorescence and qRT-PCR. We also conducted this assay with transgenic Crx-GFP ES cells, which allowed for visual identification and sorting of GFP+ photoreceptor precursors.
If first converted to primitive ectoderm, treatment with high concentrations of BMP and activin/TGFβ inhibitors resulted in a dose-dependent reduction of pluripotency markers and increase in neural and retinal progenitor markers. Interestingly, we found that 99% of the inhibitor-treated aggregates expressed markers for photoreceptors (crx), and 93% of aggregates expressed cone-specific opsins (M-opsin, S-opsin). Within these spheres, 62 ±12% of cells were crx+ photoreceptor progenitors, and that 23.8 ± 0.7% of cells expressed S-opsin after 9 days of culture.
These findings suggested that first restricting mES cells to a primitive ectoderm lineage creates an environment where BMP and activin/TGFβ inhibition can induce significant cone photoreceptor generation in vitro. These results allow a better understanding of the signaling responsible for directing cells towards photoreceptor lineages. We are currently developing a method to differentiate cone and rod progenitors in our cultures, in order to identify cone-specific signaling factors.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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