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Clonal hematopoiesis (CH),a prevalent and premalignant state in the elderly,has been detected in young individuals under selective pressures such as hematopoietic cell transplantation (HCT). However,the origin of CH and mutational processes underlying CH driver mutations in young blood systems remain unclear. Here,we used genome‐wide somatic mutation profiles to retrospectively trace the origin of DNMT3A‐mutant CH in three individuals,14–41 years after childhood HCT. Both the rate and spectrum of somatic mutations in individuals with posttransplant CH were consistent with normal age‐associated mutagenesis. Phylogenetic analysis revealed that DNMT3A‐mutant HSPCs were present in the donor before 6.8 years of age,including during fetal development,despite being undetectable with a limit of detection of variant allele frequency of 0.001 at the time of transplantation. These findings were validated by comparing the observed mutations to expected age‐dependent mutational signatures. Our results reveal that undetectable DNMT3A‐mutant clones in young donors can expand into significant CH clones within decades upon transplantation. The rapid expansion of these clones in this context indicates that specific environmental pressures,rather than solely mutation acquisition,drive the development of CH. View Publication

Background: Lung cancer is a leading cause of cancer-related mortality worldwide,with heterogeneity and acquired resistance posing major challenges to treatment. Advances in next-generation sequencing (NGS) have enabled comprehensive genomic profiling,yet there remains a need for robust patient-derived models to study tumor biology and inform precision medicine. This study aims to establish and characterize patient-derived lung cancer organoids (LCOs) using whole-genome sequencing (WGS) to explore their genomic landscape and therapeutic potential. Methods: We established a panel of LCOs from resected tumors and malignant pleural effusions (MPEs) of 14 non-small cell lung cancer (NSCLC) patients. Organoids were authenticated and subjected to WGS to profile somatic single nucleotide variants (SNVs),insertions/deletions (InDels),copy number variations (CNVs),structural variants (SVs),and microsatellite instability (MSI). Bioinformatic analyses were performed to annotate mutations,assess tumor mutation burden (TMB),and explore mutational signatures. Furthermore,deep learning-based drug response prediction and in vitro drug sensitivity assays were conducted to evaluate therapeutic potentials in the established LCOs. Results: In the established LCOs,WGS revealed recurrent mutations in TP53,TTN,MUC16,and FLG,with approximately 80% of somatic variants located in non-coding regions,highlighting the potential role of regulatory elements in lung cancer pathogenesis. Early and locally advanced-stage tumor-derived LCOs exhibited higher TMB and MSI compared to those from advanced-stage disease,suggesting greater clonal diversity prior to therapeutic intervention. Drug screening demonstrated the feasibility of using genomic data for drug prediction,but requires more advanced models to fully utilize the WGS data. Conclusions: Our comprehensive genomic characterization of patient-derived LCOs provides valuable insights into the mutational landscape and evolutionary dynamics of lung cancer. These well-annotated organoid models serve as a powerful resource for investigating tumor biology and developing genomically informed therapeutic strategies. View Publication

Older adults have decreased vaccine efficacy,but the adjuvanted recombinant VZV-gE zoster vaccine (RZV) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL),which is much less efficacious. RZV increased NK,monocyte,and DC activation in response to in vitro VZV-gE stimulation for up to 5 years post-vaccination,while ZVL increased only DC responses to VZV for up to 90 days. In purified monocyte and NK cell cocultures,RZV recipients showed increased responses to VZV-gE,HCMV and HSV antigenic stimulation post-vaccination. ATAC-seq analysis of purified monocytes revealed decreased accessibility in areas of the TGFβ1 gene. scRNA-seq and immunoproteomics confirmed decreased TGFβ1 transcription and translation,respectively. Exogenous supplementation and inhibition of TGFβ1 modulated in vitro monocyte responses to VZV-gE. In conclusion,RZV generated homologous (VZV-gE) and heterologous (HCMV,HSV) trained immunity in monocytes through genomic repression of the regulatory cytokine TGFβ-1. Cytokine modulation may represent a novel mechanism of generating trained immunity in myeloid cells. Author summaryOlder adults have decreased vaccine efficacy,but the adjuvanted recombinant varicella zoster virus (VZV)-gE zoster vaccine (RZV; Shingrix™) is highly efficacious. We investigated memory-like innate immune responses after RZV and after the zoster vaccine live (ZVL; Zostavax™),which is much less efficacious than RZV. We found that RZV increased the functionality of several innate immune cell subsets against VZV-gE other herpesviruses. The increase in functionality was associated with decreased production of the inhibitory cytokine TGFβ1,which may have resulted from decreased ability to use the TGFβ1 gene as a template for the synthesis of its product. We concluded that RZV generated homologous (VZV-gE) and heterologous (other herpesviruses) memory-like responses in innate immune cell subsets through genomic repression of the regulatory cytokine TGFβ-1. View Publication

