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Cardiac differentiation of human induced pluripotent stem cells is readily achievable,yet derivation of mature cardiomyocytes has been a recognized limitation. Here,a mesoderm priming approach was engineered to boost the maturation of cardiomyocyte progeny derived from pluripotent stem cells under standard cardiac differentiation conditions. Functional and structural hallmarks of maturity were assessed through multiparametric evaluation of cardiomyocytes derived from induced pluripotent stem cells following transfection of the mesoderm transcription factor Brachyury prior to initiation of lineage differentiation. Transfection with Brachyury resulted in earlier induction of a cardiopoietic state as hallmarked by early upregulation of the cardiac-specific transcription factors NKX2.5,GATA4,TBX20. Enhanced sarcomere maturity following Brachyury conditioning was documented by an increase in the proportion of cells expressing the ventricular isoform of myosin light chain and an increase in sarcomere length. Mesoderm primed cells displayed increased reliance on mitochondrial respiration as determined by increased mitochondrial size and a greater basal oxygen consumption rate. Further,Brachyury priming drove maturation of calcium handling enabling transfected cells to maintain calcium transient morphology at higher external field stimulation rates and augmented both calcium release and sequestration kinetics. In addition,transfected cells displayed a more mature action potential morphology with increased depolarization and repolarization kinetics. Derived cells transfected with Brachyury demonstrated increased toxicity response to doxorubicin as determined by a compromise in calcium transient morphology. Thus,Brachyury pre-treatment here achieved a streamlined strategy to promote maturity of human pluripotent stem cell-derived cardiomyocytes establishing a generalizable platform ready for deployment. View Publication

Autism spectrum disorder (ASD) and congenital heart disease (CHD) frequently co-occur,yet the underlying molecular mechanisms of this comorbidity remain unknown. Given that children with CHD are identified as newborns,understanding which CHD variants are associated with autism could help select individuals for early intervention. Autism gene perturbations commonly dysregulate neural progenitor cell (NPC) biology,so we hypothesized that CHD genes disrupting neurogenesis are more likely to increase ASD risk. Therefore,we performed an in vitro pooled CRISPR interference screen to identify CHD genes disrupting NPC biology and identified 45 CHD genes. A cluster of ASD and CHD genes are enriched for ciliary biology,and perturbing any one of seven such genes (CEP290,CHD4,KMT2E,NSD1,OFD1,RFX3 and TAOK1) impairs primary cilia formation in vitro. In vivo investigation of TAOK1 in Xenopus tropicalis reveals a role in motile cilia formation and heart development,supporting its prediction as a CHD gene. Together,our findings highlight a set of CHD genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology. View Publication

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in melty-CpG binding protein 2 (MeCP2). MeCP2 is a non-cell type-specific DNA binding protein,and its mutation influences not only neural cells but also non-neural cells in the brain,including vasculature associated with endothelial cells. Vascular integrity is crucial for maintaining brain homeostasis,and its alteration may be linked to the pathology of neurodegenerative disease,but a non-neurogenic effect,especially the relationship between vascular alternation and Rett syndrome pathogenesis,has not been shown. Here,we recapitulate a microvascular network using Rett syndrome patient-derived induced pluripotent stem (iPS) cells that carry MeCP2[R306C] mutation to investigate early developmental vascular impact. To expedite endothelial cell differentiation,doxycycline (DOX)-inducible ETV2 expression vectors were inserted into the AAVS1 locus of Rett syndrome patient-derived iPS cells and its isogenic control by CRISPR/Cas9. With these endothelial cells,we established a disease microvascular network (Rett-dMVNs) and observed higher permeability in the Rett-dMVNs compared to isogenic controls,indicating altered barrier function by MeCP2 mutation. Furthermore,we unveiled that hyperpermeability is involved in the upregulation of miR126–3p in Rett syndrome patient-derived endothelial cells by microRNA profiling and RNAseq,and rescue of miR126–3p level can recover their phenotype. We discover miR126–3p-mediated vascular impairment in Rett syndrome patients and suggest the potential application of these findings for translational medicine. View Publication

AbstractAimsDoxorubicin (DOX) is a widely used anthracycline anticancer agent; however,its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately,the pathophysiology of DICT has not yet been fully elucidated,and there are no effective strategies for its prevention or treatment. In this investigation,the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored.Methods and resultsWe observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites—D125,D136,and D215. Interestingly,overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis,effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ?-catenin. As a result,Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/?-catenin signalling,leading to DICT progression. Furthermore,the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However,these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally,in a DICT mouse model,TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation,ultimately protecting cardiomyocytes from cell death.ConclusionOur findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function,even in the context of DOX administration. Consequently,this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT. Graphical Abstract Graphical Abstract View Publication

