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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

BackgroundEmbryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs,which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation. How this primed enhancer state is established and maintained and how it affects the regulation of developmental gene networks remains poorly understood.ResultsHere,we use comparative multi-omic analyses of human and mouse early embryonic development to identify subsets of postgastrulation lineage-specific enhancers which are epigenetically primed ahead of their activation,marked by the histone modification H3K4me1 within the epiblast. We show that epigenetic priming occurs at lineage-specific enhancers for all three germ layers and that epigenetic priming of enhancers confers lineage-specific regulation of key developmental gene networks. Surprisingly in some cases,lineage-specific enhancers are epigenetically marked already in the zygote,weeks before their activation during lineage specification. Moreover,we outline a generalizable strategy to use naturally occurring human genetic variation to delineate important sequence determinants of primed enhancer function.ConclusionsOur findings identify an evolutionarily conserved program of enhancer priming and begin to dissect the temporal dynamics and mechanisms of its establishment and maintenance during early mammalian development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03658-8. 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

SUMMARY GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology,we deleted EXOC2,which encodes an essential exocyst subunit,in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2,suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to,surprisingly,the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA,making it a potential therapeutic target in C9ORF72-ALS/FTD. In brief Halim et al. deleted the gene EXOC2 from patient stem cells and then differentiated them into motor neurons. They found that several amyotrophic lateral sclerosis-related phenotypes were rescued in patient neurons when EXOC2 was deleted or knocked down by a drug. This study identifies EXOC2 as a potential therapeutic target. Graphical Abstract 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

Brain tumors are commonly treated with radiotherapy,but the efficacy of the treatment is limited by its toxicity to the normal tissue including post-irradiation contrast enhanced lesions often linked to necrosis. The poorly understood mechanisms behind such brain lesions were studied using cerebral organoids. Here we show that irradiation of such organoids leads to dose-dependent growth retardation and formation of liquid-filled cavities but is not correlated with necrosis. Instead,the radiation-induced changes comprise of an enhancement of cortical hem markers,altered neuroepithelial stem cell differentiation,and an increase of ZO1+/AQP1+/CLDN3+-choroid plexus (CP)-like structures accompanied by an upregulation of IGF2 mRNA,known to be expressed in CP and cerebrospinal fluid. The altered differentiation is attributed to changes in the WNT/BMP signaling pathways. We conclude that aberrant CP formation can be involved in radiation-induced brain lesions providing additional strategies for possible countermeasures. Human cerebral organoids provide insights into mechanisms behind the formation of choroid plexus (CP)-like structures that may contribute to radiation-induced brain image changes. View Publication