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Induced pluripotent stem cells (iPSCs) are commonly used to model human genetic diseases. Two main strategies are used. The first involves making iPSC lines from individual cases with a disease, and the second involves making disease-relevant gene edits in established iPSC lines. Because generating gene-edited lines is time consuming and expensive, most studies begin with one starting iPSC stock line and evaluate several gene-edited sublines. The current studies focus on gene-editing to model Lesch–Nyhan disease (LND), which is caused by mutations in the HPRT1 gene. The same pathogenic c.508C>T edit was made in four well-established stock lines, and three gene-edited lines were isolated from each. RNA sequencing (RNAseq) was, then, used to evaluate the impact of the gene edit. Gene-edited lines were compared to their corresponding stock lines, as well as to each other. An aggregate analysis of all lines combined was also conducted to determine the most robust findings across all lines. Results from gene editing were further compared with iPSC lines derived from individual cases with LND, to determine how closely findings from gene editing match results obtained with case-derived lines. There were two main findings. First, the same gene edit has a different impact on gene expression when starting with different starting stock lines. Second, the gene editing strategy does not produce the same results as the case-derived strategy. Potential explanations for these differences are addressed, along with the relevance of these two different strategies for disease modeling.

Background: Chronic myocarditis (CMYO) progresses to fibrosis and heart failure, yet no therapies effectively target fibrosis. Fibroblast activation protein (FAP) marks pathogenic myofibroblasts, but its therapeutic potential remains unexplored in inflammatory settings.Methods: Using bulk/scRNA-seq of human myocarditis samples, we identified FAP as a fibrosis-specific marker. We engineered FAP-targeted CAR-T (FAP.CAR-T) cells and tested their efficacy in autoimmune (EAM) and viral (CVB3) myocarditis models. Human cardiac organoids (hCOs) treated with IL-17A modeled inflammatory fibrosis.Results: FAP expression correlated with fibrosis severity in patients (r = 0.96, P = 0.0028). In EAM and CVB3 models, FAP.CAR-T cells reduced fibrosis by 65% and 55%, respectively (P < 0.001), restored ejection fraction to higher than 65%. hCOs treated with FAP.CAR-T cells showed 55% less fibrosis (P < 0.05). No toxicity was observed in healthy mice.Conclusions: FAP.CAR-T cells eliminate fibrosis-driving myofibroblasts, reversing cardiac dysfunction in chronic myocarditis. This strategy, validated in human organoids, offers translatable immunotherapy for fibrosis-driven heart disease.

Neural crest induction begins early during neural plate formation, requiring precise transcriptional control to activate lineage-specific enhancers. Here, we demonstrate that SALL4, a transcription factor associated with syndromes featuring craniofacial anomalies, plays a crucial role in early cranial neural crest (CNCC) specification. Using SALL4-het-KO human iPSCs to model clinical haploinsufficiency, we show that SALL4 directly recruits BAF to CNCC-lineage specific enhancers at the neuroectodermal stage, specifically when neural crest gene expression is induced at the neural plate border. Without functional SALL4, BAF is not loaded at chromatin, leaving CNCC enhancers inaccessible. Consequently, the cells cannot undergo proper CNCC induction and specification due to persistent enhancer repression, despite normal neuroectodermal and neural plate progression. Moreover, by performing SALL4 isoform-specific depletion, we demonstrate that SALL4A is the isoform essential for CNCC induction and specification, and that SALL4B cannot compensate for SALL4A loss in this developmental process. In summary, our findings reveal SALL4 as essential regulator of BAF-dependent enhancer activation during early stages of neural crest development, providing molecular insights into SALL4-associated craniofacial anomalies.

Osteosarcoma (OSA) is the most common primary bone tumor in children and adolescents, yet outcomes have remained largely unchanged for over 40 years. While chimeric antigen receptor (CAR) T cell therapy has shown success in blood cancers, it faces major limitations in solid tumors due to immune evasion, antigen loss, and immunosuppressive tumor microenvironments. Natural killer (NK) cells offer several advantages over T cells, including multiple killing mechanisms and lower risks of graft-versus-host disease, neurotoxicity, and cytokine release syndrome, making them promising candidates for off-the-shelf cell therapies. However, unmodified NK cells have shown limited efficacy in clinical settings due to poor engraftment, persistence, and tumor-mediated suppression. To overcome these barriers, we developed a cost-effective method to engineer CAR NK cells targeting CD70, a tumor antigen overexpressed in relapsed and metastatic OSA. We further enhanced these cells by incorporating soluble interleukin-15 (IL-15) and a dominant-negative TGF-β receptor, creating “armored” CAR NK cells. These engineered cells resist transforming growth factor β (TGF-β) suppression, secrete IL-15, and demonstrate improved cytotoxicity, persistence, and tumor homing in both in vitro and in vivo models. Our findings support CD70 CAR NK cells as a promising immunotherapeutic strategy for relapsed and metastatic OSA. Graphical abstract Engineered “armored” CAR NK cells targeting CD70 overcome immune suppression in osteosarcoma, enhancing persistence, tumor homing, and cytotoxicity. This study presents a promising off-the-shelf immunotherapy approach for relapsed and metastatic OSA, offering a potential advance where current treatments have stagnated for decades.