Systemic sclerosis (SSc) is an autoimmune disease marked by fibrosis and extensive autoantibody production. Although B-cell depletion with rituximab (RTX) has shown clinical benefit,predictive biomarkers of response remain elusive. Here,we apply proteome-wide autoantibody screening using wet protein arrays covering 13,455 human antigens in serum samples from participants of the randomized trial of RTX. We identify a significant elevation in the total autoantibody levels in SSc compared to healthy controls,with greater reductions post-treatment observed in RTX high responders than in low responders. A stepwise selection highlights 88 clinically relevant autoantibodies,including those targeting G protein-coupled receptors. Among them,anti-C-C motif chemokine receptor 8 (CCR8) autoantibodies are functionally validated by cell-based assays using CCR8-overexpressing HEK293 cells. Furthermore,in a bleomycin-induced mouse model,anti-CCR8 antibody administration exacerbates dermal fibrosis and modifies immune cell infiltration. Although external validation with multiple comparison adjustment is further required,these findings reveal an autoantibody signature associated with therapeutic response and pathogenic potential in SSc,providing a foundation for precision immunotherapy and mechanistic insights into disease progression. B-cell depletion benefits systemic sclerosis,but predictive biomarkers remain limited. The authors here map autoantibody profiles using proteome-wide screening,identify C-C motif chemokine receptor 8-targeting autoantibodies with functional impact,suggesting novel pathophysiology and precision therapy targets. View Publication

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in COL7A1. Patient-derived induced pluripotent stem cells (iPSCs) enable the personalized study of RDEB pathogenesis and potential therapies. However,current skin cell differentiation protocols via 2D culture perform suboptimally when applied to engineered 3D skin constructs (ESC). Here,we present an approach to source fibroblasts (iFBs) and keratinocytes (iKCs) from iPSC-derived skin organoids using an optimized differentiation protocol,and utilize them to engineer ESCs modeling wild-type and RDEB phenotypes. The resulting iPSC-derived skin cells display marker expression consistent with primary counterparts and produce ESCs exhibiting significant extracellular matrix remodeling,protein deposition,and epidermal differentiation. RDEB constructs recapitulated hallmark disease features,including absence of collagen VII and reduced iFB proliferation. This work establishes a robust and scalable strategy for generating physiologically-relevant,iPSC-derived skin constructs,offering a powerful model for studying RDEB mechanisms and advancing personalized regenerative medicine. View Publication

Cutaneous tuberculosis (CTB) is an infectious disease highly associated with extracellular matrix remodeling and granuloma-driven fibrosis. Fibroblasts play crucial roles in this fibrotic process,but their specific roles in Mycobacterium tuberculosis (Mtb) skin infections remain unclear due to the lack of proper in vitro models. Here,we demonstrate that skin organoids (SKOs) derived from human induced pluripotent stem cells can model CTB infected by Mtb. Single-cell RNA analyses reveal an increase in fibroblasts,upregulation of genes involved in collagen synthesis,and enhanced collagen degradation induced by MMP2 and MMP14 in Mtb-infected SKOs. This is accompanied by the destruction of nerve cells and adipocytes. Importantly,the onset of fibrosis in Mtb-infected SKOs is dependent on the activation of the PI3K-AKT signaling pathway and transcription factor AP1 in fibroblasts. Pharmacological inhibition of PI3K-AKT and AP1 alleviates fibrosis and collagen deposition. Our findings have uncovered distinct alterations in cell populations during Mtb-induced skin fibrosis,highlighting the crucial roles of PI3K-AKT and AP1. The study demonstrates the utility of SKOs for investigating CTB pathogenesis and evaluating potential antifibrotic treatments. Cutaneous tuberculosis is an infectious disease associated with extracellular matrix remodeling and granuloma-driven fibrosis. Here,the authors present an in vitro model of this disease using skin organoids infected with Mycobacterium tuberculosis,and describe infection-induced alterations in specific pathways and cell populations. View Publication

