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BackgroundPrimary cilia mediate vertebrate development and growth factor signalling. Defects in primary cilia cause inherited developmental conditions termed ciliopathies. Ciliopathies often present with cystic kidney disease,a major cause of early renal failure. Currently,only one drug,Tolvaptan,is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions are needed.MethodsWe screened clinical development compounds to identify those that reversed cilia loss due to siRNA knockdown. In parallel,we undertook a whole genome siRNA-based reverse genetics phenotypic screen to identify positive modulators of cilia formation.ResultsUsing a clinical development compound screen,we identify fasudil hydrochloride. Fasudil is a generic,off-patent drug that is a potent,broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In parallel,the siRNA screen identifies ROCK2 and we demonstrate that ROCK2 is a key mediator of cilium formation and function through its possible effects on actin cytoskeleton remodelling.ConclusionsOur results indicate that specific ROCK2 inhibitors (e.g. belumosudil) could be repurposed for cystic kidney disease treatment. We propose that ROCK2 inhibition represents a novel,disease-modifying therapeutic approach for heterogeneous ciliopathies. Plain language summaryPrimary cilia are antennae-like structures on cells that are important for early development and healthy cell function. Defects in primary cilia can cause inherited diseases called ciliopathies. Ciliopathies often cause fluid-filled sacs,called cysts,that are a major cause of kidney disease and failure. There is currently one drug licensed to slow kidney disease progression,but it is poorly tolerated in patients. Therefore,new drugs are needed. In this study,we used screening assays to identify potential drugs and their targets that are effective in promoting the formation of primary cilia. Our results identified ROCK2 (Rho-associated coiled-coil-containing protein kinase 2),an inhibitor of protein signalling,as a key mediator of cilium function. These findings suggest that drugs that specifically target ROCK2 could be a potential treatment option for cystic kidney disease. Smith et al. use clinical development screen and whole genome siRNA-reverse genetics phenotypic screen to identify ROCK2,as a modulator of cilia formation and function via its effects on actin cytoskeleton remodelling. Repurposing ROCK2 is a viable treatment for ciliopathies,for which a limited therapeutic option is available. View Publication

Human pluripotent stem cells offer a scalable platform to study genetic and signalling mechanisms governing cell lineage decisions during differentiation. Genome-wide and single-cell transcriptomics technologies likewise offer high-throughput analysis of heterogeneous cell differentiation states. While in vivo development has been extensively characterised using these technologies,there remains a need for comprehensive single-cell transcriptomic profiling of stem cell differentiation from pluripotency. Understanding gene expression changes governing differentiation in vitro is key to developing high fidelity differentiation protocols and understanding fundamental mechanisms of development. We generated a single-cell RNA sequencing time course to study the role of developmental signalling pathways on multilineage diversification from pluripotency in vitro. The combined dataset of over 60,000 cells spans cell types from a time course of differentiation across all germ layers,ranging from gastrulation cell states to progenitor and committed cell types. These data provide a diverse benchmarking reference point to compare against in vivo development and advance understanding of signalling regulation of differentiation,providing insights into protocol development,drug screening,and regenerative medicine applications. View Publication

SummaryNeonatal mouse hearts have transient renewal capacity,which is lost in juvenile and adult stages. In neonatal mouse hearts,myocardial infarction (MI) causes an initial loss of cardiomyocytes. However,it is unclear which type of regulated cell death (RCD) occurs in stressed cardiomyocytes. In the current studies,we induced MI in neonatal and juvenile mouse hearts and showed that ischemic cardiomyocytes primarily undergo ferroptosis,a non-apoptotic and iron-dependent form of RCD. We demonstrated that cardiac fibroblasts (CFs) protect cardiomyocytes from ferroptosis through paracrine effects and direct cell-cell interaction. CFs show strong resistance to ferroptosis due to high ferritin expression. The fibrogenic activity of CFs,typically considered detrimental to heart function,is negatively regulated by paired-like homeodomain 2 (Pitx2) signaling from cardiomyocytes. In addition,Pitx2 prevents ferroptosis in cardiomyocytes by regulating ferroptotic genes. Understanding the regulatory mechanisms of cardiomyocyte survival and death can identify potentially translatable therapeutic strategies for MI. Graphical abstract Highlights•Neonatal and juvenile mouse cardiomyocytes mainly undergo ferroptosis after MI•Cardiac fibroblasts protect cardiomyocytes through paracrine effect•Cardiac fibroblasts interact with cardiomyocytes to share iron burden•Pitx2 pathway protects cardiomyocytes from ferroptosis and controls fibrosis Cardiovascular medicine; Physiology; Cell biology View Publication

