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Transcriptome profiling of mouse retina organoids at defined days in culture

ABSTRACT: Methods: Mouse retinal organoids (MRO) profiles at day 20, 22, 25, and 30 in culture under control conditions. MROs were generated using the previously established trisection protocol (Völkner et al. 2016, 2019). Single-end sequencing with 30 Mio reads per sample was performed at a length of 75 bases on HiSeq2500 (Illumina). The sequence reads that passed quality filters were analyzed at the transcripts level. Results: Our histologic studies indicate that MROs complete retinogenesis at day 20, and mature including development of a stratified structure with outer and inner plexiform layers and synapses within day 20 to 30. Further, immunostaining and ultrastructural studies showed that photoreceptors neurons mature, including nascent outer segment formation. However, MRO maturation is still incomplete and MRO start to undergo some pathologic changes starting at day 30 and progressing upon longer term culture. Transcriptome analysis support and extend this based on differential changes in temporal gene expression: For example. regulators of retinal development and proliferation cease over time. Further, genes involved in the formation of photoreceptor outer segments, synaptogenesis, and phototransduction machinery increase. Starting at day 30, gene involved in reactive gliosis and retinal neuroprotection are upregulated indicating pathologic changes at the molecular level. Conclusions: Transcriptome analysis of MROs support and extend our findings at the histological level, indicating that MROs reproducibly undergo several stages in culture, i.e. complete retinogenesis, mature on the cellular and molecular level, and develop some pathologic changes.

ORGANISM(S): Mus musculus

PROVIDER: GSE168139 | GEO | 2021/05/20

REPOSITORIES: GEO

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Project description:Methods: Profiles of individual human retinal organoids (HRO) at 150, 200 and 250 days of development days were treated for 10 days with HBEGF and TNF, and compared to solvent controls (CTRL). N=6 HROs per timepoint and variable. Single-end sequencing with 30 Mio reads per sample was performed at a length of 75 bases on HiSeq2500 (Illumina). The sequence reads that passed quality filters were analyzed at the transcripts level. Results: Our data independently confirms that this protocol provides HROs with macular-like characteristics and shows low baseline variabilities for rods, cones and Müller glia. These features might be key for disease modeling, since most animal models lack a macula, and most other organoid protocols have low cone cell numbers. We found a significant reduction in the number of rod and cone photoreceptor cells after 10 days after HBEGF-TNF (HT) treatment. Specifically, some photoreceptors show pathologic changes, and some are apically displaced, with their nuclei protruding into or passing out of the retina. Histopathologic changes indicate photoreceptor extrusion of viable or dying cones and rods as a pathomechanism. Notably, photoreceptor displacement through the apical retinal border, and thus ectopy in the region of photoreceptor inner and outer segments, has been described in patients and animal models. In controls, photoreceptors were highly ordered, interspersed and interconnected with each other and with Müller glia processes, and these cell connections make up the outer limiting membrane. Upon HT, glial processes expanded in size, extended laterally along the retinal circumference, and replaced or covered photoreceptors over large areas – forming a seal-like glial scar. Taken together, HT induces photoreceptor degeneration via cell extrusion in conjunction with reactive gliosis, glial proliferation, retinal thickening/dyslamination, and glial seal-like scar formation, which reproduces a complex outer retinal atrophy. Interestingly, our transcriptome data revealed distinct gene expression patterns supporting our histological phenotype, specifically, cone and rod degeneration, reactive gliosis, glial proliferation, and photoreceptor cell extrusion. Conclusions: We show that combined application of HBEGF and TNF, two neuropathology associated factors, is sufficient to induce a complex human retinal pathology. We discovered cone and rod cell extrusion as a pathomechanism for photoreceptor degeneration and a potential interrelationship with glial pathologies. Pharmacological inhibitor experiments support this, and PIEZO1 and MAPK signaling as untapped therapeutic targets, even for complex pathologies. Thus, our model provides mechanistic access to several pathologic processes as well as pathogenic functions of inflammation and biomechanics.

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Transcriptome profiling of mouse retina organoids at defined days in culture

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