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Page 267 of 548-
CLRN1 Variants in Müller Cells Cause Mitochondrial Dysfunction in USH3A Retinal Organoids.
BACKGROUND: Usher syndrome 3A (USH3A), caused by mutations in the CLRN1 gene, leads to retinitis pigmentosa and sensorineural hearing loss. While CLRN1's role in inner ear pathology is established, its contribution to retinal degeneration remains poorly understood. METHODS: Retinal organoids derived from a USH3A patient were analyzed using single-cell RNA sequencing and multi-electrode array recording. CLRN1 expression was mapped in human fetal retina and organoids. We assessed the structural, transcriptional, and functional impact of CLRN1 variants on Müller cells and photoreceptors, and evaluated idebenone as a potential targeted therapy. RESULTS: CLRN1 was specifically expressed in Müller cells. CLRN1 variants induced severe retinal degeneration, characterized by outer nuclear layer thinning, impaired photoreceptor gene expression, activated apoptosis, and diminished electrophysiological function. Mechanistically, these variants caused mitochondrial dysfunction in Müller cells, which triggered secondary mitochondrial impairment, oxidative stress, and apoptosis in photoreceptors. Idebenone treatment partially rescued these deficits. CONCLUSIONS: CLRN1-related mitochondrial impairment in Müller cells contributes to the pathogenesis of retinitis pigmentosa in USH3A. These findings identify Müller cell mitochondrial dysfunction as a key disease mechanism and highlight potential therapeutic targets.
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Accelerated photoreceptor differentiation of hiPSC-derived retinal organoids by contact co-culture with retinal pigment epithelium.
Retinal organoids (ROs) derived from human-induced pluripotent stem cells recapitulate the three-dimensional structure of retina, mimic human retinal development, and provide cell sources for pre-clinical retinal transplantation. Retinal pigment epithelium (RPE) is crucial for normal outer retinal physiology, including phagocytosis of shed photoreceptor outer segments and secretion of neurotrophic and vasculotrophic growth factors. However, whether ROs-RPE co-culture can improve the differentiation of photoreceptors in ROs in vitro remains unknown. Herein, primary mouse RPE cells were contact co-cultured with ROs at different time points. Our results revealed that the RPE cells accelerated photoreceptor differentiation in ROs, as the cross talk between the RPE and ROs promoted the stage specific expression of photoreceptor markers at different differentiation stages. Thus, we established an improved co-culture system based on modeling of human retina-RPE dynamics during retinogenesis for the evaluation of ocular therapies.
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Extracellular Vesicles Derived from Neural Progenitor Cells--a Preclinical Evaluation for Stroke Treatment in Mice.
Stem cells such as mesenchymal stem cells (MSCs) enhance neurological recovery in preclinical stroke models by secreting extracellular vesicles (EVs). Since previous reports have focused on the application of MSC-EVs only, the role of the most suitable host cell for EV enrichment and preclinical stroke treatment remains elusive. The present study aimed to evaluate the therapeutic potential of EVs derived from neural progenitor cells (NPCs) following experimental stroke. Using the PEG technique, EVs were enriched and characterized by electron microscopy, proteomics, rt-PCR, nanosight tracking analysis, and Western blotting. Different dosages of NPC-EVs displaying a characteristic profile in size, shape, cargo protein, and non-coding RNA contents were incubated in the presence of cerebral organoids exposed to oxygen-glucose deprivation (OGD), significantly reducing cell injury when compared with control organoids. Systemic administration of NPC-EVs in male C57BL6 mice following experimental ischemia enhanced neurological recovery and neuroregeneration for as long as 3 months. Interestingly, the therapeutic impact of such NPC-EVs was found to be not inferior to MSC-EVs. Flow cytometric analyses of blood and brain samples 7 days post-stroke demonstrated increased blood concentrations of B and T lymphocytes after NPC-EV delivery, without affecting cerebral cell counts. Likewise, a biodistribution analysis after systemic delivery of NPC-EVs revealed the majority of NPC-EVs to be found in extracranial organs such as the liver and the lung. This proof-of-concept study supports the idea of EVs being a general concept of stem cell-induced neuroprotection under stroke conditions, where EVs contribute to reverting the peripheral post-stroke immunosuppression.
