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Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by sodium channel dysfunction. However,the clinical management of ARVC remains challenging. Identifying novel compounds for the treatment of ARVC is crucial for advancing drug development.PurposeIn this study,we aim to identify novel compounds for treating ARVC.MethodsMachine learning (ML) models were constructed using proteins analyzed from the scRNA-seq data of ARVC rats and their corresponding protein-protein interaction (PPI) network to predict binding affinity (BA). To validate these predictions,a series of experiments in cardiac organoids were conducted,including Western blotting,ELISA,MEA,and Masson staining to assess the effects of these compounds.ResultsWe first discovered and identified SCN5A as the most significantly affected sodium channel protein in ARVC. ML models predicted that Kaempferol binds to SCN5A with high affinity. In vitro experiments further confirmed that Kaempferol exerted therapeutic effects in ARVC.ConclusionThis study presents a novel approach for identifying potential compounds to treat ARVC. By integrating ML modeling with organoid validation,our platform provides valuable support in addressing the public health challenges posed by ARVC,with broad application prospects. Kaempferol shows promise as a lead compound for ARVC treatment. View Publication

Stargardt disease is an inherited retinopathy affecting approximately 1:8000 individuals. It is characterised by biallelic variants in ABCA4 which encodes a vital protein for the recycling of retinaldehydes in the retina. Despite its prevalence and impact,there are currently no treatments available for this condition. Furthermore,35% of STGD1 cases remain genetically unsolved. To investigate the cellular and molecular characteristics associated with STGD1,we generated iPSCs from two monoallelic unresolved (PT1 & PT2),late-onset STGD1 cases with the heterozygous complex allele - c.[5461-10?T?>?C;5603?A?>?T]. Both patient iPSCs and those from a biallelic affected control (AC) carrying -c.4892?T?>?C and c.4539+2001G?>?A,were differentiated to retinal organoids,which developed all key retinal neurons and photoreceptors with outer segments positive for ABCA4 expression. We observed patient-specific disruption to lamination with OPN1MW/LW+ cone photoreceptor retention in the retinal organoid centre during differentiation. Photoreceptor retention was more severe in the AC case affecting both cones and rods,suggesting a genotype/phenotype correlation. scRNA-Seq suggests retention may be due to the induction of stress-related pathways in photoreceptors. Whole genome sequencing successfully identified the missing alleles in both cases; PT1 reported c.-5603A?>?T in homozygous state and PT2 uncovered a rare hypomorph - c.-4685T?>?C. Furthermore,retinal organoids were able to recapitulate the retina-specific splicing defect in PT1 as shown by long-read RNA-seq data. Collectively,these results highlight the suitability of retinal organoids in STGD1 modelling. Their ability to display genotype-phenotype correlations enhances their utility as a platform for therapeutic development. View Publication

BackgroundSpinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study,we compared different isoforms of basic fibroblast growth factor 2 (FGF2),overexpressed in stably transfected Human embryonic stem cells (hESCs),following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB).MethodsIn the present work,hESCs bioengineered to overexpress 18,23,and 31 kD isoforms of FGF2,were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA,followed by Tukey’s or Dunnett’s multiple comparison tests. Significance was set at *p < 0.05,**p < 0.01,***p < 0.001,and ****p < 0.0001.ResultsFor the first set of experiments,rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival,glial reaction,and synaptic coverage,two weeks after the lesion,indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently,the second set of experiments was performed with that isoform,so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival,glial reaction,synaptic coverage,and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection,coupled with immunomodulation,attenuation of astrogliosis,and preservation of inputs to the rescued motoneurons. Behaviorally,the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery.ConclusionTransgenic hESCs were an effective delivery platform for neurotrophic factors,rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury,while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03676-6. View Publication

Cardiac hypertrophy is a cellular process characterized by the increased size of cardiomyocytes in response to a high workload or stress. 17-beta estradiol (E2) has cardioprotective and anti-hypertrophic effects by maintaining mitochondrial network and function. MUL1 is a mitochondrial ubiquitin ligase directly involved in the control of mitochondrial fission and mitophagy. Studies from our group and others have previously shown that cardiomyocyte hypertrophy is associated with mitochondrial fission and dysfunction. These findings led us to study in vitro whether E2 regulates MUL1 to prevent cardiac hypertrophy,mitochondrial fission,and dysfunction induced by the catecholamine norepinephrine (NE). Our results showed that NE induces hypertrophy in cultured rat cardiomyocytes. Pre-treatment with E2 (10-100?nM) prevented the NE-dependent increases in cell perimeter and the hypertrophic stress markers ANP and BNP at both the protein and mRNA levels. NE induced the fragmentation of the mitochondrial network and reduced ATP levels,effects that were both prevented by E2. In silico analysis suggested a putative binding site for estrogen receptors on the MUL1 gene promoter. In accordance with this finding,E2 prevented increases in MUL1 mRNA and protein levels induced by NE. Our data also showed that a siRNA MUL1 knockdown counteracted NE-induced cardiomyocyte hypertrophy and mitochondrial dysfunction,mirroring the protective effect triggered by E2. In contrast,a MUL1 adenovirus did not prevent the E2 protection from cardiomyocyte hypertrophy. Further,in vivo analysis in a transgenic mouse model overexpressing MUL1 revealed that only young male mice overexpressed the protein. Consequently,they exhibited increased levels of the hypertrophic marker ANP,an elevated heart weight,and larger cardiomyocyte size. Therefore,our data demonstrate that 17-beta estradiol prevents cardiac myocyte hypertrophy by regulating MUL1. View Publication

