Modeling inducible neuropathologies of the retina with differential phenotypes in organoids

Völkner, Manuela; Wagner, Felix; Kurth, Thomas; Sykes, Alex M.; Runzer, Claudia Del Toro; Ebner, Lynn J. A.; Kavak, Cagri; Alexaki, Vasileia Ismini; Cimalla, Peter; Mehner, Mirko; Koch, Edmund; Karl, Mike O.

doi:10.3389/fncel.2023.1106287

Cited by 2 publications

(1 citation statement)

References 113 publications

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“…However, currently, there are no appropriate techniques established to perform instance segmentation on EM images, especially in the case of highly crowded structures (Aswath et al, 2023). Deep learning approaches such as convolutional neural networks have emerged as the preferred option for automated feature extraction and segmentation in EM data, as reviewed by Aswath et al (2023). However, they state that "instance segmentation of apposing organelles, especially when the organelles contain structures that are similar to their membranes, " is still challenging (Aswath et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

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

Seidemann,

Salomon,

Hoffmann

et al. 2024

Front. Mol. Neurosci.

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The functionality of photoreceptors, rods, and cones is highly dependent on their outer segments (POS), a cellular compartment containing highly organized membranous structures that generate biochemical signals from incident light. While POS formation and degeneration are qualitatively assessed on microscopy images, reliable methodology for quantitative analyses is still limited. Here, we developed methods to quantify POS (QuaPOS) maturation and quality on retinal sections using automated image analyses. POS formation was examined during the development and in adulthood of wild-type mice via light microscopy (LM) and transmission electron microscopy (TEM). To quantify the number, size, shape, and fluorescence intensity of POS, retinal cryosections were immunostained for the cone POS marker S-opsin. Fluorescence images were used to train the robust classifier QuaPOS-LM based on supervised machine learning for automated image segmentation. Characteristic features of segmentation results were extracted to quantify the maturation of cone POS. Subsequently, this quantification method was applied to characterize POS degeneration in “cone photoreceptor function loss 1” mice. TEM images were used to establish the ultrastructural quantification method QuaPOS-TEM for the alignment of POS membranes. Images were analyzed using a custom-written MATLAB code to extract the orientation of membranes from the image gradient and their alignment (coherency). This analysis was used to quantify the POS morphology of wild-type and two inherited retinal degeneration (“retinal degeneration 19” and “rhodopsin knock-out”) mouse lines. Both automated analysis technologies provided robust characterization and quantification of POS based on LM or TEM images. Automated image segmentation by the classifier QuaPOS-LM and analysis of the orientation of membrane stacks by QuaPOS-TEM using fluorescent or TEM images allowed quantitative evaluation of POS formation and quality. The assessments showed an increase in POS number, volume, and membrane coherency during wild-type postnatal development, while a decrease in all three observables was detected in different retinal degeneration mouse models. All the code used for the presented analysis is open source, including example datasets to reproduce the findings. Hence, the QuaPOS quantification methods are useful for in-depth characterization of POS on retinal sections in developmental studies, for disease modeling, or after therapeutic interventions affecting photoreceptors.

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Section: Discussionmentioning

confidence: 99%

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

Seidemann,

Salomon,

Hoffmann

et al. 2024

Front. Mol. Neurosci.

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

Carido,

Völkner,

Steinheuer

et al. 2023

Front. Cell. Neurosci.

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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.

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Modeling inducible neuropathologies of the retina with differential phenotypes in organoids

Cited by 2 publications

References 113 publications

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

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

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

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