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Evidence for self-absorption of terminals by developing axons of retinal ganglion cells in the chick.
The appearance of membrane-bound degenerative organelles in chick optic nerve axons was studied at the electron microscopic level. A semiquantitative analysis revealed a sharp increase in the number of axons containing accumulations of such organelles during the second day after hatching. In dark-reared chicks this increase was retarded, suggesting the presence of a light-influenced event in the early post-hatch period.
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Single-Cell Transcriptomic Profiling of Human Retinal Organoids Revealed a Role of IGF1-PHLDA1 Axis in Photoreceptor Precursor Specification.
PURPOSE: Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development and degeneration results in blindness. So far, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive. METHODS: To trace and enrich the human photoreceptor lineage, we first engineered H9 human embryonic stem cell (hESC) reporter line by fusing EGFP to endogenous BLIMP1 using CRISPR/CAS9 gene-editing technology, and then used the cell line to generate 3D retinal organoids. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system, and the data were analyzed using Seurat. Immunofluorescence combined with lentivirus-mediated knockdown and overexpression experiments were used as validation approaches. RESULTS: Single-cell transcriptomic profiling revealed that retinal progenitor cells were temporally programmed to differentiate to cone and rod sequentially. We identified PHLDA1 as a novel regulator of photoreceptor specification. PHLDA1 mediated the effects of IGF1 through IGF1R, and inhibited AKT phosphorylation during photoreceptor development. CONCLUSIONS: Our data established a transcriptomic cell atlas of the human photoreceptor lineage, and identified IGF1-PHLDA1 axis to regulate human photoreceptor development.
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Loss of INPP5E affects photoreceptor outer segment membrane biogenesis in iPSC-derived human retinal organoids.
Mutations in the ciliary gene INPP5E, encoding inositol polyphosphate-5-phosphatase E (INPP5E), can cause retinal degeneration as part of the ciliopathy Joubert syndrome or non-syndromic retinitis pigmentosa (RP). INPP5E regulates the membrane makeup of the primary cilium; however, its function in the specialized sensory photoreceptor cells of the human retina remain unclear. Here, we utilize control and CRISPR/Cas9-generated INPP5E knockout (INPP5ED477N/D477N) human induced pluripotent stem cells (iPSCs) to generate retinal organoids (ROs). Through proteomic and immunofluorescence analysis, we show that INPP5E plays an important role in early retinal development and photoreceptor progenitor cell differentiation. In mature ROs, INPP5E localizes to the connecting cilium of photoreceptors, and the loss of INPP5E leads to altered localization of ARL13B and rhodopsin in mature photoreceptors. Furthermore, photoreceptor outer segment structure is affected, leading to elongated outer segment membranes in both cone and rod photoreceptors, suggesting an important role for INPP5E in photoreceptor outer segment membrane biogenesis. Together, these data underline the importance of INPP5E in retina development and photoreceptor structure and highlight the usability of ROs to study protein function in a human context.
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Neural stem cells and epilepsy: functional roles and disease-in-a-dish models.
Epilepsy is a disorder of the central nervous system characterized by spontaneous recurrent seizures. Although current therapies exist to control the number and severity of clinical seizures, there are no pharmacological cures or disease-modifying treatments available. Use of transgenic mouse models has allowed an understanding of neural stem cells in their relation to epileptogenesis in mesial temporal lobe epilepsy. Further, with the significant discovery of factors necessary to reprogram adult somatic cell types into pluripotent stem cells, it has become possible to study monogenic epilepsy-in-a-dish using patient-derived neurons. This discovery along with some of the newest technological advances in recapitulating brain development in a dish has brought us closer than ever to a platform in which to study and understand the mechanisms of this disease. These technologies will be critical in understanding the mechanism of epileptogenesis and ultimately lead to improved therapies and precision medicine for patients with epilepsy.