Acute myeloid leukemia (AML) is primarily driven by leukemic stem cells (LSCs),the main cause of relapse and therapy resistance. Here,we discover that LSCs are predominantly small and mechanically soft. These mechanical properties enable their selective isolation using microfluidic chips. Single-cell RNA-sequencing of primary human AML bone marrow identifies enrichment of LSCs within the FSClow ALDH1A1+ subpopulation,which exhibits long-term stemness in functional assays. Notably,inhibiting ALDH1A1 in these cells promotes F-actin polymerization and increases cellular stiffness,reducing their stemness while enhancing their susceptibility to natural killer (NK) cell-mediated cytotoxicity. In AML patient-derived xenograft models,the combination of ALDH1A1 inhibition with NK cell therapy markedly suppresses leukemia progression. These findings suggest that targeting the mechanical properties of LSC offers a promising strategy to overcome AML treatment resistance,providing insights into stem cell mechanobiology and paving the way for combining targeted therapies with immunotherapy to improve clinical outcomes. Leukemic stem cells (LSCs) drive relapse and therapy resistance in acute myeloid leukemia (AML). Here,the authors show that increasing the stiffness of LSCs reduces their stemness and enhances their susceptibility to natural killer cell-mediated immunotherapy in AML. View Publication

Background: Acute Lung Injury (ALI) and its most severe form,Acute Respiratory Distress Syndrome (ARDS),are critical pulmonary conditions characterized by life-threatening acute hypoxic respiratory failure,affecting over three million individuals globally each year. ALI involves alveolar inflammation and disruption of the alveolar-capillary barrier,primarily driven by neutrophil infiltration and the release of inflammatory mediators. In our previous study using a lipopolysaccharide (LPS)-induced mouse model of ALI,we demonstrated that C6,a peptide inhibitor of voltage-gated proton channels (Hv1),ameliorates lung injury,identifying Hv1 as a potential therapeutic target. However,(i) whether the anti-inflammatory effects of C6 are translatable to a clinically relevant live bacterial infection model,and (ii) the molecular mechanisms underlying these anti-inflammatory effects,remain unknown,and are a crucial next step towards targeted rational drug development. Methods: To induce ALI,we used an intratracheal Pseudomonas aeruginosa infection model,a gram-negative bacterium relevant in ventilated and immunocompromised patients. A separate group of infected mice also received intravenous treatment with C6 (4 mg/kg). Lung injury severity was evaluated using histopathological analysis. Bronchoalveolar lavage (BAL) fluid was collected to quantify neutrophil infiltration and proinflammatory cytokines concentrations. In addition,reactive oxygen species (ROS) production and intracellular calcium levels in BAL neutrophils were measured. RNA sequencing of BAL neutrophils was conducted to assess C6-induced transcriptional changes. Key findings were validated in vitro using human neutrophils. Results: C6 mitigates P. aeruginosa-induced ALI in mice by reducing neutrophil infiltration into the alveolar space by ~ 86%,improving lung injury scores,decreasing BAL fluid proinflammatory cytokine levels,and suppressing neutrophil ROS production and intracellular calcium levels. RNA sequencing of BAL neutrophils revealed 51 downregulated genes,including key regulators of neutrophil migration,cytokine release,and ROS production; only three genes were upregulated and they also have roles in neutrophil immune defense. In human neutrophils,C6 similarly inhibited chemotaxis and reduced ROS and cytokine release,and calcium influx. Conclusions: Targeting Hv1 with C6 effectively protects against P. aeruginosa-induced ALI by limiting neutrophil recruitment and activation. These findings establish C6 as a promising therapeutic candidate against infectious ALI and provide important mechanistic insights into its immunomodulatory effects on neutrophils. View Publication

How the neuroectoderm-derived eye field breaks symmetry to specify iris muscle is not well understood. Recent studies have begun to transcriptionally characterize mouse iris muscle; however,little is known about the transcriptional foundation of human iris development. Human pluripotent stem cells (hPSCs) enable the study of iris muscle specification. Here we compare iris smooth muscle from native adult iris tissues to evaluate successful specification of iris muscle from hPSC lines. We utilize a previously published eye-like organoid protocol that specified cells of the eye field to also generate iris muscle. We describe a population transcriptionally similar to native iris and describe an iris muscle gene signature. Human iris muscle not only contains pigment,but also expresses pigment synthesis genes and is responsive to acetylcholine. Integration of single-cell RNA-seq datasets confirm the similarity between the iris muscle to the adult iris,establishing the usefulness of the model in studying neuroectoderm-derived iris muscle specification,and related diseases. Single-cell RNA sequencing reveals that iris muscle,derived from neuroectoderm,can form in stem cell–derived eye organoids – enabling the modelling of iris muscle pathologies like aniridia and proliferative vitreoretinopathy. View Publication