技术资料

SummaryMutations in the DMD gene lead to Duchenne muscular dystrophy (DMD),a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2–4 years of age,but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients,which poses a serious challenge in current standards of care. Here,we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether,this work demonstrates that in vitro,dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development,which should be considered in future therapeutic strategies for DMD. Graphical abstract Highlights•Myogenic differentiation of DMD hiPSCs diverges at the somite stage•Cell junction formation is dysregulated in DMD somite cells•Somite cells from DMD hiPSCs have impaired epithelialization properties•Migration velocity of DMD-mutant somite progenitors is upregulated Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Developmental biology. View Publication

An intronic G4C2 repeat expansion in the C9ORF72 gene is the major known cause for Amyotrophic Lateral Sclerosis (ALS),with current evidence for both,loss of function and pathological gain of function disease mechanisms. We screened 96 200 small molecules in C9ORF72 patient iPS neurons for modulation of nuclear G4C2 RNA foci and identified 82 validated hits,including the Brd4 inhibitor JQ1 as well as novel analogs of Spliceostatin-A,a known modulator of SF3B1,the branch point binding protein of the U2-snRNP. Spliceosome modulation by these SF3B1 targeted compounds recruits SRSF1 to nuclear G4C2 RNA,mobilizing it from RNA foci into nucleocytoplasmic export. This leads to increased repeat-associated non-canonical (RAN) translation and ultimately,enhanced cell toxicity. Our data (i) provide a new pharmacological entry point with novel as well as known,publicly available tool compounds for dissection of C9ORF72 pathobiology in C9ORF72 ALS models,(ii) allowing to differentially modulate RNA foci versus RAN translation,and (iii) suggest that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function,counteracting translation into toxic dipeptide repeat polyproteins. Instead,our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery. View Publication

Simple SummaryAtypical teratoid rhabdoid tumors (ATRTs) and diffuse intrinsic pontine gliomas (DIPGs) are lethal pediatric brain tumors that can resist chemotherapy and be influenced by their microenvironment. Astrocytes are the key components of the brain tumor microenvironment and can support tumor growth. We investigated the effects of astrocytes on cisplatin sensitivity in pediatric brain cancer cells. The crosstalk between astrocytes and cancer cells activated astrocytes and promoted cancer cell proliferation. Moreover,the tumor cells expressed elevated levels of drug resistance genes in the presence of astrocytes. In conclusion,astrocytes can significantly improve the growth of these tumor cells and modulate their chemosensitivity,highlighting their role in therapeutic resistance. AbstractBackground: ATRTs and DIPGs are deadly pediatric brain tumors with poor prognosis. These tumors can develop resistance to chemotherapies,which may be significantly influenced by their microenvironment. Since astrocytes are the most abundant glial cell type in the brain microenvironment and may support tumor growth and chemoresistance,this study investigated the effects of induced pluripotent stem cell-derived astrocytes (iPSC-astrocytes) on cisplatin sensitivity in CHLA-05-ATRT and SF8628 (DIPG) cells. iPSCs provide an unlimited and standardized source of nascent astrocytes,which enables modeling the interaction between childhood brain tumor cells and iPSC-astrocytes within a controlled coculture system. Methods: To study the effects on tumor growth,the iPSC-astrocytes were cocultured with tumor cells. Additionally,the tumor cells were exposed to various concentrations of cisplatin to evaluate their chemosensitivity in the presence of astrocytes. Results: The paracrine interaction of iPSC-astrocytes with tumor cells upregulated astrocyte activation markers GFAP and STAT3 and promoted tumor cell proliferation. Moreover,the cisplatin treatment significantly decreased the viability of CHLA-05-ATRT and SF8628 cells. However,tumor cells exhibited reduced sensitivity to cisplatin in the coculture with iPSC-astrocytes. During cisplatin treatment,DIPG cells in particular showed upregulation of resistance markers,ERK1,STAT3,and MTDH,which are associated with enhanced proliferation and invasion. They also had increased expression of APEX1,which is involved in the base excision repair pathway following cisplatin-induced DNA damage. Conclusion: These findings underscore the significance of the tumor microenvironment in modulating tumor cell survival and chemosensitivity. View Publication

Differentiation approaches to obtain mesenchymal stem cells (MSCs) have gradually developed over the last few decades. The problem is that different protocols give different MSC types,making further research difficult. Here,we tried three different approaches to differentiate embryonic stem cells (ESCs) from early mesoderm to MSCs using serum-containing or xeno-free differentiation medium and observed differences in the cells’ morphology,doubling rate,ability to form colonies,surface marker analysis,and multilineage differentiation potential of the obtained cell lines. We concluded that the xeno-free medium best fits the criteria of MSCs’ morphology,growth kinetics,and surface marker characterization. In contrast,the serum-containing medium gives better potential for further MSC differentiation into osteogenic,chondrogenic,and adipogenic lineages. View Publication

