参考文献

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

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

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

SummaryCryosectioning remains the gold standard for antibody and transcriptomic/in situ hybridization tissue analysis. However,tissue processing is time-consuming and costly,limiting routine and diagnostic use. Currently,no commercially available protocols or products exist for multiplexing this process. Here,we introduce multiplexed tissue molds (MTMs) that enable high-throughput cryoprocessing—cutting costs and workload by up to 96% while permitting the processing of tissues of various sizes and origins. We demonstrate compatibility with heterogeneous tissues by processing 19 different adult mouse tissues in parallel. Furthermore,we process up to ?110 neural organoids of different ages and sizes simultaneously and assess their neural differentiation marker expression. MTMs allow sectioning-based tissue analysis when labor,time,and cost are limiting factors. MTMs could be used to compare high specimen numbers in histopathological settings,organism-wide antigen and antibody targeting studies,high-throughput tissue screens,and defined tissue section positioning for,e.g.,spatial transcriptomics experiments. Graphical abstract Highlights•Multiplexed tissue molds (MTMs) drastically upscale cryosectioning procedures•MTMs can simultaneously accommodate up to 19 mouse organs and ?110 cerebral organoids•MTMs reduce analysis costs and processing times of tissues by up to 96%•MTMs could be used to reduce diagnostic costs and for spatial transcriptomics MotivationEfficient cryosectioning remains a critical yet labor- and cost-intensive step for immunohistochemistry and in situ hybridization,limiting routine diagnostic and research applications. The increasing demand for high-throughput tissue analysis—driven by advances in organoid and three-dimensional (3D) culture systems and tissue analysis for diagnostics—necessitates methods capable of processing numerous heterogeneous samples simultaneously. Current protocols lack multiplexing capabilities,leading to variability and extended processing times. Our work introduces multiplexed tissue molds (MTMs),a scalable solution that drastically reduces costs and labor by up to 96% while maintaining tissue integrity and consistency,thereby enabling large-scale (>100 tissues) comparative analyses and enhanced experimental reproducibility as well as access to tissue analysis,where cost is a restrictive factor. Reumann et al. develop multiplexed tissue molds (MTMs),which allow upscaling of tissue processing (up to 19 mouse organs or ?110 cerebral organoids simultaneously) while reducing workload and associated analysis costs by up to 96%. MTMs allow cryosection-based tissue analysis when labor,time,and cost are limiting factors and could be used for patient sample analysis as well as spatial transcriptomics approaches. View Publication

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases,regenerative medicine,and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs,which involves step-by-step differentiation of hiPSCs into definitive endoderm,anterior foregut endoderm,NKX2.1+ lung progenitors,and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs,expressing basal cell markers (NGFR,KRT5,and TP63),could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs,potentially applicable for gene therapy research. View Publication

Aging is one of the most prominent risk factors for neurodegeneration,yet the molecular mechanisms underlying the deterioration of old neurons are mostly unknown. To efficiently study neurodegeneration in the context of aging,we transdifferentiated primary human fibroblasts from aged healthy donors directly into neurons,which retained their aging hallmarks,and we verified key findings in aged human and mouse brain tissue. Here we show that aged neurons are broadly depleted of RNA-binding proteins,especially spliceosome components. Intriguingly,splicing proteins—like the dementia- and ALS-associated protein TDP-43—mislocalize to the cytoplasm in aged neurons,which leads to widespread alternative splicing. Cytoplasmic spliceosome components are typically recruited to stress granules,but aged neurons suffer from chronic cellular stress that prevents this sequestration. We link chronic stress to the malfunctioning ubiquitylation machinery,poor HSP90? chaperone activity and the failure to respond to new stress events. Together,our data demonstrate that aging-linked deterioration of RNA biology is a key driver of poor resiliency in aged neurons. Rhine et al. find that neuronal aging leads to widespread dysregulation of RNA biology,including mislocalization of splicing proteins like TDP-43,chronic cellular stress and reduced resiliency. View Publication