AbstractDespite the success of combination antiretroviral therapy (ART) for individuals living with HIV,mild forms of HIV-associated neurocognitive disorder (HAND) continue to occur. Brain microglia form the principal target for HIV infection in the brain. It remains unknown how infection of these cells leads to neuroinflammation,neuronal dysfunction,and/or death observed in HAND. Utilizing two different inducible pluripotent stem cell-derived brain organoid models (cerebral and choroid plexus [ChP] organoids) containing microglia,we investigated the pathogenic changes associated with HIV infection. Infection of microglia was associated with a sharp increase in CCL2 and CXCL10 chemokine gene expression and the activation of many type I interferon stimulated genes (MX1,ISG15,ISG20,IFI27,IFITM3 and others). Production of the proinflammatory chemokines persisted at low levels after treatment of the cell cultures with ART,consistent with the persistence of mild HAND following clinical introduction of ART. Expression of multiple members of the S100 family of inflammatory genes sharply increased following HIV infection of microglia measured by single-cell RNA-seq. However,S100 gene expression was not limited to microglia but was also detected more broadly in uninfected stromal cells,mature and immature ChP cells,neural progenitor cells and importantly in bystander neurons suggesting propagation of the inflammatory response to bystander cells. Neurotransmitter transporter expression declined in uninfected neurons,accompanied by increased expression of genes promoting cellular senescence and cell death. Together,these studies underscore how an inflammatory response generated in HIV-infected microglia is propagated to multiple uninfected bystander cells ultimately resulting in the dysfunction and death of bystander neurons. View Publication

PurposeOxaliplatin-induced peripheral neuropathy (OIPN) is a chronic,debilitating late effect following oxaliplatin treatment. Neurofilament light chain (NfL) is a structural protein found in nerve axons that was investigated upon oxaliplatin exposure in vitro and in vivo correlated to symptoms of OIPN in colorectal cancer patients receiving oxaliplatin.MethodsHuman sensory neurons,derived from induced pluripotent stem cells,were exposed to clinically relevant concentrations of oxaliplatin in vitro,with NfL concentrations measured in the cell medium. The prospective clinical study included patients with colorectal cancer undergoing chemotherapy therapy with or without oxaliplatin. Possible OIPN was defined as bilateral presence of numbness and/or presence of pricking sensations in the feet documented in an interview at the time of blood sampling prior to,3,and 6 months after initiating treatment.ResultsOxaliplatin exposure led to a dose-dependent NfL increase in vitro. In the clinical cohort of 30 patients (18 in the oxaliplatin group),NfL levels rose at 3 and 6 months compared to controls. NfL level changes correlated to OIPN symptoms at the 6-month timepoint (rho 0.81,p? View Publication

Studies on MECP2 function and its implications in Rett Syndrome (RTT) have traditionally centered on neurons. Here,using human embryonic stem cell (hESC) lines,we modeled MECP2 loss-of-function to explore its effects on astrocyte (AST) development and dysfunction in the brain. Ultrastructural analysis of RTT hESC-derived cerebral organoids revealed significantly smaller mitochondria compared to controls (CTRs),particularly pronounced in glia versus neurons. Employing a multiomics approach,we observed increased gene expression and accessibility of a subset of nuclear-encoded mitochondrial genes upon mutation of MECP2 in ASTs compared to neurons. Analysis of hESC-derived ASTs showed reduced mitochondrial respiration and altered key proteins in the tricarboxylic acid cycle and electron transport chain in RTT versus CTRs. Additionally,RTT ASTs exhibited increased cytosolic amino acids under basal conditions,which were depleted upon increased energy demands. Notably,mitochondria isolated from RTT ASTs exhibited increased reactive oxygen species and influenced neuronal activity when transferred to cortical neurons. These findings underscore MECP2 mutation's differential impact on mitochondrial and metabolic pathways in ASTs versus neurons,suggesting that dysfunctional AST mitochondria may contribute to RTT pathophysiology by affecting neuronal health. View Publication

