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SummaryIn Drosophila, long noncoding RNA Hsr? rapidly assembles membraneless organelle omega speckles under heat shock with unknown biological function. Here, we identified the distribution of omega speckles in multiple tissues of adult Drosophila melanogaster and found that they were selectively distributed in differentiated enterocytes but not in the intestinal stem cells of the midgut. We mimicked the high expression level of Hsr? via overexpression or intense heat shock and demonstrated that the assembly of omega speckles nucleates TBPH for the induction of ISC differentiation. Additionally, we found that heat shock stress promoted cell differentiation, which is conserved in mammalian cells through paraspeckles, resulting in large puncta of TDP-43 (a homolog of TBPH) with less mobility and the differentiation of human induced pluripotent stem cells. Overall, our findings confirm the role of Hsr? and omega speckles in the development of intestinal cells and provide new prospects for the establishment of stem cell differentiation strategies. Graphical abstract Highlights•LncRNA Hsr? is differentially expressed in different cell types of fly midguts•Omega speckles nucleate TPBH and promote the differentiation of ISCs to ECs•Heat shock treatment induces the assembly of omega speckles and paraspeckles•Heat shock treatment accelerates the differentiation of fly midguts and human iPSCs Molecular biology; Cell biology; Developmental biology

SUMMARY Alveolar epithelial type I cells (AT1s) line the gas exchange barrier of the distal lung and have been historically challenging to isolate or maintain in cell culture. Here, we engineer a human in vitro AT1 model system via directed differentiation of induced pluripotent stem cells (iPSCs). We use primary adult AT1 global transcriptomes to suggest benchmarks and pathways, such as Hippo-LATS-YAP/TAZ signaling, enriched in these cells. Next, we generate iPSC-derived alveolar epithelial type II cells (AT2s) and find that nuclear YAP signaling is sufficient to promote a broad transcriptomic shift from AT2 to AT1 gene programs. The resulting cells express a molecular, morphologic, and functional phenotype reminiscent of human AT1 cells, including the capacity to form a flat epithelial barrier producing characteristic extracellular matrix molecules and secreted ligands. Our results provide an in vitro model of human alveolar epithelial differentiation and a potential source of human AT1s. In brief Kotton and colleagues generate human alveolar epithelial type I cells (AT1s) from induced pluripotent stem cells (iPSCs). The resulting cells can be grown as 3D organoids or in 2D air-liquid interface cultures, displaying many of the molecular, morphologic, and functional phenotypes of primary AT1s. Graphical abstract

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells, followed by the migration, differentiation, and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens, such as FGF8. Disruption of this developmental balance can lead to brain malformations, which underlie a range of complex neurodevelopmental disorders, including epilepsy, autism, and intellectual disabilities. Studying the early stages of human brain development, whether under normal or pathological conditions, remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently, human brain organoids have emerged as a powerful in vitro alternative, allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures, where neural progenitors and neurons are grown on flat surfaces, 3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However, 3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore, few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning.• To address these limitations, we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs), our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation, NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors, where they self-organize into neural rosettes and neuroepithelial structures, surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids, leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity, enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas. Key features • This protocol builds on the method established by Chambers et al. [1] for generating 2D neural progenitors, followed by dissociation and reaggregation into 3D brain organoids.• For optimal growth and maturation, telencephalic organoids are cultured in spinning mini-bioreactors [2] or on orbital shakers.• The protocol enables the generation of telencephalic neural progenitors in 10 days and produces 3D telencephalic organoids containing neocortical neurons within one month of culture.• Addition of morphogens in the culture medium (example: FGF8) enhances cellular heterogeneity, promoting the emergence of distinct brain domains within a single organoid.

Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of hepatic disorders, ranging from simple steatosis to steatohepatitis, with the most severe outcomes including cirrhosis, liver failure, and hepatocellular carcinoma. Notably, MASLD prevalence is lower in premenopausal women than in men, suggesting a potential protective role of estrogens in mitigating disease onset and progression. In this study, we utilized preclinical in vitro models—immortalized cell lines and hepatocyte-like cells derived from human embryonic stem cells—exposed to clinically relevant steatotic-inducing agents. These exposures led to lipid droplet (LD) accumulation, increased reactive oxygen species (ROS) levels, and mitochondrial dysfunction, along with decreased expression of markers associated with hepatocyte functionality and differentiation. Estrogen treatment in steatotic-induced liver cells resulted in reduced ROS levels and LD content while preserving mitochondrial integrity, mediated by the upregulation of mitochondrial thioredoxin 2 (TRX2), an antioxidant system regulated by the estrogen receptor. Furthermore, disruption of TRX2, either pharmacologically using auranofin or through genetic interference, was sufficient to counteract the protective effects of estrogens, highlighting a potential mechanism through which estrogens may prevent or slow MASLD progression.

BackgroundRecent studies highlight the critical role of microglia in neurodegenerative disorders, and emphasize the need for humanized models to accurately study microglial responses. Human-mouse microglia xenotransplantation models are a valuable platform for functional studies and for testing therapeutic approaches, yet currently those models are only available for academic research. This hampers their implementation for the development and testing of medication that targets human microglia.MethodsWe developed the hCSF1Bdes mouse line, which is suitable as a new transplantation model and available to be crossed to any disease model of interest. The hCSF1Bdes model created by CRISPR gene editing is RAG2 deficient and expresses human CSF1. Additionally, we crossed this model with two humanized App KI mice, the AppHu and the AppSAA. Flow cytometry, immunohistochemistry and bulk sequencing was used to study the response of microglia in the context of Alzheimer’s disease.ResultsOur results demonstrate the successful transplantation of iPSC-derived human microglia into the brains of hCSF1Bdes mice without triggering a NK-driven immune response. Furthermore, we confirmed the multipronged response of microglia in the context of Alzheimer’s disease. The hCSF1Bdes and the crosses with the Alzheimer’s disease knock-in model AppSAA and the humanized App knock-in control mice, AppHu are deposited with EMMA and fully accessible to the research community.ConclusionThe hCSF1Bdes mouse is available for both non-profit and for-profit organisations, facilitating the use of the xenotransplantation paradigm for human microglia to study complex human disease.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13024-025-00823-2.

Background: Pulmonary fibrosis is a major disease that leads to the progressive loss of lung function. The disease manifests early, resulting in type 2 respiratory failure. This is likely due to the bronchocentric fibrosis around the major airways, which causes airflow limitation. It affects approximately three million patients worldwide and has a poor prognosis. Skin fibroblasts isolated from patients offer valuable insights into understanding the disease mechanisms, identifying the genetic causes, and developing personalized therapies. However, the use of skin fibroblasts to study a disease that exclusively impacts the lungs is often questioned, particularly since lung fibrosis primarily affects the alveolar epithelium. Method: We report the reprogramming of skin fibroblasts from patients with an atypical early-onset form of lung fibrosis into induced pluripotent stem cells (iPSCs) and subsequently into alveolar epithelial cells. This was achieved using a Sendai virus approach. Results: We show that the reprogrammed cells carry mutations in the calcium-binding protein genes S100A3 and S100A13, leading to diminished protein expression, thus mimicking the patients’ cells. Additionally, we demonstrate that the generated patient iPSCs exhibit aberrant calcium and mitochondrial functions. Conclusions: Due to the lack of a suitable animal model that accurately resembles the human disease, generating patient lung cells from these iPSCs can provide a valuable “disease in a dish” model for studying the atypical form of inherited lung fibrosis. This condition is associated with mutations in the calcium-binding protein genes S100A3 (NM_002960) and S100A13 (NM_001024210), aiding in the understanding of its pathogenesis.

