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

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.

HBEGF-TNF induce a complex outer retinal pathology with photoreceptor cell extrusion in human organoids.

Human organoids could facilitate research of complex and currently incurable neuropathologies, such as age-related macular degeneration (AMD) which causes blindness. Here, we establish a human retinal organoid system reproducing several parameters of the human retina, including some within the macula, to model a complex combination of photoreceptor and glial pathologies. We show that combined application of TNF and HBEGF, factors associated with neuropathologies, is sufficient to induce photoreceptor degeneration, glial pathologies, dyslamination, and scar formation: These develop simultaneously and progressively as one complex phenotype. Histologic, transcriptome, live-imaging, and mechanistic studies reveal a previously unknown pathomechanism: Photoreceptor neurodegeneration via cell extrusion. This could be relevant for aging, AMD, and some inherited diseases. Pharmacological inhibitors of the mechanosensor PIEZO1, MAPK, and actomyosin each avert pathogenesis; a PIEZO1 activator induces photoreceptor extrusion. Our model offers mechanistic insights, hypotheses for neuropathologies, and it could be used to develop therapies to prevent vision loss or to regenerate the retina in patients suffering from AMD and other diseases.

A Multi-Omics Analysis of Mucosal-Associated-Invariant T Cells Reveals Key Drivers of Distinct Modes of Activation.

The function of mucosal-associated invariant T (MAIT) cells highly depends on the mode of activation, either by recognition of bacterial metabolites via their T cell receptor (TCR) or in a TCR-independent manner via cytokines. The underlying molecular mechanisms are not entirely understood. To define the activation of MAIT cells on the molecular level, we applied a multi-omics approach with untargeted transcriptomics, proteomics and metabolomics. Transcriptomic analysis of E. coli- and TCR-activated MAIT cells showed a distinct transcriptional reprogramming, including altered pathways, transcription factors and effector molecules. We validated the consequences of this reprogramming on the phenotype by proteomics and metabolomics. Thus, and to distinguish between TCR-dependent and -independent activation, MAIT cells were stimulated with IL12/IL18, anti-CD3/CD28 or both. Only a combination of both led to full activation of MAIT cells, comparable to activation by E. coli. Using an integrated network-based approach, we identified key drivers of the distinct modes of activation, including cytokines and transcription factors, as well as negative feedback regulators like TWIST1 or LAG3. Taken together, we present novel insights into the biological function of MAIT cells, which may represent a basis for therapeutic approaches to target MAIT cells in pathological conditions.