Age-related macular degeneration (AMD), a leading cause of blindness, initiates in the outer-blood-retinabarrier (oBRB) formed by Retinal pigment epithelium (RPE), Bruch's membrane, and choriocapillaris. The mechanism of AMD initiation and progression remain poorly understood due to the lack of physiologically relevant oBRB models. We engineered a native-like 3D-oBRB tissue by bioprinting endothelial cells, pericytes, and fibroblasts on the basal side of a biodegradable scaffold and establishing an RPE monolayer on top. In this 3D-oBRB, a fully-polarized RPE monolayer with apical processes and basal infoldings provides barrier resistance, induces fenestration and choroid-specific gene expression in the choriocapillaris, and supports the formation of a Bruch's-like membrane that allows tissue integration in rat eyes. Complement activation in the 3D-oBRB triggers dry-AMD phenotypes (including subRPE drusen and choriocapillaris degeneration), and hypoxia activated HIF-α induces wet-AMD phenotypes (choriocapillaris neovascularization). Anti-VEGF drug treatment suppresses neovascularizationvalidating this model for clinical translation and drug discovery.

IMPORTANCEAfter the Age-Related Eye Disease Study 2 (AREDS2) study, the beta carotene component was replaced by lutein/zeaxanthin for the development of the revised AREDS supplement. However, it is unknown if the increased risk of lung cancer observed in those assigned beta carotene persists beyond the conclusion of the AREDS2 trial and if there is a benefit of adding lutein/zeaxanthin to the original AREDS supplement that can be observed with long-term follow-up.OBJECTIVE To assess 10-year risk of developing lung cancer and late age-related macular degeneration (AMD).

Development of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types amenable for translational research. Here, we developed a highly efficient and reproducible method that differentiates hPSCs into peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest cells, followed by differentiation into sensory neurons with an in vivo-like pseudo-unipolar morphology. Detailed characterization confirmed that the hPSC-derived nociceptors displayed molecular and cellular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors, including critical pain-relevant drug targets (e.g., TRPV1, TAC1, CALCA, NAV1.7, NAV1.8). Moreover, after confirming robust functional activities and differential response to noxious stimuli and specific drugs, a robotic cell culture system was employed to produce large quantities of human sensory neurons, which can be used to develop nociceptor-selective analgesics.

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Background: In the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia-causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring-like structure that supports the platelet typical disc-shaped morphology.This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation.Objectives: To assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. Methods:We used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. Results:We identified six distinct novel mutations in TUBA8, which encodes the most-divergent α-tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8-tubulin in the microtubule network. Conclusion:This study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, and kidney) can be optimized by using the CEPT small molecule cocktail, a polypharmacology approach that ensures cytoprotection and cell survival. Application of CEPT (chroman 1, emricasan, polyamines, trans-ISRIB) for just 24 hours during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, and cellular differentiation. Various qualification methods confirmed that CEPT treatment consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

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