技术资料

SummaryCryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However,tissue processing is time-consuming and costly,limiting routine and diagnostic use. Currently,no commercially available protocols or products exist for multiplexing this process. Here,we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing—cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore,we process up to ?110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor,time,and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings,organism-wide antigen and antibody targeting studies,high-throughput tissue screens,and defined tissue section positioning for,e.g.,spatial transcriptomics experiments. Graphical abstract Highlights•Multiplexed tissue molds (MTMs) drastically upscale cryosectioning procedures•MTMs can simultaneously accommodate up to 19 mouse organs and ?110 cerebral organoids•MTMs reduce analysis costs and processing times of tissues by up to 96%•MTMs could be used to reduce diagnostic costs and for spatial transcriptomics MotivationEfficient cryosectioning remains a critical yet labor- and cost-intensive step for immunohistochemistry and in situ hybridization,limiting routine diagnostic and research applications. The increasing demand for high-throughput tissue analysis—driven by advances in organoid and three-dimensional (3D) culture systems and tissue analysis for diagnostics—necessitates methods capable of processing numerous heterogeneous samples simultaneously. Current protocols lack multiplexing capabilities,leading to variability and extended processing times. Our work introduces multiplexed tissue molds (MTMs),a scalable solution that drastically reduces costs and labor by up to 96% while maintaining tissue integrity and consistency,thereby enabling large-scale (>100 tissues) comparative analyses and enhanced experimental reproducibility as well as access to tissue analysis,where cost is a restrictive factor. Reumann et al. develop multiplexed tissue molds (MTMs),which allow upscaling of tissue processing (up to 19 mouse organs or ?110 cerebral organoids simultaneously) while reducing workload and associated analysis costs by up to 96%. MTMs allow cryosection-based tissue analysis when labor,time,and cost are limiting factors and could be used for patient sample analysis as well as spatial transcriptomics approaches. View Publication

Aging is one of the most prominent risk factors for neurodegeneration,yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging,we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons,which retained their aging hallmarks,and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins,especially spliceosome components. Intriguingly,splicing proteins—like the dementia- and ALS-associated protein TDP-43—mislocalize to the cytoplasm in aged neurons,which leads to widespread alternative splicing. Cytoplasmic spliceosome components are typically recruited to stress granules,but aged neurons suffer from chronic cellular stress that prevents this sequestration. We link chronic stress to the malfunctioning ubiquitylation machinery,poor HSP90? chaperone activity and the failure to respond to new stress events. Together,our data demonstrate that aging-linked deterioration of RNA biology is a key driver of poor resiliency in aged neurons. Rhine et al. find that neuronal aging leads to widespread dysregulation of RNA biology,including mislocalization of splicing proteins like TDP-43,chronic cellular stress and reduced resiliency. View Publication

SummaryDysfunction of the sympathetic nervous system and increased epicardial adipose tissue (EAT) have been independently associated with the occurrence of cardiac arrhythmia. However,their exact roles in triggering arrhythmia remain elusive. Here,using an in vitro coculture system with sympathetic neurons,cardiomyocytes,and adipocytes,we show that adipocyte-derived leptin activates sympathetic neurons and increases the release of neuropeptide Y (NPY),which in turn triggers arrhythmia in cardiomyocytes by interacting with the Y1 receptor (Y1R) and subsequently enhancing the activity of the Na+/Ca2+ exchanger (NCX) and calcium/calmodulin-dependent protein kinase II (CaMKII). The arrhythmic phenotype can be partially blocked by a leptin neutralizing antibody or an inhibitor of Y1R,NCX,or CaMKII. Moreover,increased EAT thickness and leptin/NPY blood levels are detected in atrial fibrillation patients compared with the control group. Our study provides robust evidence that the adipose-neural axis contributes to arrhythmogenesis and represents a potential target for treating arrhythmia. Graphical abstract Highlights•Stem cell-based coculture model can simulate the pathogenesis of cardiac arrhythmia•The adipose-neural axis plays critical roles in cardiac arrhythmias•Leptin,NPY/Y1R,NCX,and CaMKII are potential intervention targets for arrhythmia•Increased EAT thickness and leptin/NPY levels are detected in CS blood of AF patients Fan et al. establish a stem cell-based coculture model to mimic the in vivo cardiac microenvironment and elucidate that the adipose-neural interaction plays a critical role in epicardial adipose tissue-related cardiac arrhythmia through leptin-NPY axis. Their results may provide potential therapeutic targets for treating arrhythmia. View Publication

