技术资料

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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 View Publication

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 View Publication

CSMD1 (Cub and Sushi Multiple Domains 1) is a well-recognized regulator of the complement cascade,an important component of the innate immune response. CSMD1 is highly expressed in the central nervous system (CNS) where emergent functions of the complement pathway modulate neural development and synaptic activity. While a genetic risk factor for neuropsychiatric disorders,the role of CSMD1 in neurodevelopmental disorders is unclear. Through international variant sharing,we identified inherited biallelic CSMD1 variants in eight individuals from six families of diverse ancestry who present with global developmental delay,intellectual disability,microcephaly,and polymicrogyria. We modeled CSMD1 loss-of-function (LOF) pathogenesis in early-stage forebrain organoids differentiated from CSMD1 knockout human embryonic stem cells (hESCs). We show that CSMD1 is necessary for neuroepithelial cytoarchitecture and synchronous differentiation. In summary,we identified a critical role for CSMD1 in brain development and biallelic CSMD1 variants as the molecular basis of a previously undefined neurodevelopmental disorder. View Publication

Neural stem cells have intact innate immune responses that protect them from virus infection and cell death. Yet,viruses can antagonize such responses to establish neuropathogenesis. Using a forebrain organoid model system at two developmental time points,we identified that neural stem cells,in particular radial glia,are basally primed to respond to virus infection by upregulating several antiviral interferon-stimulated genes. Infection of these organoids with a neuropathogenic Enterovirus-D68 strain,demonstrated the ability of this virus to impede immune activation by blocking interferon responses. Together,our data highlight immune gene signatures present in different types of neural stem cells and differential viral capacity to block neural-specific immune induction.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-024-03275-5. View Publication

Understanding how embryonic progenitors decode extrinsic signals and transform into lineage-specific regulatory networks to drive cell fate specification is a fundamental,yet challenging question. Here,we develop a new model of surface epithelium (SE) differentiation induced by human embryonic stem cells (hESCs) using retinoic acid (RA),and identify BMP4 as an essential downstream signal in this process. We show that the retinoid X receptors,RXRA and RXRB,orchestrate SE commitment by shaping lineage-specific epigenetic and transcriptomic landscapes. Moreover,we find that TCF7,as a RA effector,regulates the transition from pluripotency to SE initiation by directly silencing pluripotency genes and activating SE genes. MSX2,a downstream activator of TCF7,primes the SE chromatin accessibility landscape and activates SE genes. Our work reveals the regulatory hierarchy between key morphogens RA and BMP4 in SE development,and demonstrates how the TCF7-MSX2 axis governs SE fate,providing novel insights into RA-mediated regulatory principles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05525-4. View Publication

Astroglia are integral to brain development and the emergence of neurodevelopmental disorders. However,studying the pathophysiology of human astroglia using brain organoid models has been hindered by inefficient astrogliogenesis. In this study,we introduce a robust method for generating astroglia-enriched organoids through BMP4 treatment during the neural differentiation phase of organoid development. Our RNA sequencing analysis reveals that astroglia developed within these organoids exhibit advanced developmental characteristics and enhanced synaptic functions compared to those grown under traditional two-dimensional conditions,particularly highlighted by increased neurexin (NRXN)-neuroligin (NLGN) signaling. Cell adhesion molecules,such as NRXN and NLGN,are essential in regulating interactions between astroglia and neurons. We further discovered that brain organoids derived from human embryonic stem cells (hESCs) harboring the autism-associated NLGN3 R451C mutation exhibit increased astrogliogenesis. Notably,the NLGN3 R451C astroglia demonstrate enhanced branching,indicating a more intricate morphology. Interestingly,our RNA sequencing data suggest that these mutant astroglia significantly upregulate pathways that support neural functions when compared to isogenic wild-type astroglia. Our findings establish a novel astroglia-enriched organoid model,offering a valuable platform for probing the roles of human astroglia in brain development and related disorders.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-024-00219-5. View Publication

ABSTRACTLoss of Cdx2 in vivo leads to stunted development of the allantois,an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here,we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT),heterozygous (CDX2-Het),and homozygous null CDX2 (CDX2-KO) genotypes,differentiating these cells in a 2D gastruloid model,and subjecting these cells to single-nucleus RNA and ATAC sequencing,we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile,suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo,we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together,these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo. Summary: Using 2D human gastruloids,CDX2 is shown to dose-dependently influence genes related to tissue permeability,cell-cell adhesions,and cytoskeletal architecture during extraembryonic mesoderm development. View Publication