技术资料

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality,inter-batch consistency,cryopreservation and scale remain,reducing experimental reproducibility and clinical translation. Here,we report a robust stirred suspension cardiac differentiation protocol,and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines,the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs,bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks,which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible,scalable,and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications,and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology,Induced pluripotent stem cells,Cardiovascular models View Publication

microRNAs (miRNAs) are small,non-coding RNAs that regulate expression of multiple genes. MiR-193a-3p functions as a tumor suppressor in many cancer types,but its effect on inducing specific anti-tumor immune responses is unclear. Therefore,we examined the effect of our lipid nanoparticle (LNP) formulated,chemically modified,synthetic miR-193a-3p mimic (INT-1B3) on anti-tumor immunity. INT-1B3 inhibited distant tumor metastasis and significantly prolonged survival. INT-1B3-treated animals were fully protected against challenge with autologous tumor cells even in absence of treatment indicating long-term immunization. Protection against autologous tumor cell challenge was hampered upon T cell depletion and adoptive T cell transfer abrogated tumor growth. Transfection of tumor cells with our miR-193a-3p mimic (1B3) resulted in tumor cell death and apoptosis accompanied by increased expression of DAMPs. Co-culture of 1B3-transfected tumor cells and immature DC led to DC maturation and these mature DC were able to stimulate production of type 1 cytokines by CD4+ and CD8+ T cells. CD4-CD8- T cells also produced type 1 cytokines,even in response to 1B3-transfected tumor cells directly. Live cell imaging demonstrated PBMC-mediated cytotoxicity against 1B3-transfected tumor cells. These data demonstrate for the first time that miR-193a-3p induces long-term immunity against tumor development via modulation of the tumor microenvironment and induction of immunogenic cell death. View Publication

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features,we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT),a specialized DNA polymerase intrinsic to VDJ recombination,broadly expressed within CD34 + progenitors prior to B/T cell emergence. While these TdT + cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype,their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84 lo GMPs demonstrates robust lymphoid potentials ex vivo,while still retaining significant myeloid differentiation capacity,akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors,further defining the lympho-myeloid axis in human hematopoiesis. Subject terms: Lymphopoiesis,Systems analysis,Proteomic analysis,Myelopoiesis View Publication

Haemophilus influenzae is a human respiratory pathogen and inhabits the human respiratory tract as its only niche. Despite this,the molecular mechanisms that allow H . influenzae to establish persistent infections of human epithelia are not well understood. Here,we have investigated how H . influenzae adapts to the host environment and triggers the host immune response using a human primary cell-based infection model that closely resembles human nasal epithelia (NHNE). Physiological assays combined with dualRNAseq revealed that NHNE from five healthy donors all responded to H . influenzae infection with an initial,‘unproductive’ inflammatory response that included a strong hypoxia signature but did not produce pro-inflammatory cytokines. Subsequently,an apparent tolerance to large extracellular and intraepithelial burdens of H . influenzae developed,with NHNE transcriptional profiles resembling the pre-infection state. This occurred in parallel with the development of intraepithelial bacterial populations,and appears to involve interruption of NFκB signalling. This is the first time that large-scale,persistence-promoting immunomodulatory effects of H . influenzae during infection have been observed,and we were able to demonstrate that only infections with live,but not heat-killed H . influenzae led to immunomodulation and reduced expression of NFκB-controlled cytokines such as IL-1β,IL-36γ and TNFα. Interestingly,NHNE were able to re-activate pro-inflammatory responses towards the end of the 14-day infection,resulting in release of IL-8 and TNFα. In addition to providing first molecular insights into mechanisms enabling persistence of H . influenzae in the host,our data further indicate the presence of infection stage-specific gene expression modules,highlighting fundamental similarities between immune responses in NHNE and canonical immune cells,which merit further investigation. View Publication