Cell signaling plays a critical role in neurodevelopment,regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing,scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq,an integrated approach that aims to quantify cytoplasmic and nuclear proteins,including those with post-translational modifications,and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells,alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines,induced pluripotent stem cells,and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories,shed light on gene regulatory relationships,and help explore the causes and effects of diverse cell signaling in neurodevelopment. Here,the authors demonstrate Phospho-seq,a single-cell multiomics method capable of quantifying chromatin accessibility alongside intracellular proteins,including post-translationally modified proteins. Then,they apply Phospho-seq to organoid models of neurodevelopment. View Publication

Citron Kinase (CITK) is a protein encoded by the CIT gene,whose pathogenic variants underlie microcephalic phenotypes that characterize MCPH17 syndrome. In neural progenitors,CITK loss leads to microtubule instability,resulting in mitotic spindle positioning defects,cytokinesis failure,and accumulation of DNA double strand breaks (DSBs),ultimately resulting in TP53-dependent senescence and apoptosis. Although DNA damage accumulation has been associated with impaired homologous recombination (HR),the role of CITK in this process and whether microtubule dynamics are involved is still unknown. In this report we show that CITK is required for proper BRCA1 localization at sites of DNA DSBs. We found that CITK’s scaffolding,rather than its catalytic activity,is necessary for maintaining BRCA1 interphase levels in progenitor cells during neurodevelopment. CITK regulates the nuclear levels of HDAC6,a modulator of both microtubule stability and DNA damage repair. Targeting HDAC6 in CITK-deficient cells increases microtubule stability and recovers BRCA1 localization defects and DNA damage levels to that detected in controls. In addition,the CIT-HDAC6 axis is functionally relevant in a MCPH17 zebrafish model,as HDAC6 targeting recovers the head size phenotype produced by interfering with the CIT orthologue gene. These data provide novel insights into the functional interplay between HR and microtubule dynamics and into the pathogenesis of CITK based MCPH17,which may be relevant for development of therapeutic strategies. View Publication

SummaryHuman cerebral organoids derived from induced pluripotent stem cells can recapture early developmental processes and reveal changes involving neurodevelopmental disorders. Mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome,and disease severity varies depending on the location and type of mutation. Here,we focused on neuronal activity in Rett syndrome patient-derived organoids,analyzing two types of MeCP2 mutations – a missense mutation (R306C) and a truncating mutation (V247X) - using calcium imaging with three-photon microscopy. Compared to isogenic controls,we found abnormal neuronal activity in Rett organoids and altered network function based on graph theoretic analyses,with V247X mutations impacting functional responses and connectivity more severely than R306C mutations. These changes paralleled EEG data obtained from patients with comparable mutations. Labeling DLX promoter-driven inhibitory neurons demonstrated differences in activity and functional connectivity of inhibitory and excitatory neurons in the two types of mutation. Transcriptomic analyses revealed HDAC2-associated impairment in R306C organoids and decreased GABAA receptor expression in excitatory neurons in V247X organoids. These findings demonstrate mutation-specific mechanisms of vulnerability in Rett syndrome and suggest targeted strategies for their treatment. View Publication

AbstractTGF-? signaling family plays an essential role to regulate fate decisions in pluripotency and lineage specification. How the action of TGF-? family signaling is intrinsically executed remains not fully elucidated. Here,we show that HBO1,a MYST histone acetyltransferase (HAT) is an essential cell intrinsic determinant for TGF-? signaling in human embryonic stem cells (hESCs). HBO1?/? hESCs fail to response to TGF-? signaling to maintain pluripotency and spontaneously differentiate into neuroectoderm. Moreover,HBO1 deficient hESCs show complete defect in mesendoderm specification in BMP4-triggered gastruloids or teratomas. Molecularly,HBO1 interacts with SMAD4 and co-binds the open chromatin labeled by H3K14ac and H3K4me3 in undifferentiated hESCs. Upon differentiation,HBO1/SMAD4 co-bind and maintain the mesoderm genes in BMP4-triggered mesoderm cells while lose chromatin occupancy in neural cells induced by dual-SMAD inhibition. Our data reveal an essential role of HBO1,a chromatin factor to determine the action of SMAD in both human pluripotency and mesendoderm specification. Graphical Abstract Graphical Abstract View Publication

To investigate intestinal health and its potential disruptors in vitro,representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However,the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently,the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge,IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model,while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately,comparison to in vivo transcriptomics data showed limited similarities,which could be explained by essential differences in the study setups. Altogether,hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-74802-w. View Publication