Uncovering the stimulus-response histories that give rise to cell fates and behaviors is an area of great interest in developmental biology, tissue engineering, and regenerative medicine. A comprehensive accounting of cell experiences that lead to the development of organs and tissues can help us to understand developmental anomalies that may underly disease. Perhaps more provocatively, such a record can also reveal clues as to how to drive cell collective decision-making processes, which may yield predictable cell-based therapies or facilitate production of tissue substitutes for transplantation or in vitro screening of prospective therapies to mitigate disease. Toward this end, various methods have been applied to molecularly trace developmental trajectories and record interaction histories of cells. Typical methods involve artificial gene circuits based on recombinases that activate a suite of fluorescent reporters or CRISPR-Cas9 genome writing technologies whose nucleic acid-based record keeping serves to chronicle cell-cell interactions or past exposure to stimuli of interests. Exciting expansions of the synthetic biology toolkit with artificial receptors that permit establishment of defined input-to-output linkages of cell decision-making processes opens the door to not only record cell-cell interactions, but to also potentiate directed manipulation of the outcomes of such interactions via regulation of carefully selected transgenes. Here, we combine CRISPR-based strategies to genetically and epigenetically manipulate cells to express components of the synthetic Notch receptor platform, a widely used artificial cell signaling module. Our approach gives rise to the ability to conditionally record interactions between human cells, where the record of engagement depends on expression of a state-specific marker of a subset of cells in a population. Further, such signal-competent interactions can be used to direct differentiation of human embryonic stem cells toward pre-selected fates based on assigned synNotch outputs. We also implemented CRISPR-based manipulation of native gene expression profiles to bias outcomes of cell engagement histories in a targeted manner. Thus, we present a useful strategy that gives rise to both state-specific recording of cell-cell interactions as well as methods to intentionally influence products of such cell-cell exchanges.

Animal models have proven integral to broadening our understanding of complex cardiac diseases but have been hampered by significant species-dependent differences in cellular physiology. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have shown great promise in the modelling of cardiac diseases despite limitations in functional and structural maturity. 3D stem cell-derived cardiac models represent a step towards mimicking the intricate microenvironment present in the heart as an in vitro model. Incorporation of non-myocyte cell types, such as cardiac fibroblasts, into engineered heart tissue models (EHTs) can help better recapitulate the cell-to-cell and cell-to-matrix interactions present in the human myocardium. Integration of human-induced pluripotent stem cell-derived cardiac fibroblasts (hiPSC-CFs) and hiPSC-CM into EHT models enables the generation of a genetically homogeneous modelling system capable of exploring the abstruse structural and electrophysiological interplay present in cardiac pathophysiology. Furthermore, the construction of more physiologically relevant 3D cardiac models offers great potential in the replacement of animals in heart disease research. Here we describe efficient and reproducible protocols for the differentiation of hiPSC-CMs and hiPSC-CFs and their subsequent assimilation into EHTs. The resultant EHT consists of longitudinally arranged iPSC-CMs, incorporated alongside hiPSC-CFs. EHTs with both hiPSC-CMs and hiPSC-CFs exhibit slower beating frequencies and enhanced contractile force compared to those composed of hiPSC-CMs alone. The modified protocol may help better characterise the interplay between different cell types in the myocardium and their contribution to structural remodelling and cardiac fibrosis.

Human trophoblast stem (TS) cells are an informative in vitro model for the generation and testing of biologically meaningful hypotheses. The goal of this project was to derive patient-specific TS cell lines from clinically available chorionic villus sampling biopsies. Cell outgrowths were captured from human chorionic villus tissue specimens cultured in modified human TS cell medium. Cell colonies emerged early during the culture and cell lines were established and passaged for several generations. Karyotypes of the newly established chorionic villus-derived trophoblast stem (TSCV) cell lines were determined and compared to initial genetic diagnoses from freshly isolated chorionic villi. Phenotypes of TSCV cells in the stem state and following differentiation were compared to cytotrophoblast-derived TS (TSCT) cells. TSCV and TSCT cells uniformly exhibited similarities in the stem state and following differentiation into syncytiotrophoblast and extravillous trophoblast cells. Chorionic villus tissue specimens provide a valuable source for TS cell derivation. They expand the genetic diversity of available TS cells and are associated with defined clinical outcomes. TSCV cell lines provide a new set of experimental tools for investigating trophoblast cell lineage development.