A large number of synaptic proteins have been recurrently associated with complex brain disorders. One of these proteins,the Traf and Nck interacting kinase (TNIK),is a postsynaptic density (PSD) signaling hub,with many variants reported in neurodevelopmental disorder (NDD) and psychiatric disease. While rodent models of TNIK dysfunction have abnormal spontaneous synaptic activity and cognitive impairment,the role of mutations found in patients with TNIK protein deficiency and TNIK protein kinase activity during early stages of neuronal and synapse development has not been characterized. Here,using hiPSC-derived excitatory neurons,we show that TNIK mutations dysregulate neuronal activity in human immature synapses. Moreover,the lack of TNIK protein kinase activity impairs MAPK signaling and protein phosphorylation in structural components of the PSD. We show that the TNIK interactome is enriched in NDD risk factors and TNIK lack of function disrupts signaling networks and protein interactors associated with NDD that only partially overlap to mature mouse synapses,suggesting a differential role of TNIK in immature synapsis in NDD. View Publication

N6-methyladenosine (m6A) is an abundant internal RNA modification that can impact gene expression at both post-transcriptional and transcriptional levels. However,the landscapes and functions of m6A in human brains and neurodegenerative diseases,including Alzheimer’s disease (AD),are under-explored. Here,we examined RNA m6A methylome using total RNA-seq and meRIP-seq in middle frontal cortex of post-mortem brains from individuals with or without AD,which revealed m6A alteration on both mRNAs and various noncoding RNAs. Notably,many promoter-antisense RNAs (paRNAs) displayed cell-type-specific expression and changes in AD,including one produced adjacent to MAPT that encodes the Tau protein. MAPT-paRNA is highly expressed in neurons,and m6A positively controls its expression. In iPSC-derived human excitatory neurons,MAPT-paRNA does not impact the nearby MAPT mRNA,but instead promotes expression of hundreds of neuronal and synaptic genes,and is protective against excitotoxicity. Analysis of single nuclei RNA-DNA interactome in human brains supports that brain paRNAs interact with both cis- and trans-chromosomal target genes to impact their transcription. These data reveal landscapes and functions of noncoding RNAs and m6A in brain gene regulation and AD pathogenesis. This study characterised the landscapes and changes of RNA m6A in brains of individuals with or without Alzheimer’s disease,and revealed roles of a promoter antisense RNA next to MAPT in neuronal gene regulation that promote neuronal survival. View Publication

Biallelic mutations in PIGK cause GPI biosynthesis defect 22 (GPIBD22),characterized with developmental delay,hypotonia,and cerebellar atrophy. The understanding of the underlying causes is limited due to the lack of suitable disease models. To address this gap,we generated a mouse model with PIGK deficits,specifically in Purkinje cells (Pcp2-cko) and an induced pluripotent stem cell (iPSC) model using the c.87dupT mutant (KI) found in GPIBD22 patients. Pcp2-cko mice demonstrated cerebellar atrophy,ataxia and progressive Purkinje cells loss which were accompanied by increased apoptosis and neuroinflammation. Similarly,KI iPSCs exhibited increased apoptosis and accelerated neural rosette formation,indicating that PIGK defects could impact early neural differentiation that confirmed by the RNA-Seq results of neural progenitor cells (NPCs). The increased apoptosis and accelerated NPC differentiation in KI iPSCs are associated with excessive unfolded protein response (UPR) pathway activation,and can be rescued by UPR pathway inhibitor. Our study reveals potential pathogenic mechanism of GPIBD22 and providing new insights into the therapeutic strategy for GPIBD. View Publication

ABSTRACTHuman induced pluripotent stem cells (iPSCs) are an invaluable endless cell source for generating various therapeutic cells and tissues. However,their differentiation into specific cell lineages,such as definitive endoderm (DE) and pancreatic progenitor (PP),often suffers from poor reproducibility,due partially to their pluripotency. In this work,we investigated the impact of iPSC confluency during cell self?renewal and seeding density on cell metabolic activity,glycolysis to oxidative phosphorylation shift,and differentiation potential toward DE and PP lineages. Our findings demonstrated that cell seeding strategy influences cellular metabolic activity and the robustness of iPSC differentiation. iPSCs maintained at higher seeding density exhibited lower initial oxygen consumption rate (OCR) and metabolic activity. There is an optimal seeding density to ensure sufficient oxygen consumption during differentiation and to yield high expression of SOX17 in the DE lineage and high PDX1/NKX6.1 dual?positive cells in PPs. Interestingly,we found that cell confluency at the time of harvest has less impact on the efficacy of pancreatic lineage formation or metabolic activity. This study sheds light on the interplay between metabolic activity and iPSC lineage specification,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. Graphical Abstract and Lay SummaryHuman induced pluripotent stem cell (iPSC) differentiation into specific cell lineages often shows poor reproducibility due to cell pluripotency. This study demonstrated impact of iPSC seeding strategy on metabolic activity and differentiation potential,offering new insights into the robustness of iPSC self?renewal and differentiation for creating human tissues. View Publication