Alzheimer’s disease (AD) is a devastating neurodegenerative condition that affects memory and cognition,characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression,the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined iPSC-derived neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E,alongside CRISPR-corrected isogenic cell lines,to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls,suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally,both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs,while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding disease pathogenesis. View Publication

Epicardium,the most outer mesothelium,exerts crucial functions in fetal heart development and adult heart regeneration. Here we use a three-step manipulation of WNT signalling entwined with BMP and RA signalling for generating a self-organized epicardial organoid that highly express with epicardium makers WT1 and TCF21 from human embryonic stem cells. After 8-days treatment of TGF-beta following by bFGF,cells enter into epithelium-mesenchymal transition and give rise to smooth muscle cells. Epicardium could also integrate and invade into mouse heart with SNAI1 expression,and give birth to numerous cardiomyocyte-like cells. Single-cell RNA seq unveils the heterogeneity and multipotency exhibited by epicardium-derived-cells and fetal-like epicardium. Meanwhile,extracellular matrix and growth factors secreted by epicardial organoid mimics the ecology of subepicardial space between the epicardium and cardiomyocytes. As such,this epicardial organoid offers a unique ground for investigating and exploring the potential of epicardium in heart development and regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01339-w. View Publication

Periventricular heterotopia (PH),a common form of gray matter heterotopia associated with developmental delay and drug-resistant seizures,poses a challenge in understanding its neurophysiological basis. Human cerebral organoids (hCOs) derived from patients with causative mutations in FAT4 or DCHS1 mimic PH features. However,neuronal activity in these 3D models has not yet been investigated. Here we show that silicon probe recordings reveal exaggerated spontaneous spike activity in FAT4 and DCHS1 hCOs,suggesting functional changes in neuronal networks. Transcriptome and proteome analyses identify changes in neuronal morphology and synaptic function. Furthermore,patch-clamp recordings reveal a decreased spike threshold specifically in DCHS1 neurons,likely due to increased somatic voltage-gated sodium channels. Additional analyses reveal increased morphological complexity of PH neurons and synaptic alterations contributing to hyperactivity,with rescue observed in DCHS1 neurons by wild-type DCHS1 expression. Overall,we provide new comprehensive insights into the cellular changes underlying symptoms of gray matter heterotopia. Periventricular heterotopia (PH) is associated with neurodevelopmental delay. Here authors report patient-derived organoids with FAT4 and DCHS1 mutations mimic PH features,showing hyperactivity,synaptic changes and cell morphological alterations. View Publication

Highlights•Preformation of aggregates tuned by cell density enable cultivation of hiPSCs in scale-down shear environments.•Scale-down systems utilizing preformation protocols achieve comparable fold expansion with commercial systems.•Expression of pluripotency markers and functional differentiation capacity is maintained following passage in scale-down culture.•Successful application of hiPSC protocols at < 20 mL scales enable rapid and cost-effective research into cell phenotype under dynamic conditions. Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further,costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research,and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing. Graphical abstractImplementation of adapted protocols enable scale-down systems as a tool for high-throughput iPSC biomanufacturing research,in platforms conducive to scale-up for clinical manufacturing.Image,graphical abstract View Publication

