June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Synchronous BMP and Activin/TGFβ inhibition and FGF activation drives rapid generation of photoreceptor-like cells by in mouse embryonic stem cell cultures
Author Affiliations & Notes
  • Kimberly A Wong
    Neuroscience & Physiology, SUNY Upstate Medical University, Syracuse, New York, United States
    Center for Vision Research, SUNY Upstate Medical University, Syracuse, New York, United States
  • Michael Trembley
    Pharmacology & Physiology, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States
  • Ichiro Hiratani
    RIKEN Center for Developmental Biology, Kobe, Japan
  • David M. Gilbert
    Biological Science, Florida State University, Tallahassee, Florida, United States
  • Michael Zuber
    Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
    Center for Vision Research, SUNY Upstate Medical University, Syracuse, New York, United States
  • Andrea Sophia Viczian
    Ophthalmology, SUNY Upstate Medical University, Syracuse, New York, United States
    Center for Vision Research, SUNY Upstate Medical University, Syracuse, New York, United States
  • Footnotes
    Commercial Relationships   Kimberly Wong, None; Michael Trembley, None; Ichiro Hiratani, None; David Gilbert, None; Michael Zuber, None; Andrea Viczian, None
  • Footnotes
    Support  NEI Grant EY019517, Research to Prevent Blindness Unrestricted Award, and the Lions of District 20-Y1
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4575. doi:
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      Kimberly A Wong, Michael Trembley, Ichiro Hiratani, David M. Gilbert, Michael Zuber, Andrea Sophia Viczian; Synchronous BMP and Activin/TGFβ inhibition and FGF activation drives rapid generation of photoreceptor-like cells by in mouse embryonic stem cell cultures. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4575.

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      © ARVO (1962-2015); The Authors (2016-present)

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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.

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