Mantle cell lymphoma (MCL) is a clinically aggressive and incurable form of non-Hodgkin lymphoma with very heterogeneous clinical and biological behaviors. Dysregulation of RNA splicing machinery is common in various types of cancer, including hematologic malignancies, and is associated with cancer progression. However, whether and how splicing factors, the spliceosome components responsible for pre-mRNA splicing, regulate MCL aggressive behaviors remain largely unknown. Bioinformatics analyses were employed to profile the mRNA expression of two key families of splicing factors, that are serine/arginine-rich (SR) and heterogenous nuclear ribonucleoprotein (hnRNP) families, in clinical specimens of MCL patients in comparison to normal B cells. Functional roles of splicing factors in MCL aggressive phenotypes defined as hallmarks of cancer were investigated in MCL cell lines. Kaplan–Meier survival analyses were performed to determine the clinical significance of splicing factors according to their gene expression level. Depletion of SRSF1, hnRNP F, and PTBP1, which are highly expressed in MCL clinical specimens, by CRISPR/Cas9 system significantly inhibit cell growth and proliferation, motility, and angiogenesis of MCL cells. SRSF1, hnRNP F (for Z-138 cells), and PTBP1 were found to mediate BTZ sensitivity in MCL cells, in agreement with an increase in caspase-3 activation and the occurrence of splicing events that favor the expression of pro-apoptotic isoforms of apoptosis regulatory genes. We also found that depletion of SRSF1, hnRNP F, and PTBP1 induces the expression of autophagy-related genes and the accumulation of autophagic vacuoles, which may act as one of the tumor-suppressive mechanisms. Survival analyses revealed that co-high expression of SRSF1, HNRNPF, or PTBP1 and oncogenic MYC predicts poor clinical outcomes in MCL patients. Our data describe the clinical significance of aberrant SRSF1, hnRNP F, and PTBP1 in MCL and their tumor-promoting roles via the regulation of cancer hallmarks, which could be important in understanding MCL pathogenesis and therapeutic development.

Over the past decade, significant advancements have been made in understanding the developmental mechanisms involved in human gastrointestinal formation, with organoids emerging as key experimental models. These three‐dimensional in vitro cellular structures mimic the organization and functions of various gut regions, providing a powerful tool for research. By replicating critical stages of gut development, we can now direct the differentiation of cells into specific gastrointestinal tissues. In this protocol, we outline how to generate two types of organoids derived from human pluripotent stem cells (hPSCs): human intestinal organoids (HIOs) and human colonic organoids (HCOs). First, we induce definitive endoderm formation to produce these organoids and specify midgut/hindgut tissues. Three‐dimensional spheroids form spontaneously, can be collected, embedded in an extracellular matrix, and cultured over time. During this phase, the organoid epithelium develops, supported by a mesenchymal layer that promotes maturation and differentiation. After a month of culture, HIOs and HCOs reach a developmental and maturation stage comparable to that of the human fetal intestine. These organoids can be used to study human gastrointestinal development, model diseases, and test therapeutic agents. Human pluripotent stem cells can be guided through a stepwise differentiation process to produce self‐organizing intestinal and colonic organoids. The resulting organoids recapitulate fetal‐stage epithelial and mesenchymal organization, offering a powerful model to explore human gastrointestinal development and its disorders.

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.

We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ, MMEJ), and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1), affecting several key DNA repair proteins, such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling, gene therapy, chimeric antigen receptor immunotherapy, and cancer treatment. DNA repair pathways shape CRISPR editing outcomes. Here, authors identified FDA approved drugs that can be repurposed as repair modulators or to induce synthetic lethality, and uncovered new roles for ESR2 and AOX1 in DNA repair, enhancing editing and offering potential therapeutic applications.

Despite the progressive extension of CFTR variant eligibility to the triple combination of elexacaftor/tezacaftor/ivacaftor (ETI), most rare CFTR pathogenic variants remain ineligible for CFTR modulators. It is crucial to determine whether unexplored variants are rescuable by clinical modulators and to identify innovative therapeutic strategies for rescuing non-responder variants. The approach known as “theratyping” (in vitro testing of genotypes) has been accepted by the Food and Drug Administration (FDA) for the extension of clinical modulators’ approval for in vitro responding genotypes. We used one of the most advanced models for theratyping: organoids derived from nasal epithelia of people with cystic fibrosis (pwCF). We optimized the forskolin-induced swelling (FIS) of organoids to assess CFTR basal or modulator-restored function. Nasal organoids mimicked the original epithelial tissue, CFTR residual activity, and modulator response. We set up the FIS assay using nasal organoids with reference genotypes and theratyped 38 rare (non-F508del) CFTR genotypes, either eligible or non-eligible for FDA approval, for treatment with ETI or ivacaftor. We found strong correspondence between the in vitro response of CFTR variants to modulators and their FDA approval status. Additionally, some previously uncharacterized CFTR variants have proven responsive to clinical modulators, with significant therapeutic implications. These results suggest that the nasal organoid FIS assay, pending confirmation of the prediction in the corresponding pwCF, might be considered as a powerful in vitro tool to predict modulator efficacy in each pwCF, guiding out-of-label prescription in CF, and to identify uncharacterized variants responsive to modulators. This approach may allow comparison of the efficacy of different therapeutics or the identification of innovative strategies for non-responding genotypes, improving personalized therapy and quality of life for pwCF.

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.

The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations. Genome editing often requires marker genes for selection of edited cells. Here, the authors present SNIPE, a marker-free method that selects cells based on DNA sequence, enabling precise enrichment of edited cells and applications from evolutionary research to the elimination of cancer cells.

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.