SUMMARY Elevated interferon (IFN) signaling is associated with kidney diseases including COVID-19,HIV,and apolipoprotein-L1 (APOL1) nephropathy,but whether IFNs directly contribute to nephrotoxicity remains unclear. Using human kidney organoids,primary endothelial cells,and patient samples,we demonstrate that IFN-? induces pyroptotic angiopathy in combination with APOL1 expression. Single-cell RNA sequencing,immunoblotting,and quantitative fluorescence-based assays reveal that IFN-?-mediated expression of APOL1 is accompanied by pyroptotic endothelial network degradation in organoids. Pharmacological blockade of IFN-? signaling inhibits APOL1 expression,prevents upregulation of pyroptosis-associated genes,and rescues vascular networks. Multiomic analyses in patients with COVID-19,proteinuric kidney disease,and collapsing glomerulopathy similarly demonstrate increased IFN signaling and pyroptosis-associated gene expression correlating with accelerated renal disease progression. Our results reveal that IFN-? signaling simultaneously induces endothelial injury and primes renal cells for pyroptosis,suggesting a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. In brief Juliar et al. address interferon signaling in kidney disease. Organoids,primary cells,and clinical datasets reveal that interferon signaling simultaneously induces APOL1 expression and endothelial cell pyroptosis. This suggests a combinatorial mechanism for APOL1-mediated collapsing glomerulopathy,which can be targeted therapeutically. The findings may also be relevant in other organs. Graphical Abstract View Publication

Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations,small insertions,and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA),which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs,especially when the genetic context precludes the screening of multiple pegRNAs,and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system,we generated other isogenic models of inherited retinal diseases (IRDs),including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure,such as plasmid concentrations,PE component ratios,and delivery method settings,showing that our improved workflow increased the hiPSC editing efficiency. View Publication

Doublecortin is a neuronal microtubule-associated protein that regulates microtubule structure in neurons. Mutations in Doublecortin cause lissencephaly and subcortical band heterotopia by impairing neuronal migration. We use CRISPR/Cas9 to knock-out the Doublecortin gene in induced pluripotent stem cells and differentiate the cells into cortical neurons. DCX-KO neurons show reduced velocities of nuclear movements and an increased number of neurites early in neuronal development,consistent with previous findings. Neurite branching is regulated by a host of microtubule-associated proteins,as well as by microtubule polymerization dynamics. However,EB comet dynamics are unchanged in DCX-KO neurons. Rather,we observe a significant reduction in ?-tubulin polyglutamylation in DCX-KO neurons. Polyglutamylation levels and neuronal branching are rescued by expression of Doublecortin or of TTLL11,an ?-tubulin glutamylase. Using U2OS cells as an orthogonal model system,we show that DCX and TTLL11 act synergistically to promote polyglutamylation. We propose that Doublecortin acts as a positive regulator of ?-tubulin polyglutamylation and restricts neurite branching. Our results indicate an unexpected role for Doublecortin in the homeostasis of the tubulin code. Lissencephaly is a severe neurodevelopmental disease often caused by mutations in the Dcx gene. Using a human cellular model of lissencephaly,the authors report that DCX restricts neuronal branching by activating tubulin polyglutamylation. View Publication

SummaryVascular complications caused by diabetes mellitus contribute a major threat to increased disability and mortality of diabetic patients,which are characterized by damaged endothelial cells and angiogenesis. Human umbilical cord-derived mesenchymal stem cells (hucMSCs) have been demonstrated to alleviate endothelial cell damage and improve angiogenesis. However,these investigations overlooked the pivotal role of vasculogenesis. In this study,we utilized blood vessel organoids (BVOs) to investigate the impact of high glucose on vasculogenesis and subsequent angiogenesis. We found that BVOs in the vascular lineage induction stage were more sensitive to high glucose and more susceptible to affect endothelial cell differentiation and function. Moreover,hucMSCs can alleviate the high glucose-induced inhibition of endothelial cell differentiation and dysfunction through MAPK signaling pathway downregulation,with the MAPK activator dimethyl fumarate further illustrating the results. Thereby,we demonstrated that high glucose can lead to abnormal vasculogenesis and impact subsequent angiogenesis,and hucMSCs can alleviate this effect. Graphical abstract Highlights•The induction process of BVOs can be divided into vasculogenesis and angiogenesis•The formation of VI-BVOs is more vulnerable to damage from high glucose than MI-BVOs•HucMSCs can improve vasculogenesis through the MAPK signaling pathway Pathophysiology; Stem cells research; Vascular remodeling View Publication

