技术资料

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation,but whether and how metabolic sensor O -GlcNAcylation,which can be modulated via an inhibition of its cycling enzymes O -GlcNAcase (OGA) and O -GlcNAc transferase (OGT),contributes to hematopoiesis remains largely unknown. Herein,isogenic,single-cell clones of OGA -depleted (OGAi) and OGT -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A,which were reprogrammed from CD34 + hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O -GlcNAcylation concomitant with their loss of OGA and OGT,respectively,appeared normal in phenotype and karyotype,and retained pluripotency,although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition,we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O -GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors,thus confirming their functional characteristics. Altogether,the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O -GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation,i.e.,by small molecules,allowing the molecular dynamics studies of O -GlcNAcylation. View Publication

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem‐like cancer cells,called glioblastoma stem cells (GSCs). GSCs have the capacity for self‐renewal and tumorigenesis,leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem‐like cancer cells,their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation,novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image‐based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs,facilitating the development of NK cell‐based immunotherapy for improved GBM treatment. View Publication

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however,an associated loss of mitochondrial efficiency in effector T cells ultimately limits anti-tumor immunity. Memory phenotype (MP) T cells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4 + MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity,but MP cells retain less compromised mitochondria compared to effector CD4 + T cells,and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak T cell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells. Subject areas: immunity,cell biology View Publication

Early childhood tumours arise from transformed embryonic cells,which often carry large copy number alterations (CNA). However,it remains unclear how CNAs contribute to embryonic tumourigenesis due to a lack of suitable models. Here we employ female human embryonic stem cell (hESC) differentiation and single-cell transcriptome and epigenome analysis to assess the effects of chromosome 17q/1q gains,which are prevalent in the embryonal tumour neuroblastoma (NB). We show that CNAs impair the specification of trunk neural crest (NC) cells and their sympathoadrenal derivatives,the putative cells-of-origin of NB. This effect is exacerbated upon overexpression of MYCN,whose amplification co-occurs with CNAs in NB. Moreover,CNAs potentiate the pro-tumourigenic effects of MYCN and mutant NC cells resemble NB cells in tumours. These changes correlate with a stepwise aberration of developmental transcription factor networks. Together,our results sketch a mechanistic framework for the CNA-driven initiation of embryonal tumours. Subject terms: Paediatric cancer,Stem cells,Disease model,Cancer genomics,Embryonal neoplasms View Publication

Kidney organoids are an innovative tool in transplantation research. The aim of the present study was to investigate whether kidney organoids are susceptible for allo-immune attack and whether they can be used as a model to study allo-immunity in kidney transplantation. Human induced pluripotent stem cell-derived kidney organoids were co-cultured with human peripheral blood mononuclear cells (PBMC),which resulted in invasion of allogeneic T-cells around nephron structures and macrophages in the stromal cell compartment of the organoids. This process was associated with the induction of fibrosis. Subcutaneous implantation of kidney organoids in immune-deficient mice followed by adoptive transfer of human PBMC led to the invasion of diverse T-cell subsets. Single cell transcriptomic analysis revealed that stromal cells in the organoids upregulated expression of immune response genes upon immune cell invasion. Moreover,immune regulatory PD-L1 protein was elevated in epithelial cells while genes related to nephron differentiation and function were downregulated. This study characterized the interaction between immune cells and kidney organoids,which will advance the use of kidney organoids for transplantation research. View Publication

The use of genetic engineering to generate point mutations in induced pluripotent stem cells (iPSCs) is essential for studying a specific genetic effect in an isogenic background. We demonstrate that a combination of p53 inhibition and pro-survival small molecules achieves a homologous recombination rate higher than 90% using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in human iPSCs. Our protocol reduces the effort and time required to create isogenic lines. View Publication

CRISPR-Cas9 genome editing often induces unintended,large genomic rearrangements,posing potential safety risks. However,there are no methods for mitigating these risks. Using long-read individual-molecule sequencing (IDMseq),we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs,while depleting or overexpressing RPA increases or reduces LD frequency,respectively. Interestingly,small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells. Our findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR,suggesting new strategies for safer and more precise genome editing. The online version contains supplementary material available at 10.1186/s12915-024-01896-z. View Publication

Allogeneic cell therapies,such as those involving macrophages or Natural Killer (NK) cells,are of increasing interest for cancer immunotherapy. However,the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges,such as required cell pre-activation and inefficiency in transduction,which hinder the assessment of preclinical efficacy and clinical translation. In our study,we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein,which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors,this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood,as well as freshly obtained monocytes,which were differentiated to M1 macrophages as well as B cells from multiple donors,achieving up to 80% reporter gene expression within three days post-transduction. Importantly,KoRV-based transduction does not compromise the expression of crucial immune cell receptors,nor does it impair immune cell functionality,including NK cell viability,proliferation,cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion,our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics,requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics,expediting their availability to patients in need. Subject terms: Genetic transduction,Tumour immunology,Immunotherapy,Genetic vectors,Innate immune cells View Publication

SARS-CoV-2 Omicron variant has evolved into sublineages. Here,we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However,there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population. Subject areas: Immunology,Immune response,Virology View Publication

N6‐methyladenosine (m6A) is an RNA modification involved in RNA processing and widely found in transcripts. In cancer cells,m6A is upregulated,contributing to their malignant transformation. In this study,we analyzed gene expression and m6A modification in cancer tissues,ducts,and acinar cells derived from pancreatic cancer patients using MeRIP‐seq. We found that dozens of RNAs highly modified by m6A were detected in cancer tissues compared with ducts and acinar cells. Among them,the m6A‐activated mRNA TCEAL8 was observed,for the first time,as a potential marker gene in pancreatic cancer. Spatially resolved transcriptomic analysis showed that TCEAL8 was highly expressed in specific cells,and activation of cancer‐related signaling pathways was observed relative to TCEAL8‐negative cells. Furthermore,among TCEAL8‐positive cells,the cells expressing the m6A‐modifying enzyme gene METTL3 showed co‐activation of Notch and mTOR signaling,also known to be involved in cancer metastasis. Overall,these results suggest that m6A‐activated TCEAL8 is a novel marker gene involved in the malignant transformation of pancreatic cancer. View Publication

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 ( V606M ; R453C ),MYBPC3 ( G148R ) or digenic variants ( MYBPC3 P955fs,TNNI3 A157V ). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls,variant-positive CMs show sarcomere disorganization,higher contractility,calcium transients,and ATPase activity. However,only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM. View Publication

Johnson,Ogishi,and Domingo-Vila et al. describe two siblings with inherited PD-L1 deficiency. Human PD-L1 deficiency underlies early-onset T1D,like PD-1 deficiency,but does not lead to fatal autoimmunity with extensive leukocytic dysregulation,unlike PD-1 deficiency. View Publication