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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 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

AbstractSpinal 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. Graphical Abstract Graphical Abstract View Publication

?-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

The ability to robustly predict guide RNA (gRNA) activity is a long-standing goal for CRISPR applications,as it would reduce the need to pre-screen gRNAs. Quantification of formation of short insertions and deletions (indels) after DNA cleavage by transcribed gRNAs has been typically used to measure and predict gRNA activity. We evaluate the effect of chemically synthesized Cas9 gRNAs on different cellular DNA cleavage outcomes and find that the activity of different gRNAs is largely similar and often underestimated when only indels are scored. We provide a simple linear model that reliably predicts synthetic gRNA activity across cell lines,robustly identifies inefficient gRNAs across different published datasets,and is easily accessible via online genome browser tracks. In addition,we develop a homology-directed repair efficiency prediction tool and show that unintended large-scale repair events are common for Cas9 but not for Cas12a,which may be relevant for safety in gene therapy applications. Reliable prediction of guide RNA (gRNA) activity is key for efficient CRISPR gene editing. Here,the authors show that efficiency of gRNAs is often underestimated when only indels are scored and introduce tools for predicting activity of chemically synthesized gRNAs and HDR efficiency. View Publication

To accurately study human organ function and disease ‘in the dish’,it is necessary to develop reliable cell-based models that closely track human physiology. Our interest lay with the liver,which is the largest solid organ in the body. The liver is a multifunctional and highly regenerative organ; however,severe liver damage can have dire consequences for human health. A common cause of liver damage is adverse reactions to prescription drugs. Therefore,the development of predictive liver models that capture human drug metabolism patterns is required to optimise the drug development process. In our study,we aimed to identify compounds that could improve the metabolic function of stem cell-derived liver tissue. Therefore,we screened a compound library to identify additives that improved the maturity of in vitro-engineered human tissue,with the rationale that by taking such an approach,we would be able to fine-tune neonatal and adult cytochrome P450 metabolic function in stem cell-derived liver tissue. View Publication

During gastrulation,the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT),which is marked by cadherin switching,where E-Cadherin is downregulated,and N-Cadherin is upregulated. To understand the relationships between differentiation,EMT,and cadherin switching,we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols,as well as variants in which signaling through key pathways including Activin,BMP,and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus,cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently,E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall,cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation. View Publication

AbstractDespite being one of the most prevalent RNA modifications,the role of N6?methyladenosine (m6A) in amyotrophic lateral sclerosis (ALS) remains ambiguous. In this investigation,we explore the contribution of genetic defects of m6A?related genes to ALS pathogenesis. We scrutinized the mutation landscape of m6A genes through a comprehensive analysis of whole?exome sequencing cohorts,encompassing 508 ALS patients and 1660 population?matched controls. Our findings reveal a noteworthy enrichment of RNA binding motif protein X?linked (RBMX) variants among ALS patients,with a significant correlation between pathogenic m6A variants and adverse clinical outcomes. Furthermore,Rbmx knockdown in NSC?34 cells overexpressing mutant TDP43Q331K results in cell death mediated by an augmented p53 response. Similarly,RBMX knockdown in ALS motor neurons derived from induced pluripotent stem cells (iPSCs) manifests morphological defects and activation of the p53 pathway. Transcriptional analysis using publicly available single?cell sequencing data from the primary motor cortex indicates that RBMX?regulated genes selectively influence excitatory neurons and exhibit enrichment in ALS?implicated pathways. Through integrated analyses,our study underscores the emerging roles played by RBMX in ALS,suggesting a potential nexus between the disease and dysregulated m6A?mediated mRNA metabolism. The dysregulation of m6A modification has gained recognition as a crucial factor in the development of amyotrophic lateral sclerosis (ALS). Among the m6A reader proteins,RNA binding motif protein X?linked (RBMX) stands out with a notable enrichment of variants in ALS patients,and the presence of pathogenic RBMX variants is associated with a faster disease progression. In vitro experiments have provided evidence that reducing RBMX levels can result in neuronal defects. Additionally,bioinformatic analyses have supported these findings by revealing that RBMX?associated genes specifically impact excitatory neurons. Furthermore,these genes are involved in the regulation of pathways and genes associated with neurodegeneration and RNA metabolism,underscoring the relevance of RBMX in ALS pathogenesis. View Publication

BackgroundCertain structural variants (SVs) including large-scale genetic copy number variants,as well as copy number-neutral inversions and translocations may not all be resolved by chromosome karyotype studies. The identification of genetic risk factors for Parkinson’s disease (PD) has been primarily focused on the gene-disruptive single nucleotide variants. In contrast,larger SVs,which may significantly influence human phenotypes,have been largely underexplored. Optical genomic mapping (OGM) represents a novel approach that offers greater sensitivity and resolution for detecting SVs. In this study,we used induced pluripotent stem cell (iPSC) lines of patients with PD-linked SNCA and PRKN variants as a proof of concept to (i) show the detection of pathogenic SVs in PD with OGM and (ii) provide a comprehensive screening of genetic abnormalities in iPSCs.ResultsOGM detected SNCA gene triplication and duplication in patient-derived iPSC lines,which were not identified by long-read sequencing. Additionally,various exon deletions were confirmed by OGM in the PRKN gene of iPSCs,of which exon 3–5 and exon 2 deletions were unable to phase with conventional multiplex-ligation-dependent probe amplification. In terms of chromosomal abnormalities in iPSCs,no gene fusions,no aneuploidy but two balanced inter-chromosomal translocations were detected in one line that were absent in the parental fibroblasts and not identified by routine single nucleotide variant karyotyping.ConclusionsIn summary,OGM can detect pathogenic SVs in PD-linked genes as well as reveal genomic abnormalities for iPSCs that were not identified by other techniques,which is supportive for OGM’s future use in gene discovery and iPSC line screening.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-024-10902-1. View Publication