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Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins,in particular albumin,which is prone to quality variations and presents a substantial cost factor,hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations,we have developed chemically defined,entirely protein-free media based on RPMI,supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). Both media compositions enable the efficient,directed differentiation of embryonic and induced hPSCs,matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture,producing > 1.3 × 10 9 cardiomyocytes in a single process run. Transcriptome analysis,flow cytometry,electrophysiology,and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. This study promotes the robustness and upscaling of the cardiomyogenic differentiation process,substantially reduces media costs,and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration. The online version contains supplementary material available at 10.1186/s13287-024-03826-w. View Publication

Mucus stasis is a pathologic hallmark of muco-obstructive diseases,including cystic fibrosis (CF). Mucins,the principal component of mucus,are extensively modified with hydroxyl (O)-linked glycans,which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however,the consequences of reduced sialylation on mucus clearance have not been fully determined. Here,we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin,MUC5B,and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways,and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally,we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall,this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases. Subject terms: Biophysical chemistry,Glycobiology,Respiration View Publication

Therapy-related myeloid neoplasms (t-MN) arise as a complication of chemo- and/or radiotherapy. Although t-MN can occur both in adult and childhood cancer survivors,the mechanisms driving therapy-related leukemogenesis likely vary across different ages. Chemotherapy is thought to induce driver mutations in children,whereas in adults pre-existing mutant clones are selected by the exposure. However,selective pressures induced by chemotherapy early in life are less well studied. Here,we use single-cell whole genome sequencing and phylogenetic inference to show that the founding cell of t-MN in children starts expanding after cessation of platinum exposure. In patients with Li-Fraumeni syndrome,characterized by a germline TP53 mutation,we find that the t-MN already expands during treatment,suggesting that platinum-induced growth inhibition is TP53- dependent. Our results demonstrate that germline aberrations can interact with treatment exposures in inducing t-MN,which is important for the development of more targeted,patient-specific treatment regimens and follow-up. Subject terms: Cancer genomics,Cancer genomics,Haematological cancer,Paediatric cancer 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

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

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

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

The development of haematopoiesis involves the coordinated action of numerous genes,some of which are implicated in haematological malignancies. However,the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here,we report a previously uncharacterised gene in haematopoiesis,identified by screening mutant embryonic stem cells. The gene,‘ attenuated haematopoietic development ( Ahed )’,encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward,leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed -deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model,we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways,and published databases present cancer patients with somatic mutations in Ahed . Collectively,our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis,implicating its association with malignancies. Subject terms: Lymphopoiesis,Development,Haematopoietic stem cells,Differentiation View Publication