SummaryGlioblastomas are the most common malignant brain tumors in adults; they are highly aggressive and heterogeneous and show a high degree of plasticity. Here,we show that methyltransferase-like 7B (METTL7B) is an essential regulator of lineage specification in glioblastoma,with an impact on both tumor size and invasiveness. Single-cell transcriptomic analysis of these tumors and of cerebral organoids derived from expanded potential stem cells overexpressing METTL7B reveal a regulatory role for the gene in the neural stem cell-to-astrocyte differentiation trajectory. Mechanistically,METTL7B downregulates the expression of key neuronal differentiation players,including SALL2,via post-translational modifications of histone marks. Graphical abstract Highlights•METTL7B is highly expressed in human glioblastoma stem cells•METTL7B regulates tumor size and invasiveness in an in vivo xenograft model•METTL7B controls the neural stem cell-to-astrocyte differentiation trajectory•METTL7B regulates SALL2 expression via H3K27me3 modulation Constantinou et al. identify METTL7B as an essential regulator of lineage specification and a modulator of the expression of the transcription factor SALL2 with wide-ranging impacts on invasion and tumor growth in glioblastoma. View Publication

Stargardt disease (STGD),predominantly caused by mutations in the ABCA4 gene,is a leading cause of inherited retinal degeneration. Although several lines of mice expressing disease-causing variants have been produced,mice due to the lack of macular may not be the perfect model to mimic the characteristics of STGD. To address this knowledge gap,we generated retinal organoids from patient-derived induced pluripotent stem cells (iPSCs) harboring ABCA4 mutations and performed biological validation. The generated retinal organoids were subjected to single-cell RNA sequencing (scRNA-seq) at major developmental stages (40,90,150,200,and 260 days),and we additionally compared the transcriptomics with our recently published control retinal organoids to further confirm the reliability of the dataset. By using iPSCs carrying most common variant in Chinese STGD patients,the dataset not only provides a powerful resource for studying STGD,but also offers novels insight into the developmental mechanisms underlying ABCA4-associated pathological changes in the retinal organoid system. View Publication

Disease modeling with isogenic Induced Pluripotent Stem Cell (iPSC)-differentiated organoids serves as a powerful technique for studying disease mechanisms. Multiplexed coculture is crucial to mitigate batch effects when studying the genetic effects of disease-causing variants in differentiated iPSCs or organoids,and demultiplexing at the single-cell level can be conveniently achieved by assessing natural genetic barcodes. Here,to enable cost-efficient time-series experimental designs via multiplexed bulk and single-cell RNA-seq of hybrids,we introduce a computational method in our Vireo Suite,Vireo-bulk,to effectively deconvolve pooled bulk RNA-seq data by genotype reference,and thereby quantify donor abundance over the course of differentiation and identify differentially expressed genes among donors. Furthermore,with multiplexed scRNA-seq and bulk RNA-seq,we demonstrate the usefulness and necessity of a pooled design to reveal donor iPSC line heterogeneity during macrophage cell differentiation and to model rare WT1 mutation-driven kidney disease with chimeric organoids. Our work provides an experimental and analytic pipeline for dissecting disease mechanisms with chimeric organoids. IPSC-derived organoids model diseases. Multiplexed coculture and demultiplexing natural genetic barcodes aid in studying genetic effects. Here,authors introduce Vireo-bulk to deconvolve bulk RNA-seq data,quantify donor abundance and identify differentially expressed genes. View Publication

Human genetics analysis has identified many noncoding SNPs associated with diabetic traits,but whether and how these variants contribute to diabetes is largely unknown. Here,we focus on a noncoding variant,rs6048205,and report that the risk-G variant impairs the generation of PDX1+/NKX6-1+ pancreatic progenitor cells and further results in the abnormal decrease of functional ? cells during pancreatic differentiation. Mechanistically,this risk-G variant greatly enhances RXRA binding and over-activates FOXA2 transcription,specifically in the pancreatic progenitor stage,which in turn represses NKX6-1 expression. Consistently,inducible FOXA2 overexpression could phenocopy the differentiation defect. More importantly,mice carrying risk-G exhibit abnormal pancreatic islet architecture and are more sensitive to streptozotocin or a high-fat diet to develop into diabetes eventually. This study not only identifies a causal noncoding variant in diabetes susceptibility but also dissects the underlying gain-of-function mechanism by recruiting stage-specific factors. How GWAS-annotated noncoding SNPs contribute to diabetes remains unclear. Here,the authors report that the noncoding SNP rs6048205 drives stage-specific defects in human pancreatic differentiation and increases diabetes susceptibility in mice. View Publication