Targeted genome editing of human pluripotent stem cells (hPSCs) is critical for basic and translational research and can be achieved with site-specific endonucleases. Cpf1 (CRISPR from Prevotella and Francisella) is a programmable DNA endonuclease with AT-rich PAM sequences. In this protocol,we describe procedures for using a single vector system to deliver Cpf1 and CRISPR RNA (crRNA) for genome editing in hPSCs. This protocol enables indel formation and homologous recombination-mediated precise editing at multiple loci. With the delivery of Cpf1 and a single U6 promoter-driven guide RNA array composed of an AAVS1-targeting and a MAFB-targeting crRNA array,efficient multiplex genome editing at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs could be achieved in a single experiment. The edited hPSCs expressed pluripotency markers and could differentiate into neurons in vitro. This system also generated INS reporter hPSCs with a 6 kb cassette knockin at the INS locus. The INS reporter cells can differentiate into β-cells that express tdTomato and luciferase,permitting fluorescence-activated cell sorting of hPSC-β-cells. By targeted screening of potential off-target sequences that are most homologous to crRNA sequences,no off-target mutations were detected in any of the tested sequences. This work provides an efficient and flexible system for precise genome editing in mammalian cells including hPSCs with the benefits of less off-target effects. Key features • A single-vector system to deliver Cpf1 and crRNA enables the sorting of transfected cells • Efficient and simultaneous multi-modular genome editing exemplified by mutation of MAFB and knockin of AAVS1 loci in a single experiment • Edited PSCs showed minimal off-target effects and can be differentiated into multiple cell types. View Publication

BackgroundHuman pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) show tremendous promise for cardiac regeneration following myocardial infarction (MI),but their transplantation gives rise to transient ventricular tachycardia (VT) in large-animal MI models,representing a major hurdle to translation. Our group previously reported that these arrhythmias arise from a focal mechanism whereby graft tissue functions as an ectopic pacemaker; therefore,we hypothesized that hPSC-CMs engineered with a dominant negative form of the pacemaker ion channel HCN4 (dnHCN4) would exhibit reduced automaticity and arrhythmogenic risk following transplantation.MethodsWe used CRISPR/Cas9-mediated gene-editing to create transgenic dnHCN4 hPSC-CMs,and their electrophysiological behavior was evaluated in vitro by patch-clamp recordings and optical mapping. Next,we transplanted WT and homozygous dnHCN4 hPSC-CMs in a pig MI model and compared post-transplantation outcomes including the incidence of spontaneous arrhythmias and graft structure by immunohistochemistry.ResultsIn vitro dnHCN4 hPSC-CMs exhibited significantly reduced automaticity and pacemaker funny current (If) density relative to wildtype (WT) cardiomyocytes. Following transplantation with either dnHCN4 or WT hPSC-CMs,all recipient hearts showed transmural infarct scar that was partially remuscularized by scattered islands of human myocardium. However,in contrast to our hypothesis,both dnHCN4 and WT hPSC-CM recipients exhibited frequent episodes of ventricular tachycardia (VT).ConclusionsWhile genetic silencing of the pacemaker ion channel HCN4 suppresses the automaticity of hPSC-CMs in vitro,this intervention is insufficient to reduce VT risk post-transplantation in the pig MI model,implying more complex mechanism(s) are operational in vivo. View Publication

Efficient tumor T-cell infiltration is crucial for the effectiveness of T-cell-based therapies against solid tumors. Eosinophils play crucial roles in recruiting T cells in solid tumors. Our group has previously generated induced eosinophils (iEOs) from human pluripotent stem cells and exhibited synergistic efficacy with CAR-T cells in solid tumor inhibition. However,administrated eosinophils might influx into inflammatory lungs,posing a potential safety risk. Mitigating the safety concern and enhancing efficacy is a promising development direction for further application of eosinophils.MethodsWe developed a new approach to generate eosinophils with enhanced potency from human chemically reprogrammed induced pluripotent stem cells (hCiPSCs) with the Toll-like receptor (TLR) 7/8 signaling agonist R848.ResultsR848-activated iEOs (R-iEOs) showed significantly decreased influx to the inflamed lungs,indicating a lower risk of causing airway disorders. Furthermore,these R-iEOs had enhanced anti-tumor functions,preferably accumulated at tumor sites,and further increased T-cell infiltration. The combination of R-iEOs and CAR-T cells suppressed tumor growth in mice. Moreover,the chemo-trafficking signaling increased in R-iEOs,which may contribute to the decreased lung influx of R-iEOs and the increased tumor recruitment of T cells.ConclusionOur study provides a novel approach to alleviate the potential safety concerns associated with eosinophils while increasing T-cell infiltration in solid tumors. This finding offers a prospective strategy for incorporating eosinophils to improve CAR-T-cell immunotherapy for solid tumors in the future. View Publication

