Abstract
Purpose :
Photoreceptor cells derived from mouse embryonic stem (mES) cells are an attractive source for studying formation of photoreceptor cells in vitro. While human stem cell cultures take up to 5 months to generate photoreceptors, we have developed a culture system that can form photoreceptors in two weeks. This study was done to determine the optimal concentration of inhibitors to maximize the production of these cells.
Methods :
Mouse D3 or primary transgenic Crx>GFP ES cells were cultured in suspension with hepatocellular carcinoma conditioned media for 5 days and transformed into pluripotent primitive ectoderm-like (EPL) organoids. Cultures were then supplemented with inhibitors, including Noggin, or dorsomorphin and SB431542, to inhibit BMP and Activin/TGFβ signaling. After differentiation, the presence of neural and retinal cell types were observed using immunofluorescence and qRT-PCR.
Results :
Noggin, or dorsomorphin and SB431542, treatment resulted in a dose-dependent induction of markers for retinal progenitor cells, post-mitotic ganglion cells, and cone photoreceptors. After random selection of organoids at day 9, 95% of the inhibitor-treated organoids expressed markers for photoreceptors (Crx) and cone-specific opsins (M-opsin, S-opsin), and 23.8±0.7% of cells within each organoid were cone photoreceptor-like (Crx+, S-opsin+). Similar results were also observed with the crx>GFP ES cell line: after inhibitor treatment, crx>GFP promoter activation was detected as early as day 6 in 99% of organoids. Using a dose-response assay, we have optimized culture conditions to produce an average of 62±12% GFP-positive photoreceptor progenitor cells after simultaneous treatment with dorsomorphin, SB431542, and FGF2. By day 9, crx>GFP expression was sustained and Otx2 and M-opsin expression was observed in 99±1% and 74±2% of organoids, respectively.
Conclusions :
These findings suggested that our culture system creates an environment where simultaneous BMP and Activin/TGFβ inhibition and FGF activation can bias mouse embryonic stem cells towards photoreceptor lineages in vitro, which will be tested for light sensitivity in the future.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.