技术资料

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

BackgroundPhotoreceptor death leads to inherited blinding retinal diseases,such as retinitis pigmentosa (RP). As disease progression often outpaces therapeutic advances,developing effective treatments is urgent. This study evaluates the efficacy of small peptides derived from pigment epithelium-derived factor (PEDF),which are known to restrict common cell death pathways associated with retinal diseases.MethodsWe tested chemically synthesized peptides (17-mer and H105A) with affinity for the PEDF receptor,PEDF-R,delivered as eye drops to two RP mouse models: rd10 (phosphodiesterase 6b mutation) and RhoP23H/+ (rhodopsin P23H mutation). Additionally,we engineered AAV-H105A vectors for intravitreal delivery in RhoP23H/+ mice. To assess peptide effects in human tissue,we used retinal organoids exposed to cigarette smoke extract,a model of oxidative stress. Photoreceptor survival,morphology and function were evaluated.ResultsHere we show that peptides 17-mer and H105A delivered via eye drops successfully reach the retina,promote photoreceptor survival,and improve retinal function in both RP mouse models. Intravitreal delivery of a AAV-H105A vector delays photoreceptor degeneration in RhoP23H/+ mice up to six months. In human retinal organoids,peptide H105A specifically prevents photoreceptor death induced by oxidative stress,a contributing factor to RP progression.ConclusionsPEDF peptide-based eye drops offer a promising,minimally invasive therapy to prevent photoreceptor degeneration in retinal disorders,with a favorable safety profile. Plain language summaryRetinitis pigmentosa (RP) is a rare inherited condition that causes the gradual death of photoreceptors (light-sensing cells) in the eye,leading to vision loss. There is currently no cure. This study tested a potential treatment using small protein fragments (peptides) from PEDF,a protective protein naturally found in the eye. Researchers delivered these peptides through eye drops or gene therapy in mouse models of RP and to human retinal organoids (lab-grown retina tissue). Mice treated early maintained healthy vision cells,while untreated mice experienced rapid cell loss and vision decline. These results suggest that peptide-based eye drops could be a simple,safe,and effective way to slow vision loss in patients with RP. Bernardo-Colón et al. evaluate small peptides derived from the neurotrophic region of pigment epithelium-derived factor (PEDF) as potential therapeutics for retinitis pigmentosa using mouse models and human retinal organoids. A significant delay in photoreceptor death with eye drop or gene therapy delivery is seen. View Publication

SummaryHuman pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here,we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion,purification,banking,and quality assessment.For complete details on the use and execution of this protocol,please refer to Wells et al.1 Graphical abstract Highlights•Brief pulse of Ngn2 induces neural progenitor cells from human stem cells•Guidance on expanding,freezing,and thawing SNaP cells for future use•Immunostaining-based assays assess cell identity and differentiation potential Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here,we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells—termed stem cell-derived Ngn2-accelerated progenitor cells—followed by steps for expansion,purification,banking,and quality assessment. 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

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by profound neuronal and cognitive decline,with increasing evidence implicating astrocyte dysfunction in disease pathology. While traditional therapeutic approaches have primarily targeted neurons,the crucial role of astrocytes in metabolism,neurotransmission,amyloid-beta clearance,and neuroinflammation underscores their potential as therapeutic targets. In this study,we employed a multiomic integrative analysis combining transcriptomic and kinomic profiling of human induced pluripotent stem cell (hiPSC)-derived astrocytes from patients with familial AD (fAD) compared to healthy controls (HCs). Our transcriptomic analysis identified 1249 significantly differentially expressed genes,highlighting a pronounced upregulation of inflammatory genes (SERPINA3,IL6R,IL1RAP,TNFRSF11A) and a concomitant downregulation of genes essential for synaptic support and ion channel function (STMN2,NMNAT2,SCN2A,GRIN1). Kinomic profiling revealed dysregulated kinase activities within DYRK,GSK,and MAPK families,further implicating altered kinase signaling pathways in astrocyte dysfunction. Integration of these datasets pinpointed critical molecular hubs,notably within the PI3K signaling and inflammatory pathways,highlighting targets such as JAK2,STAT3,and AKT1 as potential modulators of disease progression. Furthermore,leveraging the Library of Integrated Network-Based Cellular Signatures (LINCS) platform,we identified chemical perturbagens,including fluticasone propionate and Akt inhibitors,capable of reversing the transcriptomic signatures associated with fAD astrocytes. This integrative multiomic approach not only enhances our understanding of astrocyte-specific molecular mechanisms in AD but also provides novel targets for therapeutic intervention aimed at mitigating astrocyte-driven neurodegeneration. Highlights•Familial AD astrocytes display significant pro-inflammatory transcriptomic and kinomic dysregulation.•PI3K and inflammatory signaling pathways are highly dysregulated in familial AD astrocytes.•Expression of inflammatory markers such as SERPINA3,IL6R,and TNFRSF11A is increased in familial AD astrocytes.•Kinase activity analysis identifies DYRK,GSK,and MAPK pathways as key dysregulated axes in familial AD astrocytes.•Potential astrocyte-specific therapeutic approaches to AD include targeting PI3K,JAK,and STAT3. 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