Acute myeloid leukaemia (AML) is a disease of the haematopoietic stem cells(HSCs) that is characterised by the uncontrolled proliferation and impaired differentiation of normal haematopoietic stem/progenitor cells. Several pathways that control the proliferation and differentiation of HSCs are impaired in AML. Activation of the Wnt/beta-catenin signalling pathway has been shown in AML and beta-catenin,which is thought to be the key element of this pathway,has been frequently highlighted. The present study was designed to determine beta-catenin expression levels and beta-catenin-related genes in AML. In this study,beta-catenin gene expression levels were determined in 19 AML patients and 3 controls by qRT-PCR. Transcriptome analysis was performed on AML grouped according to beta-catenin expression levels. Differentially expressed genes(DEGs) were investigated in detail using the Database for Annotation Visualisation and Integrated Discovery(DAVID),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),STRING online tools. The transcriptome profiles of our AML samples showed different molecular signature profiles according to their beta-catenin levels(high-low). A total of 20 genes have been identified as hub genes. Among these,TTK,HJURP,KIF14,BTF3,RPL17 and RSL1D1 were found to be associated with beta-catenin and poor survival in AML. Furthermore,for the first time in our study,the ELOV6 gene,which is the most highly up-regulated gene in human AML samples,was correlated with a poor prognosis via high beta-catenin levels. It is suggested that the identification of beta-catenin-related gene profiles in AML may help to select new therapeutic targets for the treatment of AML. View Publication

Autoimmune thyroid disease (AITD) is a common autoimmune disease. In a GWAS meta-analysis of 110,945 cases and 1,084,290 controls,290 sequence variants at 225 loci are associated with AITD. Of these variants,115 are previously unreported. Multiomics analysis yields 235 candidate genes outside the MHC-region and the findings highlight the importance of genes involved in T-cell regulation. A rare 5’-UTR variant (rs781745126-T,MAF = 0.13% in Iceland) in LAG3 has the largest effect (OR = 3.42,P = 2.2 × 10 −16 ) and generates a novel start codon for an open reading frame upstream of the canonical protein translation initiation site. rs781745126-T reduces mRNA and surface expression of the inhibitory immune checkpoint LAG-3 co-receptor on activated lymphocyte subsets and halves LAG-3 levels in plasma among heterozygotes. All three homozygous carriers of rs781745126-T have AITD,of whom one also has two other T-cell mediated diseases,that is vitiligo and type 1 diabetes. rs781745126-T associates nominally with vitiligo (OR = 5.1,P = 6.5 × 10 −3 ) but not with type 1 diabetes. Thus,the effect of rs781745126-T is akin to drugs that inhibit LAG-3,which unleash immune responses and can have thyroid dysfunction and vitiligo as adverse events. This illustrates how a multiomics approach can reveal potential drug targets and safety concerns. Subject terms: Genetics research,Disease genetics,Thyroid diseases,Genome-wide association studies,Gene expression View Publication

DNA methylation plays an important role in various biological processes,including cell differentiation,ageing,and cancer development. The most important methylation in mammals is 5-methylcytosine mostly occurring in the context of CpG dinucleotides. Sequencing methods such as whole-genome bisulfite sequencing successfully detect 5-methylcytosine DNA modifications. However,they suffer from the serious drawbacks of short read lengths and might introduce an amplification bias. Here we present Rockfish,a deep learning algorithm that significantly improves read-level 5-methylcytosine detection by using Nanopore sequencing. Rockfish is compared with other methods based on Nanopore sequencing on R9.4.1 and R10.4.1 datasets. There is an increase in the single-base accuracy and the F1 measure of up to 5 percentage points on R.9.4.1 datasets,and up to 0.82 percentage points on R10.4.1 datasets. Moreover,Rockfish shows a high correlation with whole-genome bisulfite sequencing,requires lower read depth,and achieves higher confidence in biologically important regions such as CpG-rich promoters while being computationally efficient. Its superior performance in human and mouse samples highlights its versatility for studying 5-methylcytosine methylation across varied organisms and diseases. Finally,its adaptable architecture ensures compatibility with new versions of pores and chemistry as well as modification types. Subject terms: Genome informatics,Epigenomics,Computational models,DNA sequencing,DNA methylation View Publication

