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Visual Abstract Generation of human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) offers an unprecedented approach to modeling movement disorders such as dystonia and amyotrophic lateral sclerosis. However,achieving survival poses a significant challenge when culturing induced MNs,especially when aiming to reach late maturation stages. Utilizing hiPSC-derived motor neurons and primary mouse astrocytes,we assembled two types of coculture systems: direct coculturing of neurons with astrocytes and indirect coculture using culture inserts that physically separate neurons and astrocytes. Both systems significantly enhance neuron survival. Compared with these two systems,no significant differences in neurodevelopment,maturation,and survival within 3 weeks,allowing to prepare neurons at maturation stages. Using the indirect coculture system,we obtained highly pure MNs at the late mature stage from hiPSCs. Transcriptomic studies of hiPSC-derived MNs showed a typical neurodevelopmental switch in gene expression from the early immature stage to late maturation stages. Mature genes associated with neurodevelopment and synaptogenesis are highly enriched in MNs at late stages,demonstrating that these neurons achieve maturation. This study introduces a novel tool for the preparation of highly pure hiPSC-derived neurons,enabling the determination of neurological disease pathogenesis in neurons at late disease onset stages through biochemical approaches,which typically necessitate highly pure neurons. This advancement is particularly significant in modeling age-related neurodegeneration. View Publication

IntroductionThe inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states,however,the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype,which is one of the reasons why macrophage transplantation has failed.MethodsIn this study,we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover,we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor,sensory and autonomic functions. Instead of transplanting the cells,we injected the paracrine factors and extracellular vesicles that they secrete,avoiding the loss of the phenotype of the transplanted cells due to local environmental cues.ResultsWe demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival,namely,the alternative activation with IL-10 and TGF-?1 (M(IL-10+TGF-?1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-?1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally,the M(IL-10+TGF-?1) secretome supported the recovery of bladder function and decreased microglial activation,astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-?1)-derived secretome identified clusters of proteins involved in axon extension,dendritic spine maintenance,cell polarity establishment,and regulation of astrocytic activation.DiscussionOverall,our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications. View Publication

BackgroundAbnormalities in T cell activation play an important role in the pathogenesis of myocarditis,and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation,as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models,the specific mechanism for human cardiomyocytes has not been investigated.MethodsIn this study,we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation.ResultsBy combination of quantitative proteomics with tissue and cell immunofluorescence examination,we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content,creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway.ConclusionsThe data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03791-4. View Publication

Polycomb repressive complex 1 (PRC1) modifies chromatin through catalysis of histone H2A lysine 119 monoubiquitination (H2AK119ub1). RING1 and RNF2 interchangeably serve as the catalytic subunit within PRC1. Pathogenic missense variants in PRC1 core components reveal functions of these proteins that are obscured in knockout models. While Ring1a knockout models remain healthy,the microcephaly and neuropsychiatric phenotypes associated with a pathogenic RING1 missense variant implicate unappreciated functions. Using an in vitro model of neurodevelopment,we observe that RING1 contributes to the broad placement of H2AK119ub1,and that its targets overlap with those of RNF2. PRC1 complexes harboring hypomorphic RING1 bind target loci but do not catalyze H2AK119ub1,reducing H2AK119ub1 by preventing catalytically active complexes from accessing the locus. This results in delayed DNA damage repair and cell cycle progression in neural progenitor cells (NPCs). Conversely,reduced H2AK119ub1 due to hypomorphic RING1 does not generate differential expression that impacts NPC differentiation. In contrast,hypomorphic RNF2 generates a greater reduction in H2AK119ub1 that results in both delayed DNA repair and widespread transcriptional changes. These findings suggest that the DNA damage response is more sensitive to H2AK119ub1 dosage change than is regulation of gene expression. Here,the authors establish a human in vitro model of neurodevelopment to investigate an allelic series of clinically relevant RING1 and RNF2 missense variants. The observations reveal that missense variants function according to a dominant-negative genetic mechanism. View Publication

Human-induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) have been shown to be useful for the development of cell-based regenerative strategies and for modelling drug discovery. However,stem cell-derived HLCs are not identical in nature to primary human hepatocytes (PHHs),which could affect the cell phenotype and,potentially,model reliability. Therefore,we employed the in-depth gene expression profiling of HLCs and other important and relevant cell types,which led to the identification of clear similarities and differences between them at the transcriptional level. Through gene set enrichment analysis,we identified that genes that are critical for immune signalling pathways become downregulated upon HLC differentiation. Our analysis also found that TAV.HLCs exhibit a mild gene signature characteristic of acute lymphoblastic leukaemia,but not other selected cancers. Importantly,HLCs present significant similarity to PHHs,making them genuinely valuable for modelling human liver biology in vitro and for the development of prototype cell-based therapies for pre-clinical testing. View Publication

