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

Frontiers in Cellular Neuroscience

Neuroscience

Shahryar Khattak¹  Thomas Kurth²  Selen Ulusoy³  Marius Ader³  Madalena Carido³  Anabel Villanueva Norniella³  Felix Wagner³  Maria Scamozzi³  Natalie Dumler³  Mike O. Karl⁴  Manuela Völkner⁴  Sebastian Canzler⁵  Jörg Hackermüller⁶  Lisa Maria Steinheuer⁶  Katja Zoschke⁷  Stephanie Wieneke⁷  Bruno Schönfelder⁷  Cristina Golfieri⁷ 

[1] Center for Molecular and Cellular Bioengineering (CMCB), Stem Cell Engineering Facility, TU Dresden, Dresden, Germany;Center for Molecular and Cellular Bioengineering (CMCB), Technology Platform, Core Facility Electron Microscopy and Histology, TU Dresden, Dresden, Germany;Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany;Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Dresden, Germany;German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany;Department Computational Biology, Helmholtz Centre for Environmental Research—UFZ, Leipzig, Germany;Department Computational Biology, Helmholtz Centre for Environmental Research—UFZ, Leipzig, Germany;Department of Computer Science, Leipzig University, Leipzig, Germany;German Center for Neurodegenerative Diseases (DZNE) Dresden, Dresden, Germany;

关键词: organoid;    human;    retina;    hiPSC;    stem cells;    neurodevelopment;    pathology;    gliosis;   

DOI  :  10.3389/fncel.2023.1166641

 received in 2023-02-15, accepted in 2023-09-18,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Carido, Völkner, Steinheuer, Wagner, Kurth, Dumler, Ulusoy, Wieneke, Norniella, Golfieri, Khattak, Schönfelder, Scamozzi, Zoschke, Canzler, Hackermüller, Ader and Karl.