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Models for assessment of angiogenesis in gliomas.
In the last two decades, much attention has been focussed on mechanisms of glioma vascularization including the investigation of growth factors and receptors involved. Recently, these efforts resulted in various approaches for antiangiogenic treatment of experimental brain tumors. These basic science and preclinical trials need an assortment of models, which should allow investigating a variety of questions. Several objectives concerning basic endothelial cell (EC) characteristics can adequately be studied in vitro using EC monolayer assays. Three-dimensional spheroid techniques respect the more complex cell-cell and cell-environment interplay within a three-dimensional culture. To optimize the imitation of the crucial interaction of human gliomas with host endothelial cells, immunological cells and extracellular matrix, animal models are mandatory. An essential rule is to utilize an orthotopic model, since tumor-host interaction is organ specific. To avoid alloimmunogenic responses, it is desirable to use weakly or not immunogenic glioma grafts, what is best accomplished in a syngeneic model. However, since rat gliomas poorly resemble human glioma growth patterns, human glioma xenografting into immunocompromized animals should be considered. In vivo monitoring techniques like videoscopy via a cranial window or magnetic resonance imaging (MRI) allow for functional studies and improve the validity of the model employed. Finally, it is essentially to recognize the limitations of each model considered and to select that model, which seems to be most appropriate for the objectives to be investigated.
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A differential requirement for ciliary transition zone proteins in human and mouse neural progenitor fate specification.
Studying ciliary genes in the context of the human central nervous system is crucial for understanding the underlying causes of neurodevelopmental ciliopathies. Here, we use pluripotent stem cell-derived spinal organoids to reveal distinct functions of the ciliopathy gene RPGRIP1L in humans and mice, and uncover an unexplored role for cilia in human axial patterning. Previous research has emphasized Rpgrip1l critical functions in mouse brain and spinal cord development through the regulation of SHH/GLI pathway. Here, we show that RPGRIP1L is not required for SHH activation or motoneuron lineage commitment in human spinal progenitors and that this feature is shared by another ciliopathy gene, TMEM67. Furthermore, human RPGRIP1L-mutant motoneurons adopt hindbrain and cervical identities instead of caudal brachial identity. Temporal transcriptome analysis reveals that this antero-posterior patterning defect originates in early axial progenitors and correlates with cilia loss. These findings provide important insights into the role of cilia in human neural development.
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Functional expression of the mechanosensitive PIEZO1 channel in primary endometrial epithelial cells and endometrial organoids.
Successful pregnancy requires the establishment of a complex dialogue between the implanting embryo and the endometrium. Knowledge regarding molecular candidates involved in this early communication process is inadequate due to limited access to primary human endometrial epithelial cells (EEC). Since pseudo-pregnancy in rodents can be induced by mechanical scratching of an appropriately primed uterus, this study aimed to investigate the expression of mechanosensitive ion channels in EEC. Poking of EEC provoked a robust calcium influx and induced an increase in current densities, which could be blocked by an inhibitor of mechanosensitive ion channels. Interestingly, RNA expression studies showed high expression of PIEZO1 in EEC of mouse and human. Additional analysis provided further evidence for the functional expression of PIEZO1 since stimulation with Yoda1, a chemical agonist of PIEZO1, induced increases in intracellular calcium concentrations and current densities in EEC. Moreover, the ion channel profile of human endometrial organoids (EMO) was validated as a representative model for endometrial epithelial cells. Mechanical and chemical stimulation of EMO induced strong calcium responses supporting the hypothesis of mechanosensitive ion channel expression in endometrial epithelial cells. In conclusion, EEC and EMO functionally express the mechanosensitive PIEZO1 channel that could act as a potential target for the development of novel treatments to further improve successful implantation processes.