Tribbles pseudokinase 3 (TRIB3) expression significantly increases during terminal erythropoiesis in vivo. However,we found that TRIB3 expression remained relatively low during human embryonic stem cell (hESC) erythropoiesis,particularly in the late stage,where it is typically active. TRIB3 was expressed in megakaryocyte-erythrocyte progenitor cells and its low expression was necessary for megakaryocyte differentiation. Thus,we proposed that the high expression during late stage of erythropoiesis could be the clue for promotion of maturation of hESC-derived erythroid cells. To our knowledge,the role of TRIB3 in the late stage of erythropoiesis remains ambiguous. To address this,we generated inducible TRIB3 overexpression hESCs,named TRIB3tet-on OE H9,based on a Tet-On system. Then,we analyzed hemoglobin expression,condensed chromosomes,organelle clearance,and enucleation with or without doxycycline treatment. TRIB3tet-on OE H9 cells generated erythrocytes with a high proportion of orthochromatic erythroblast in flow cytometry,enhanced hemoglobin and related protein expression in Western blot,decreased nuclear area size,promoted enucleation rate,decreased lysosome and mitochondria number,more colocalization of LC3 with LAMP1 (lysosome marker) and TOM20 (mitochondria marker) and up-regulated mitophagy-related protein expression after treatment with 2 ?g/mL doxycycline. Our results showed that TRIB3 overexpression during terminal erythropoiesis may promote the maturation of erythroid cells. Therefore,our study delineates the role of TRIB3 in terminal erythropoiesis,and reveals TRIB3 as a key regulator of UPS and downstream mitophagy by ensuring appropriate mitochondrial clearance during the compaction of chromatin. Highlights•TRIB3 boosts erythroid cell maturation.•Key insights into erythropoiesis from hESCs.•Enhanced ubiquitin-proteasome system and downstream mitophagy in erythroid differentiation. View Publication

Angelman syndrome (AS) and Prader-Willi syndrome (PWS),two distinct neurodevelopmental disorders,result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele,respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless,gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences,reprogramming methods,and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here,we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally,these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS. View Publication

Concentrations of RNAs and proteins provide important determinants of cell fate. Robust gene circuit design requires an understanding of how the combined actions of individual genetic components influence both messenger RNA (mRNA) and protein levels. Here,we simultaneously measure mRNA and protein levels in single cells using hybridization chain reaction Flow-FISH (HCR Flow-FISH) for a set of commonly used synthetic promoters. We find that promoters generate differences in both the mRNA abundance and the effective translation rate of these transcripts. Stronger promoters not only transcribe more RNA but also show higher effective translation rates. While the strength of the promoter is largely preserved upon genome integration with identical elements,the choice of polyadenylation signal and coding sequence can generate large differences in the profiles of the mRNAs and proteins. We used long-read direct RNA sequencing to define the transcription start and splice sites of common synthetic promoters and independently vary the defined promoter and 5? UTR sequences in HCR Flow-FISH. Together,our high-resolution profiling of transgenic mRNAs and proteins offers insight into the impact of common synthetic genetic components on transcriptional and translational mechanisms. By developing a novel framework for quantifying expression profiles of transgenes,we have established a system for building more robust transgenic systems. View Publication

Current culture systems available for studying hepatitis D virus (HDV) are suboptimal. In this study,we demonstrate that hepatocyte-like cells (HLCs) derived from human pluripotent stem cells (hPSCs) are fully permissive to HDV infection across various tested genotypes. When co-infected with the helper hepatitis B virus (HBV) or transduced to express the HBV envelope protein HBsAg,HLCs effectively release infectious progeny virions. We also show that HBsAg-expressing HLCs support the extracellular spread of HDV,thus providing a valuable platform for testing available anti-HDV regimens. By challenging the cells along the differentiation with HDV infection,we have identified CD63 as a potential HDV co-entry factor that was rate-limiting for HDV infection in immature hepatocytes. Given their renewable source and the potential to derive hPSCs from individual patients,we propose HLCs as a promising model for investigating HDV biology. Our findings offer new insights into HDV infection and expand the repertoire of research tools available for the development of therapeutic interventions. View Publication

Understanding the multicellular organization of stem cells is vital for determining the mechanisms that coordinate cell fate decision-making during differentiation; these mechanisms range from neighbor-to-neighbor communication to tissue-level biochemical gradients. Current methods for quantifying multicellular patterning tend to capture the spatial properties of cell colonies at a fixed scale and typically rely on human annotation. We present a computational pipeline that utilizes topological data analysis to generate quantitative,multiscale descriptors which capture the shape of data extracted from 2D multichannel microscopy images. By applying our pipeline to certain stem cell colonies,we detected subtle differences in patterning that reflect distinct spatial organization associated with loss of pluripotency. These results yield insight into putative directed cellular organization and morphogen-mediated,neighbor-to-neighbor signaling. Because of its broad applicability to immunofluorescence microscopy images,our pipeline is well-positioned to serve as a general-purpose tool for the quantitative study of multicellular pattern formation. View Publication