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 ( NCF1 ) gene,resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context,the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore,we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery,to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox,an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs,with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery,with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy,as it leads to the co-expression of p47 phox and p67 phox,ensuring spatiotemporal and near-physiological transgene expression in myeloid cells. View Publication

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly,laborious,time-consuming,complex,and require skilled personnel. Hence,utilisation of most diagnostics for AAT is impracticable in rural areas,where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection,without relying on expensive equipment,would facilitate AAT detection. In turn,early management,would reduce disease incidence and severity. Today,several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context,we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T . congolense infections,which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here,originated from a past study. Briefly,the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T . congolense glycosomal fructose-1,6-bisphosphate aldolase ( Tco ALD) was discovered as the cognate Ag of Nb474H. In this study,splenocytes were harvested from a mouse immunized with recombinant Tco ALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on Tco ALD retrieved a lone binder,designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native Tco ALD released during infection by T . congolense parasites. Hitherto,development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) “hybrid” sandwich technology offers prospects for exploration,using the unique specificity of Nb as a key determinant in Ag capturing,while using the versatility of monoclonal Ab to adapt to various detection conditions. View Publication

RNA constitutes a large fraction of chromatin. Spatial distribution and functional relevance of most of RNA-chromatin interactions remain unknown. We established a landscape analysis of RNA-chromatin interactions in human acute myeloid leukemia (AML). In total more than 50 million interactions were captured in an AML cell line. Protein-coding mRNAs and long non-coding RNAs exhibited a substantial number of interactions with chromatin in cis suggesting transcriptional activity. In contrast,small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) associated with chromatin predominantly in trans suggesting chromatin specific functions. Of note,snoRNA-chromatin interaction was associated with chromatin modifications and occurred independently of the classical snoRNA-RNP complex. Two C/D box snoRNAs,namely SNORD118 and SNORD3A,displayed high frequency of trans -association with chromatin. The transcription of SNORD118 and SNORD3A was increased upon leukemia transformation and enriched in leukemia stem cells,but decreased during myeloid differentiation. Suppression of SNORD118 and SNORD3A impaired leukemia cell proliferation and colony forming capacity in AML cell lines and primary patient samples. Notably,this effect was leukemia specific with less impact on healthy CD34+ hematopoietic stem and progenitor cells. These findings highlight the functional importance of chromatin-associated RNAs overall and in particular of SNORD118 and SNORD3A in maintaining leukemia propagation. Subject terms: Acute myeloid leukaemia,Cancer epigenetics View Publication

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise,with a negligible affinity for mismatched substrates,but its low cellular targeting efficiency limits therapeutic use. Here,we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency,knock-in rates,and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally,we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor,highlighting its therapeutic utility. Subject terms: CRISPR-Cas9 genome editing,Molecular medicine,Genetic engineering,CRISPR-Cas9 genome editing View Publication

Current protocols generate highly pure human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet,a risk of arrhythmias exists when hiPSC‐CMs are injected into large animal models. Thus,understanding hiPSC‐CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC‐CM maturation. Here,we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC‐CM maturation in 3‐dimensional suspension culture. Three‐dimensional cultures of hiPSC‐CMs were treated with Nutlin‐3a (p53 activator,10 μM),LOM612 (FOXO relocator,5 μM),AS1842856 (FOXO inhibitor,1 μM),or RCM‐1 (FOXM1 inhibitor,1 μM),starting 2 days after onset of beating,with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC‐CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation,in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac‐specific markers such as TNNT2. In contrast,FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility,oxygen consumption,and voltage peak maximum upstroke velocity,in n=3 to 6 per group for all assays. Further,by single‐cell RNA sequencing of n=2 LOM612‐treated cells compared with dimethyl sulfoxide,LOM612‐mediated FOXO activation promoted expression of cardiac maturational pathways. We show that p53 activation promotes FOXO and suppresses FOXM1 during 3‐dimensional hiPSC‐CM maturation. These results expand our understanding of hiPSC‐CM maturational mechanisms in a clinically‐relevant 3‐dimensional culture system. View Publication