技术资料

In this study,Chen et al. describe two independent mechanisms that control ?-catenin levels in neuroglial cells and drive their proliferation. The work provides mechanistic insight into the impact of MEK activation resulting from the biallelic loss of NF1 or BRAF rearrangement in pediatric gliomas. The two major genomic alterations in pediatric pilocytic astrocytoma (PA) are NF1 loss and KIAA1549:BRAF rearrangement. Although these molecular changes result in increased MEK activity and tumor growth,it is not clear exactly how MEK controls human neuroglial cell proliferation. Leveraging human-induced pluripotent stem cells harboring these PA-associated alterations,we used a combination of genetic and pharmacological approaches to demonstrate that MEK-regulated cell growth is mediated by ?-catenin through independent mechanisms involving IRX2 control of CTNNB1 transcription and NPTX1 stabilization of ?-catenin protein levels. These results provide new mechanistic insights into MEK regulation of human brain cell function. 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

BackgroundOsteoarthritis (OA) is a complex,age-related multifactorial degenerative disease of diarthrodial joints marked by impaired mobility,joint stiffness,pain,and a significant decrease in quality of life. Among other risk factors,such as genetics and age,hyper-physiological mechanical cues are known to play a critical role in the onset and progression of the disease (Guilak in Best Pract Res Clin Rheumatol 25:815–823,2011). It has been shown that post-mitotic cells,such as articular chondrocytes,heavily rely on methylation at CpG sites to adapt to environmental cues and maintain phenotypic plasticity. However,these long-lasting adaptations may eventually have a negative impact on cellular performance. We hypothesize that hyper-physiologic mechanical loading leads to the accumulation of altered epigenetic markers in articular chondrocytes,resulting in a loss of the tightly regulated balance of gene expression that leads to a dysregulated state characteristic of the OA disease state.ResultsWe showed that hyper-physiological loading evokes consistent changes in CpGs associated with expression changes (ML-tCpGs) in ITGA5,CAV1,and CD44,among other genes,which together act in pathways such as anatomical structure morphogenesis (GO:0009653) and response to wound healing (GO:0042060). Moreover,by comparing the ML-tCpGs and their associated pathways to tCpGs in OA pathophysiology (OA-tCpGs),we observed a modest but particular interconnected overlap with notable genes such as CD44 and ITGA5. These genes could indeed represent lasting detrimental changes to the phenotypic state of chondrocytes due to mechanical perturbations that occurred earlier in life. The latter is further suggested by the association between methylation levels of ML-tCpGs mapped to CD44 and OA severity.ConclusionOur findings confirm that hyper-physiological mechanical cues evoke changes to the methylome-wide landscape of chondrocytes,concomitant with detrimental changes in positional gene expression levels (ML-tCpGs). Since CAV1,ITGA5,and CD44 are subject to such changes and are central and overlapping with OA-tCpGs of primary chondrocytes,we propose that accumulation of hyper-physiological mechanical cues can evoke long-lasting,detrimental changes in set points of gene expression that influence the phenotypic healthy state of chondrocytes. Future studies are necessary to confirm this hypothesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13148-024-01676-0. 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

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

The formation of mammalian synapses entails the precise alignment of presynaptic release sites with postsynaptic receptors but how nascent cell–cell contacts translate into assembly of presynaptic specializations remains unclear. Guided by pioneering work in invertebrates,we hypothesized that in mammalian synapses,liprin-? proteins directly link trans-synaptic initial contacts to downstream steps. Here we show that,in human neurons lacking all four liprin-? isoforms,nascent synaptic contacts are formed but recruitment of active zone components and accumulation of synaptic vesicles is blocked,resulting in ‘empty’ boutons and loss of synaptic transmission. Interactions with presynaptic cell adhesion molecules of either the LAR-RPTP family or neurexins via CASK are required to localize liprin-? to nascent synaptic sites. Liprin-? subsequently recruits presynaptic components via a direct interaction with ELKS proteins. Thus,assembly of human presynaptic terminals is governed by a hierarchical sequence of events in which the recruitment of liprin-? proteins by presynaptic cell adhesion molecules is a critical initial step. This paper identifies the evolutionarily conserved liprin-? protein family as key mediators of presynaptic assembly in human neurons. Their recruitment to sites formed by contacting neurons is the critical initial step that triggers presynaptic differentiation. 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

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