SummaryCardiac fibrosis is a key characteristic of heart failure. CTPR390,an experimental anti-fibrotic inhibitor targeting Hsp90,has shown success in animal models,but remains unexplored in human cardiac models. This study evaluated an engineered cardiac connective tissue (ECCT) model,focusing on changes in the extracellular matrix and fibroblasts. Results showed that CTPR390 prevented architectural changes in TGF?1-activated ECCT,preserving tissue perimeter,collagen fibers alignment while reducing structured areas and degree of collagen structuration. CTPR390 treatment reduced cell area of fibroblasts under tension,without changes in the internal rounded cells devoid of tension. Fibroblast recruitment to tension areas was diminished,showing biomechanical behavior similar to control ECCT. This treatment also lowered the gene and protein expression of key pro-fibrotic markers. Here,advanced biotechnology was employed to detect the detailed structure of tissue fibrosis reduction after administering CTPR390,representing a significant advancement toward clinical application for cardiac fibrosis treatment. Graphical abstract Highlights•CTPR390 prevented architectural changes in TGF?1-activated ECCT•CTPR390 preserves tissue perimeter,collagen fibers alignment•CTPR390 reduces structured areas and degree of collagen structuration•CTPR390-trested ECCTs presented a biomechanical behavior similar to control ECCT Molecular biology; Cell biology View Publication

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here,using a candidate-based shRNA screen,we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors,or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells,neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2,for which an FDA-approved small molecule inhibitor,EPZ6438 (also known as tazemetostat),is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately,EZH2 inhibitors do not efficiently cross the blood-brain barrier,limiting their therapeutic use for FXS. Recently,antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons,and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively,our results establish EZH2 inhibition in general,and EZH2 ASOs in particular,as a therapeutic approach for FXS. View Publication

In mammals,early embryonic gastrulation process is high energy demanding. Previous studies showed that,unlike endoderm and mesoderm cells,neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process,which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/?-catenin signaling during neuroectoderm differentiation. Notably,histone lactylation,especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments,we further identified that four genes,including PAX6,were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05510-x. View Publication

Pancreatic islets are densely packed cellular aggregates containing various hormonal cell types essential for blood glucose regulation. Interactions among these cells markedly affect the glucoregulatory functions of islets along with the surrounding niche and pancreatic tissue-specific geometrical organization. However,stem cell (SC)-derived islets generated in vitro often lack the three-dimensional extracellular microenvironment and peri-vasculature,which leads to the immaturity of SC-derived islets,reducing their ability to detect glucose fluctuations and insulin release. Here,we bioengineer the in vivo-like pancreatic niches by optimizing the combination of pancreatic tissue-specific extracellular matrix and basement membrane proteins and utilizing bioprinting-based geometrical guidance to recreate the spatial pattern of islet peripheries. The bioprinted islet-specific niche promotes coordinated interactions between islets and vasculature,supporting structural and functional features resembling native islets. Our strategy not only improves SC-derived islet functionality but also offers significant potential for advancing research on islet development,maturation,and diabetic disease modeling,with future implications for translational applications. The glucoregulatory functions of pancreatic islets are affected by their surrounding niche and spatial organization. Here,bioengineered stem-cell derived islet niches use bioprinting-based geometrical guidance to promote islet maturation for improved functionality and diabetes research. View Publication

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract,referred to as mutant ataxin-3. In this study,we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First,we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently,the pluripotency of GE-iPSCs was confirmed,and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons,restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells),and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion,our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8. View Publication

Ribosome profiling,which is based on deep sequencing of ribosome footprints,has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However,the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here,we overcome these hurdles through the development of “Ribo-FilterOut”,which is based on the separation of footprints from ribosome subunits by ultrafiltration,and “Ribo-Calibration”,which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript,the translation initiation rate,and the overall number of translation events before its decay,all in a genome-wide manner. Moreover,our method reveals the allocation of ribosomes under heat shock stress,during aging,and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome. Ribosome profiling faces issues with rRNA contamination and measurements of ribosome numbers on transcripts. Here,the authors develop Ribo-FilterOut and Ribo-Calibration,methods which can be used to estimate kinetic and stoichiometric parameters of translation under various conditions. View Publication

BackgroundCHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders,including CHARGE (Coloboma,Heart defects,Atresia of the choanae,Retardation of growth and development,Genital hypoplasia,and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus,liver,pancreas,and intestine.MethodsIn this study,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7?/?),heterozygous mutant (CHD7+/?),and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers,mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).ResultsWe found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.ConclusionsCHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04437-9. View Publication