The glucocorticoid receptor as a master regulator of the Müller cell response to diabetic conditions in mice.

Diabetic retinopathy (DR) is considered a primarily microvascular complication of diabetes. Müller glia cells are at the centre of the retinal neurovascular unit and play a critical role in DR. We therefore investigated Müller cell-specific signalling pathways that are altered in DR to identify novel targets for gene therapy. Using a multi-omics approach on purified Müller cells from diabetic db/db mice, we found the mRNA and protein expression of the glucocorticoid receptor (GR) to be significantly decreased, while its target gene cluster was down-regulated. Further, oPOSSUM TF analysis and ATAC- sequencing identified the GR as a master regulator of Müller cell response to diabetic conditions. Cortisol not only increased GR phosphorylation. It also induced changes in the expression of known GR target genes in retinal explants. Finally, retinal functionality was improved by AAV-mediated overexpression of GR in Müller cells. Our study demonstrates an important role of the glial GR in DR and implies that therapeutic approaches targeting this signalling pathway should be aimed at increasing GR expression rather than the addition of more ligand.

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.

Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

Transplanted human cones incorporate into the retina and function in a murine cone degeneration model.

Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.

Multimodal profiling of the transcriptional regulatory landscape of the developing mouse cortex identifies Neurog2 as a key epigenome remodeler.

How multiple epigenetic layers and transcription factors (TFs) interact to facilitate brain development is largely unknown. Here, to systematically map the regulatory landscape of neural differentiation in the mouse neocortex, we profiled gene expression and chromatin accessibility in single cells and integrated these data with measurements of enhancer activity, DNA methylation and three-dimensional genome architecture in purified cell populations. This allowed us to identify thousands of new enhancers, their predicted target genes and the temporal relationships between enhancer activation, epigenome remodeling and gene expression. We characterize specific neuronal transcription factors associated with extensive and frequently coordinated changes across multiple epigenetic modalities. In addition, we functionally demonstrate a new role for Neurog2 in directly mediating enhancer activity, DNA demethylation, increasing chromatin accessibility and facilitating chromatin looping in vivo. Our work provides a global view of the gene regulatory logic of lineage specification in the cerebral cortex.

Morpho-Rheological Fingerprinting of Rod Photoreceptors Using Real-Time Deformability Cytometry.

Distinct cell-types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High-throughput analysis of morpho-rheological features has recently been introduced using real-time deformability cytometry (RT-DC) providing new insights into the properties of different cell-types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell-derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell-derived retinal organoids, we characterized the inherent morpho-mechanical properties of mouse rod photoreceptors during development based on RT-DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label-free sorting of photoreceptors. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Organoid technology for retinal repair.

A major cause for vision impairment and blindness in industrialized countries is the loss of the light-sensing retinal tissue in the eye. Photoreceptor damage is one of the main characteristics found in retinal degeneration diseases, such as Retinitis Pigmentosa or age-related macular degeneration. The lack of effective therapies to stop photoreceptor loss together with the absence of significant intrinsic regeneration in the human retina converts such degenerative diseases into permanent conditions that are currently irreversible. Cell replacement by means of photoreceptor transplantation has been proposed as a potential approach to tackle cell loss in the retina. Since the first attempt of photoreceptor transplantation in humans, about twenty years ago, several research groups have focused in the development and improvement of technologies necessary to bring cell transplantation for retinal degeneration diseases to reality. Progress in recent years in the generation of human tissue derived from pluripotent stem cells (PSCs) has significantly improved our tools to study human development and disease in the dish. Particularly the availability of 3D culture systems for the generation of PSC-derived organoids, including the human retina, has dramatically increased access to human material for basic and medical research. In this review, we focus on important milestones towards the generation of transplantable photoreceptor precursors from PSC-derived retinal organoids and discuss recent pre-clinical transplantation studies using organoid-derived photoreceptors in context to related in vivo work using primary photoreceptors as donor material. Additionally, we summarize remaining challenges for developing photoreceptor transplantation towards clinical application.

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs.

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing.

UNLABELLED: To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. SIGNIFICANCE: The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs.

Characterization of a mouse model with complete RPE loss and its use for RPE cell transplantation.

PURPOSE: Age-related macular degeneration (AMD) is a major leading cause of visual impairment and blindness with no cure currently established. Cell replacement of RPE is discussed as a potential therapy for AMD. Previous studies were performed in animal models with severe limitations in recapitulating the disease progression. In detail, we describe the effect of systemic injection of sodium iodate in the mouse retina. We further evaluate the usefulness of this animal model to analyze cell-specific effects following transplantation of human embryonic stem cell (hESC)-derived RPE cells. METHODS: Morphologic, functional, and behavioral changes following sodium iodate injection were monitored by histology, gene expression analysis, electroretinography, and optokinetic head tracking. Human embryonic stem cell-derived RPE cells were transplanted 1 week after sodium iodate injection and experimental retinae were analyzed 3 weeks later. RESULTS: Injection of sodium iodate caused complete RPE cell loss, photoreceptor degeneration, and altered gene and protein expression in outer and inner nuclear layers. Retinal function was severely affected by day 3 and abolished from day 14. Following transplantation, donor hESC-derived RPE cells formed extensive monolayers that displayed wild-type RPE cell morphology, organization, and function, including phagocytosis of host photoreceptor outer segments. CONCLUSIONS: Systemic injection of sodium iodate has considerable effects on RPE, photoreceptors, and inner nuclear layer neurons, and provides a model to assay reconstitution and maturation of RPE cell transplants. The availability of an RPE-free Bruch's membrane in this model likely allows the unprecedented formation of extensive polarized cell monolayers from donor hESC-derived RPE cell suspensions.

Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

Synthesis of capsular polysaccharide at the division septum of Streptococcus pneumoniae is dependent on a bacterial tyrosine kinase.

One of the main virulence factors of the pathogenic bacterium Streptococcus pneumoniae is the capsule, present at the bacterial surface, surrounding the entire cell. Virtually all the 90 different capsular serotypes of S. pneumoniae, which vary in their chemical composition, express two conserved proteins, Wzd and Wze, which regulate the rate of the synthesis of capsule. In this work, we show that Wzd, a membrane protein, and Wze, a cytoplasmic tyrosine kinase, localize at the bacterial division septum, when expressed together in pneumococcal cells, without requiring the presence of additional proteins encoded in the capsule operon. The interaction between the two proteins and their consequent septal localization was dependent on a functional ATP binding domain of Wze. In the absence of either Wzd or Wze, capsule was still produced, linked to the cell surface, but it was absent from the division septum. We propose that Wzd and Wze are spatial regulators of capsular polysaccharide synthesis and, in the presence of ATP, localize at the division site, ensuring that capsule is produced in co-ordination with cell wall synthesis, resulting in full encapsulation of the pneumococcal cells.