Reliability of human retina organoid generation from hiPSC-derived neuroepithelial cysts

Abstract

The possible applications for human retinal organoids (HROs) derived from human induced pluripotent stem cells (hiPSC) rely on the robustness and transferability of the methodology for their generation. Standardized strategies and parameters to effectively assess, compare, and optimize organoid protocols are starting to be established, but are not yet complete. To advance this, we explored the efficiency and reliability of a differentiation method, called CYST protocol, that facilitates retina generation by forming neuroepithelial cysts from hiPSC clusters. Here, we tested seven different hiPSC lines which reproducibly generated HROs. Histological and ultrastructural analyses indicate that HRO differentiation and maturation are regulated. The different hiPSC lines appeared to be a larger source of variance than experimental rounds. Although previous reports have shown that HROs in several other protocols contain a rather low number of cones, HROs from the CYST protocol are consistently richer in cones and with a comparable ratio of cones, rods, and Müller glia. To provide further insight into HRO cell composition, we studied single cell RNA sequencing data and applied CaSTLe, a transfer learning approach. Additionally, we devised a potential strategy to systematically evaluate different organoid protocols side-by-side through parallel differentiation from the same hiPSC batches: In an explorative study, the CYST protocol was compared to a conceptually different protocol based on the formation of cell aggregates from single hiPSCs. Comparing four hiPSC lines showed that both protocols reproduced key characteristics of retinal epithelial structure and cell composition, but the CYST protocol provided a higher HRO yield. So far, our data suggest that CYST-derived HROs remained stable up to at least day 200, while single hiPSC-derived HROs showed spontaneous pathologic changes by day 200. Overall, our data provide insights into the efficiency, reproducibility, and stability of the CYST protocol for generating HROs, which will be useful for further optimizing organoid systems, as well as for basic and translational research applications.

Automated quantification of photoreceptor outer segments in developing and degenerating retinas on microscopy images across scales

“…Mice were sacrificed at different postnatal ages (P8-P30, Figures 1A,E), and eyes were enucleated and dissected as described in 2.2.1. For TEM analyses, eyes were prefixed in 4% formaldehyde (prepared from paraformaldehyde prills, Electron Microscopy Sciences [EMS] # 19208) in 100 mM phosphate buffer (PB), washed in PBS, further dissected to small (0.5-1 mm) blocks, and postfixed with modified Karnovsky's fixative (2% PFA/2% glutaraldehyde (EMS #16220) in 50 mM HEPES (pH 7.4, EMS #16782) or in 100 mM PB) at 4°C overnight (Kurth et al, 2010;Gasparini et al, 2022;Völkner et al, 2023). After aldehyde fixation, the tissue pieces were washed and further postfixed in 2% aqueous OsO 4 solution containing 1.5% potassium ferrocyanide and 2 mM CaCl 2 , followed by several washes in water, incubation in 1% thiocarbohydrazide, washes in water, and a second contrasting step in 2% osmium/water (OTO procedure, Hanker et al, 1966;Deerinck et al, 2010).…”

“…POS formation can be analyzed ex vivo using extracted retinas as flat mounts and sections (Hollyfield and Rayborn, 1979;Steinberg et al, 1980;Obata and Usukura, 1992;Guerin et al, 1993;Mayhew and Astle, 1997;Burgoyne et al, 2015;Ding et al, 2015;Hsu et al, 2015;Daum et al, 2017;Rakshit et al, 2017;Tu et al, 2019;Corral-Serrano et al, 2020). More recently, the generation of retinal organoids from mouse and human pluripotent stem cells containing high numbers of photoreceptors adds another source to assess early POS formation, though full photoreceptor and thus POS maturation have not been observed yet (Zhong et al, 2014;Gonzalez-Cordero et al, 2017;West et al, 2022;Carido et al, 2023;Völkner et al, 2023). Additionally, POS degeneration was studied in various disease models, particularly using inherited retinal degeneration mouse models (Busskamp et al, 2014;Datta et al, 2015;Veleri et al, 2015;Conley et al, 2018;Baehr et al, 2019;Collin et al, 2020;Corral-Serrano et al, 2020).…”

“…Important insights in outer segment structure and formation have been achieved using qualitative microscopic observations and manual quantification of POS (Carter-Dawson and Lavail, 1979;Hollyfield and Rayborn, 1979;Guerin et al, 1993;Mayhew and Astle, 1997;Rakshit et al, 2017). This includes representative images of immunohistochemical staining of photopigments (e.g., rhodopsin and S-or M/L-opsin), proteins involved in the signal transduction cascade (e.g., recoverin, transducin, arrestin, phosphodiesterase), or structural markers of POS (e.g., peripherin, ROM1; Assawachananont et al, 2014;Daum et al, 2017;Shindou et al, 2017;Tu et al, 2019;Gasparini et al, 2022;West et al, 2022;Yamasaki et al, 2022;Völkner et al, 2023). Electron microscopy or tomography images were used to assess POS structure, volume, disc assembly, and size (Carter-Dawson and Lavail, 1979;Obata and Usukura, 1992;Eberle et al, 2012;Gilliam et al, 2012;Burgoyne et al, 2015;Ding et al, 2015;Goldberg et al, 10.3389/fnmol.2024.1398447 Frontiers in Molecular Neuroscience 03 frontiersin.org 2016; Daum et al, 2017;Ribeiro et al, 2021;Gasparini et al, 2022).…”