技术资料

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

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor screens have enabled efficient production of a few cell types; however,engineering cell types that require complex transcription factor combinations remains challenging. Here,we report an iterative,high-throughput single-cell transcription factor screening method that enables the identification of transcription factor combinations for specialized cell differentiation,which we validated by differentiating human microglia-like cells. We found that the expression of six transcription factors,SPI1,CEBPA,FLI1,MEF2C,CEBPB,and IRF8,is sufficient to differentiate human iPSC into cells with transcriptional and functional similarity to primary human microglia within 4 days. Through this screening method,we also describe a novel computational method allowing the exploration of single-cell RNA sequencing data derived from transcription factor perturbation assays to construct causal gene regulatory networks for future cell fate engineering. Liu et al. developed a platform to identify transcription factors (TFs) that turn stem cells into desired cell types. They discovered six key TFs that produce microglia efficiently,enhancing cell differentiation methods. View Publication

Spinal cord injury (SCI) elicits a hostile microenvironment characterized by inflammation,gliosis,and disrupted signaling pathways that collectively impede neural repair. Neural progenitor cells (NPCs) represent a promising regenerative approach,yet their survival and differentiation are often compromised in this setting. Here,we investigated whether engineering NPCs to overexpress the Notch pathway modulator Delta-like non-canonical Notch ligand 1 (DLK1) could overcome these limitations and improve functional outcomes after cervical SCI in rats. NPCs were engineered to express DLK1 under a Pax6 promoter-driven expression system,ensuring elevated DLK1 levels during the progenitor state. Following transplantation of DLK1-overexpressing NPCs or control NPCs,we assessed graft survival,lineage differentiation,behavioral performance,and electrophysiological integration over 12 weeks. DLK1-expressing NPCs exhibited significantly greater retention in the injured spinal cord and showed enhanced neuronal differentiation alongside reduced astrocytic commitment compared to controls. Behavioral tests—including forelimb grip strength and CatWalk gait assessments—demonstrated that DLK1-modified NPCs conferred robust improvements in forelimb motor coordination and overall locomotion. Concordantly,electrophysiological recordings revealed increased motor-evoked potential amplitudes and area-under-the-curve values in animals receiving DLK1-transduced NPC grafts,indicative of strengthened synaptic integration within the host motor circuitry. View Publication

β-propeller protein-associated neurodegeneration (BPAN) is a rare X-linked neurodegenerative disorder caused by mutations in the WDR45 gene,yet its molecular mechanisms remain poorly understood. Here,we identify a role for WDR45 in stress granule (SG) disassembly,mediated through its phase separation with Caprin-1. We demonstrate that WDR45 forms gel-like condensates via its WD5 domain,which competitively displaces G3BP1 from Caprin-1 to promote SG disassembly. BPAN-associated WDR45 mutations impair condensate formation and Caprin-1 interaction,leading to delayed SG disassembly,which correlates with earlier disease onset. WDR45 depletion also exacerbates amyotrophic lateral sclerosis-associated pathological SGs,highlighting its broader relevance to neurodegenerative diseases. Using iPSC-derived midbrain neurons from a BPAN patient,we demonstrate delayed SG recovery,directly linking WDR45 dysfunction to neurodegeneration. These findings establish WDR45 as a critical regulator of SG dynamics,uncover a potential molecular basis of BPAN pathogenesis,and identify therapeutic targets for neurodegenerative diseases associated with SG dysregulation. BPAN is a rare neurodegenerative disease caused by WDR45 mutations. Here,the authors discover that WDR45 can competitively displace G3BP1 from Caprin-1 to promote stress granule disassembly,a function that is disrupted by BPAN-associated WDR45 mutations. View Publication