Title: Porcine iPSCs (piPSCs) reproducibly and efficiently generate retinal organoids (ROs) containing an abundance of photoreceptors using a modified human PSC-RO differentiation protocol.
Legend: (A) Schematic showing the timing of key steps in the piPSC-RO differentiation protocol used for this study compared to that of an established hPSC-RO differentiation protocol (Capowski et al., 2019). (B) Representative light microscopic live images of (from left to right) piPSC colonies grown on a MEF feeder layer, d2 embryoid bodies (EBs) in suspension, d12 adherent RO colonies prior to dissection, and free-floating d40 and d120 ROs in 3D culture after dissection. (C) Plot showing the number of piPSC-ROs obtained from each of 30 consecutive differentiations (mean = 217 ± 19 ROs/differentiation). (D) Low magnification, confocal images of whole piPSC-RO sections at d40, d80, and d120 of differentiation showing the location of RCVRN+ photoreceptor cells. The arrowhead indicates RCVRN+ photoreceptors primarily localized within a deeper layer at d40, with progressive formation of a dense outer nuclear layer (arrow) by d120. (E) Percentage of RCVRN+ photoreceptors in fixed and immunostained cells of piPSC-ROs at d40, d80, and d120 as determined via flow cytometry. ****p <0.0001, *** p= 0.0004; ANOVA post hoc Tukey’s multiple comparisons test. Scale bars shown.
Citation: Kimberly L. Edwards, Bethany M. Moore, Tyler-Serie Ganser, Praveen Joseph Susaimanickam, Kai Sovell, Yolana Martin, Lindsey D. Jager, Ashley M. Willes, Tyra H. Moyer, Lydia Bowar, M. Joseph Phillips, Ron Stewart, Li-Fang Chu, David M. Gamm. Robust generation of photoreceptor-dominant retinal organoids from porcine induced pluripotent stem cells. Stem cell reports (In press).
Abstract: Outer retinal degenerative diseases (RDDs) and injuries leading to photoreceptor (PR) loss are prevailing causes of blindness worldwide. While significant progress has been made in the manufacture of human pluripotent stem cell (hPSC)-derived PRs, robust production of PSC-PRs from swine, a popular preclinical large animal model, would provide an avenue to collect conspecific functional and safety data to complement human xenograft studies. Toward this goal, we describe the highly efficient generation of PR-dominant porcine induced PSC (piPSC)-derived retinal organoids (ROs) using modifications of our established hPSC-RO differentiation protocol. Porcine iPSC-ROs were characterized using immunocytochemistry (ICC) and single cell RNA-sequencing (scRNA-seq), which revealed the presence and maturation of major neural retina cell types, including PRs and retinal ganglion cells, that possess molecular signatures akin to those found in hPSC-ROs. In late piPSC-ROs, a highly organized outer neuroepithelium was observed with rods and cones possessing outer segments and axon terminals expressing pre-synaptic markers adjacent to dendritic terminals of bipolar cells. The existence of piPSC lines and protocols that support reproducible, scalable production of female and male ROs will facilitate transplant studies in porcine models of retinal injury and RDD unconfounded by immunological and evolutionary incompatibilities inherent to human xenografts.
Slide Author: Kimberly L. Edwards
Investigator: David Gamm, MD, PhD
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About the Lab: David Gamm’s laboratory at the Waisman Center uses stem cell technology to investigate the cellular and molecular events that occur during human retinal differentiation and to generate cells for use in human retinal disease modeling and cell-based rescue or replacement strategies. To meet these goals, Gamm utilizes a variety of human cell types, including ES and iPS cells, which have the capacity to mimic retinal development and disease, as well as to delineate the genetic “checkpoints” necessary to produce particular retinal cell types. By understanding the behavior of these cell types in vitro and in vivo, Gamm hopes to optimize strategies to delay or reverse the effects of blinding disorders such as retinitis pigmentosa and age–related macular degeneration.