June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Robust generation of retinal organoids from porcine induced pluripotent stem cells.
Author Affiliations & Notes
  • Kimberly L. Edwards
    Department of Cellular and Molecular Pathology, University of Wisconsin-Madison, Madison, Wisconsin, United States
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Li-Fang Chu
    Department of Comparative Biology and Experimental Medicine, University of Calgary, Calgary, Alberta, Canada
  • Yolana C. Martin
    Waisman Center, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • James A. Thomson
    Morgridge Insitute for Research, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • David M Gamm
    McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Kimberly Edwards None; Li-Fang Chu None; Yolana Martin None; James Thomson None; David Gamm None
  • Footnotes
    Support  W81XWH-20-1-0655, NICHHD U54 HD090256
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3727 – F0333. doi:
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    • Get Citation

      Kimberly L. Edwards, Li-Fang Chu, Yolana C. Martin, James A. Thomson, David M Gamm; Robust generation of retinal organoids from porcine induced pluripotent stem cells.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3727 – F0333.

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

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Abstract

Purpose : Tremendous advances have been made in human pluripotent stem cell (PSC)-derived retinal cell transplantation in mammalian model species, but evolutionary divergence in synaptic proteins may limit what can be learned from xenogeneic transplants. Allografts with species-specific PSC-derived retinal cells from pigs, a popular large animal pre-clinical model, can provide valuable anatomic, functional, and safety data for future cell replacement trials. We optimized the timeline of our established human retinal organoid (RO) differentiation protocol to align with porcine development and generated a retinal differentiation protocol that produces an abundance of porcine induced pluripotent stem cell (piPSC)-derived ROs containing a high percentage of photoreceptors.

Methods : To generate piPSC-ROs, we shortened the timing of our human PSC-RO protocol (Capowski et al., Development 2019) to better align with pig gestation. Embryoid bodies (EBs) were weaned into Neural Induction Media (NIM) over 24 hours for pig cultures vs. 4 days for human cultures, then treated with NIM + BMP4 for retinal specification and plating on Matrigel on d2 for pig vs. d6-7 for human. Pig cultures were switched to Retinal Differentiation Medium (RDM) with daily feeding on d5 (vs. d16 for human) until manual lifting of 3D ROs on d11-13 (vs. d25-30 for human). ROs were maintained with twice-weekly feeding of 3D-RDM. Differentiation of ROs was assessed by brightfield microscopy for morphology and structural organization and immunocytochemistry (ICC) for cell type-specific protein expression and localization.

Results : Light microscopic images demonstrated the phase-bright outer rim characteristic of ROs. ICC of piPSC-ROs revealed highly organized outer laminae with an abundant photoreceptor population evidenced by RECOVERIN+ and CRX+ cells, including both rods (NRL+ and RHODOPSIN+) and cones (RP-1+). As expected, piPSC-ROs contained additional retinal cell types such as CRALBP+ muller glia and G0alpha+ bipolar cells.

Conclusions : We present a robust and reproducible protocol for generating retinal organoids from pig iPSCs in a manner that parallels production of human PSC-derived ROs and photoreceptors. We anticipate that piPSC-ROs will provide a highly useful, conspecific donor cell source for assessing the potential for synaptic connectivity and functional responses to photoreceptor transplantation in pig preclinical models.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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