SUMMARY Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis,how ALK contributes to tumor formation remains unclear. Here,we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic,while ALK/MYCN tumors resembled mesenchymal,neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling,particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN,but not FN1,delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore,loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally,inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus,ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling. In brief Huang et al. used a human stem cell model to elucidate the mechanism for cooperation between MYCN and ALK. ALK contributes to tumor growth by upregulating the extracellular matrix protein periostin and activating WNT signaling. Periostin and WNT signal through a feedforward loop. Graphical Abstract View Publication

The Y-linked SRY gene initiates mammalian testis-determination. However,how the expression of SRY is regulated remains elusive. Here,we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5’ regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination,including a large multigenerational family,identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing,in a novel in vitro cellular model recapitulating human Sertoli cell formation,resulted in a significant reduction in expression of SRY. Therefore,human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression,a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare,the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process. Disease-causing variants define a conserved and unique NR5A1 responsive enhancer for SRY expression to initiate testis-determination in humans. Modelling regulatory variants causing sex-reversal provides a tool to understand global enhancer activity. View Publication

The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases,their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients,enabling the study of disease-associated mutations and,when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately,the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study,we optimized our previously published protocol by fine-tuning the initial cell number,exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple,cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB,HNF4?,and CYP3A4) and protein (ALB) expression,as well as significantly elevated inducible CYP3A4 activity. Graphical Abstract View Publication

Dominant optic atrophy (DOA) is the most commonly inherited optic neuropathy. The majority of DOA is caused by mutations in the OPA1 gene,which encodes a dynamin-related GTPase located to the mitochondrion. OPA1 has been shown to regulate mitochondrial dynamics and promote fusion. Within the mitochondrion,proteolytically processed OPA1 proteins form complexes to maintain membrane integrity and the respiratory chain complexity. Although OPA1 is broadly expressed,human OPA1 mutations predominantly affect retinal ganglion cells (RGCs) that are responsible for transmitting visual information from the retina to the brain. Due to the scarcity of human RGCs,DOA has not been studied in depth using the disease affected neurons. To enable studies of DOA using stem-cell-derived human RGCs,we performed CRISPR-Cas9 gene editing to generate OPA1 mutant pluripotent stem cell (PSC) lines with corresponding isogenic controls. CRISPR-Cas9 gene editing yielded both OPA1 homozygous and heterozygous mutant ESC lines from a parental control ESC line. In addition,CRISPR-mediated homology-directed repair (HDR) successfully corrected the OPA1 mutation in a DOA patient’s iPSCs. In comparison to the isogenic controls,the heterozygous mutant PSCs expressed the same OPA1 protein isoforms but at reduced levels; whereas the homozygous mutant PSCs showed a loss of OPA1 protein and altered mitochondrial morphology. Furthermore,OPA1 mutant PSCs exhibited reduced rates of oxygen consumption and ATP production associated with mitochondria. These isogenic PSC lines will be valuable tools for establishing OPA1-DOA disease models in vitro and developing treatments for mitochondrial deficiency associated neurodegeneration. View Publication

Brain organoids offer unprecedented insights into brain development and disease modeling and hold promise for drug screening. Significant hindrances,however,are morphological and cellular heterogeneity,inter-organoid size differences,cellular stress,and poor reproducibility. Here,we describe a method that reproducibly generates thousands of organoids across multiple hiPSC lines. These High Quantity brain organoids (Hi-Q brain organoids) exhibit reproducible cytoarchitecture,cell diversity,and functionality,are free from ectopically active cellular stress pathways,and allow cryopreservation and re-culturing. Patient-derived Hi-Q brain organoids recapitulate distinct forms of developmental defects: primary microcephaly due to a mutation in CDK5RAP2 and progeria-associated defects of Cockayne syndrome. Hi-Q brain organoids displayed a reproducible invasion pattern for a given patient-derived glioma cell line. This enabled a medium-throughput drug screen to identify Selumetinib and Fulvestrant,as inhibitors of glioma invasion in vivo. Thus,the Hi-Q approach can easily be adapted to reliably harness brain organoids’ application for personalized neurogenetic disease modeling and drug discovery. Human brain organoids are plagued by heterogeneity and poor reproducibility,critical parameters for reliable disease modeling and drug testing. Here,the authors report on Hi-Q organoids which solve these limitations and can be cryopreserved in large quantities. View Publication