June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Direct comparison of in vitro retinal organoids with in vivo eye development using Rax-Cre;tdTomato mice and iPSC-derived retinal organoids
Author Affiliations & Notes
  • Mikaela Louie
    Cellular Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Adam Miltner
    Cellular Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Simranjeet K. Cheema
    Cellular Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Anna La Torre
    Cellular Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Nadean L Brown
    Cellular Biology and Human Anatomy, University of California Davis, Davis, California, United States
  • Footnotes
    Commercial Relationships   Mikaela Louie, None; Adam Miltner, None; Simranjeet Cheema, None; Anna La Torre, None; Nadean Brown, None
  • Footnotes
    Support  R01EY026942
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 1699. doi:
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      Mikaela Louie, Adam Miltner, Simranjeet K. Cheema, Anna La Torre, Nadean L Brown; Direct comparison of in vitro retinal organoids with in vivo eye development using Rax-Cre;tdTomato mice and iPSC-derived retinal organoids. Invest. Ophthalmol. Vis. Sci. 2021;62(8):1699.

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

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Abstract

Purpose : Our understanding of the molecular steps of mammalian eye development remains incomplete. This holds especially true for early eye development where in vivo experiments are difficult due to the small size of the tissue. Here, we directly compared in vitro differentiation of stem cells into retinal tissue or, retinal organoids (ROs), to in vivo eye development, using BAC transgenic Rax-Cre and flox-stop tdTomato (Ai9) lineage reporter mice. Embryos of the same genotype were used to make the stem cell lines for RO differentiation. The Rax/Rx transcription factor is one of the earliest genes to demarcate the eye field. The time-course and characteristics of ROs arising from these lines were directly compared to in vivo eye formation.

Methods : Embryos containing both Rax-Cre and Ai9 transgenes were previously reported to recapitulate Rax spatiotemporal expression during early eye formation eye (Bosze et al., 2020). We extended the time course of Ai9 expression during embryogenesis using live imaging and immunofluorescence. The morphologic and molecular progression of ROs, derived from double-transgenic iPSC lines, using established protocols (Sasai et al 2011, La Torre et al. 2016), was analyzed by the same methods, plus RT-qPCR.

Results : Rax-Cre;Ai9 (Rc;Ai9) embryos show Ai9 expression from the embryonic day 7.5 (E7.5). At E9.5 and E11.5, the developing ventral diencephalon -including the optic vesicle- and telencephalon express high levels of Ai9. Rc;Ai9 undifferentiated stem cell colonies are indistinguishable from mouse embryonic stem cells (mESCs). When differentiated into ROs, retinal markers increase in expression while markers of pluripotency are down-regulated, comparable to differentiated mESCs. Ai9 expression began on day 4 and, by day 6, all ROs expressed Ai9. Interestingly, these cells respond to well-known morphogens such as Sonic Hedgehog (Shh). Hence, the addition of Shh changes proliferation rates compared to control ROs.

Conclusions : The Rc;Ai9 mice have been shown a to be a powerful tool for retinal lineage tracing. Similarly, Rc;Ai9 ROs mimic normal developing retinas in gene expression, cell morphology, and Ai9 expression. Importantly, these ROs can be used to study molecular mechanism early development, and also to inform strategies to advance the current protocols to generate ROs.

This is a 2021 ARVO Annual Meeting abstract.

 

Timeline of Rax-cre;tdTomato organoid differentiation

Timeline of Rax-cre;tdTomato organoid differentiation

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