Abstract ETV6::RUNX1, the most common oncogenic fusion in pediatric B cell precursor acute lymphoblastic leukemia (BCP?ALL), induces a clinically silent preleukemic state that can persist in carriers for over a decade and may progress to overt leukemia upon acquisition of secondary lesions. The mechanisms contributing to quiescence of ETV6::RUNX1+ preleukemic cells still remain elusive. In this study, we identify linker histone H1?0 as a critical mediator of the ETV6::RUNX1+ preleukemic state by employing human ?induced pluripotent stem cell (hiPSC) models engineered by using CRISPR/Cas9 gene editing. Global gene expression analysis revealed upregulation of H1?0 in ETV6::RUNX1+ hiPSCs that was preserved upon hematopoietic differentiation. Moreover, whole transcriptome data of 1,727 leukemia patient samples showed significantly elevated H1?0 levels in ETV6::RUNX1+?BCP?ALL compared to other leukemia entities. Using dual?luciferase promoter assays, we show that ETV6::RUNX1 induces H1?0 promoter activity. We further demonstrate that depletion of H1?0 specifically inhibits ETV6::RUNX1 signature genes, including RAG1 and EPOR. Single?cell sequencing showed that H1?0 is highly expressed in quiescent hematopoietic cells. Importantly, H1?0 protein levels correspond to susceptibility of BCP?ALL cells towards histone deacetylase inhibitors (HDACis) and combinatorial treatment using the H1?0?inducing HDACi Quisinostat showed promising synergism with established chemotherapeutic drugs. Taken together, our data identify H1?0 as a key regulator of the ETV6::RUNX1+ transcriptome and indicate that the addition of Quisinostat may be beneficial to target non?responsive or relapsing ETV6::RUNX1+?BCP?ALL.

Induced pluripotent stem cell (iPSC) derived endothelial cells (iECs) have emerged as a promising tool for studying vascular biology and providing a platform for modelling various vascular diseases, including those with genetic origins. Currently, primary ECs are the main source for disease modelling in this field. However, they are difficult to edit and have a limited lifespan. To study the effects of targeted mutations on an endogenous level, we generated and characterized an iPSC derived model for venous malformations (VMs). CRISPR-Cas9 technology was used to generate a novel human iPSC line with an amino acid substitution L914F in the TIE2 receptor, known to cause VMs. This enabled us to study the differential effects of VM causative mutations in iECs in multiple in vitro models and assess their ability to form vessels in vivo. The analysis of TIE2 expression levels in TIE2L914F iECs showed a significantly lower expression of TIE2 on mRNA and protein level, which has not been observed before due to a lack of models with endogenous edited TIE2L914F and sparse patient data. Interestingly, the TIE2 pathway was still significantly upregulated and TIE2 showed high levels of phosphorylation. TIE2L914F iECs exhibited dysregulated angiogenesis markers and upregulated migration capability, while proliferation was not affected. Under shear stress TIE2L914F iECs showed reduced alignment in the flow direction and a larger cell area than TIE2WT iECs. In summary, we developed a novel TIE2L914F iPSC-derived iEC model and characterized it in multiple in vitro models. The model can be used in future work for drug screening for novel treatments for VMs.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10456-024-09925-9.

Glioblastoma (GBM) is a malignant brain tumor with diffuse infiltration. Here, we demonstrate how GBM cells usurp guidance receptor Plexin-B2 for confined migration through restricted space. Using live-cell imaging to track GBM cells negotiating microchannels, we reveal endocytic vesicle accumulation at cell front and filamentous actin assembly at cell rear in a polarized manner. These processes are interconnected and require Plexin-B2 signaling. We further show that Plexin-B2 governs membrane tension and other membrane features such as endocytosis, phospholipid composition, and inner leaflet surface charge, thus providing biophysical mechanisms by which Plexin-B2 promotes GBM invasion. Together, our studies unveil how GBM cells regulate membrane tension and mechano-electrical coupling to adapt to physical constraints and achieve polarized confined migration. The biomechanical mechanisms enabling the invasive growth of brain tumors remain opaque. Here, Junqueira Alves et al. reveal that the guidance receptor Plexin-B2 controls membrane tension, facilitating confined migration of brain tumor cells.