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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)
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.
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.
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.
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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