ObjectiveGene discovery studies in individuals with diabetes diagnosed within 6 months of life (neonatal diabetes,NDM) can provide unique insights into the development and function of human pancreatic beta-cells.MethodsWe performed genome sequencing in a cohort of 43 consanguineous individuals with NDM in whom all the known genetic causes had previously been excluded. We used quantitative PCR and RNA-sequencing in CRISPR-edited human induced pluripotent stem cells (iPSCs),and CUT&RUN-sequencing in EndoC-?H1 cells to investigate the effect of PAX4 loss on human pancreatic development.ResultsWe describe the identification of homozygous PAX4 loss-of-function variants in 2 individuals with transient NDM: a p.(Arg126?) stop-gain variant and a c.-352_104del deletion affecting the first 4 PAX4 exons. We confirmed the p.(Arg126?) variant causes nonsense mediated decay in CRISPR-edited iPSC-derived pancreatic endoderm cells. Integrated analysis of CUT&RUN-sequencing in EndoC-?H1 cells and RNA-sequencing in PAX4-depleted islet stem cell models identified genes directly regulated by PAX4 involved in both pancreatic islet development and glucose-stimulated insulin secretion.ConclusionWe report the first human cases of complete loss of PAX4,establishing it as a novel cause of NDM and highlighting its role in human beta cell development. Both probands had transient NDM which remitted in early infancy but relapsed at the ages of 2.4 and 6.7 years,demonstrating that in contrast to mouse models,PAX4 is not essential for the development of human pancreatic beta-cells. Highlights•Homozygous loss-of-function variants in PAX4 are a novel genetic cause of transient neonatal diabetes.•PAX4 directly regulates genes involved in pancreatic beta cell development and glucose-sensitive insulin secretion.•The role of PAX4 in humans differs to that observed in mouse and is not essential for beta cell development. View Publication

Cathepsin B (CatB),a protease in endosomal and lysosomal compartments,plays a key role in neuronal protein processing and degradation,but its function in brain development remains unclear. In this study,we found that CatB is highly expressed in the cortex of E12.5–E16.5 mice. Morphological analysis revealed significant defects in cortical development in CatB knockout (KO) mice,particularly in layer 6. In vitro experiments showed that CatB deficiency notably impaired neuronal migration and development. Behaviorally,CatB KO mice displayed prominent depressive-like behaviors,and electrophysiological recordings demonstrated significantly reduced neuronal activity in layer 6 of the medial prefrontal cortex. Mechanistically,proteomics analysis revealed that CatB KO affected neuronal migration and axonal growth,and decreased the expression of key transcription factors involved in neuronal development,particularly PEG3. Deficiency of PEG3 also significantly impaired neuronal migration and development. Our findings uncover a role for CatB in cortical development and suggest a mechanism linking CatB deficiency with depression and developmental defects through the destabilization of PEG3. Cathepsin B (CatB) is essential for cortical development. Its deficiency impairs neuronal migration,reduces PEG3 expression,and leads to layer 6 defects and depression-like behaviors,revealing a novel link between CatB and brain development. View Publication

IntroductionDual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal VGF gene network that regulates late-onset Alzheimer’s disease (AD). Importantly,decreased DUSP6 levels are correlated with an increased clinical dementia rating (CDR) in human subjects,and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model.MethodsTo investigate the role of DUSP6 in AD,we stereotactically injected AAV5-DUSP6 or AAV5-GFP (control) into the dorsal hippocampus (dHc) of both female and male 5xFAD or wild type mice,to induce overexpression of DUSP6 or GFP.ResultsBarnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load,Aß1–40 and Aß1–42 levels,and amyloid precursor protein processing enzyme BACE1,in male but not in female mice. Microglial activation,which was increased in 5xFAD mice,was significantly reduced by dHc DUSP6 overexpression in both males and females,as was the number of “microglial clusters,” which correlated with reduced amyloid plaque size. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulation of inflammatory and extracellular signal-regulated kinase pathways,while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. Gene ontology analysis of DEGs (p < 0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD.DiscussionIn summary,DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice,whereas reduced neuroinflammation and microglial activation were observed in both males and females,suggesting that DUSP6-induced reduction of microglial activation did not contribute to sex-dependent improvement in memory deficits. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression,however,correlated with the improvement of spatial memory deficits in male but not female 5xFAD. View Publication