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ObjectiveThyroid hormone (TH) action is mediated by thyroid hormone receptor (THR) isoforms. While THR?1 is likely the main isoform expressed in liver,its role in human hepatocytes is not fully understood.MethodsTo elucidate the role of THR?1 action in human hepatocytes we used CRISPR/Cas9 editing to knock out THR?1 in induced pluripotent stem cells (iPSC). Following directed differentiation to the hepatic lineage,iPSC-derived hepatocytes were then interrogated to determine the role of THR?1 in ligand-independent and -dependent functions.ResultsWe found that the loss of THR?1 promoted alterations in proliferation rate and metabolic pathways regulated by T3,including gluconeogenesis,lipid oxidation,fatty acid synthesis,and fatty acid uptake. We observed that key genes involved in liver metabolism are regulated through both T3 ligand-dependent and -independent THR?1 signaling mechanisms. Finally,we demonstrate that following THR?1 knockout,several key metabolic genes remain T3 responsive suggesting they are THR? targets.ConclusionsThese results highlight that iPSC-derived hepatocytes are an effective platform to study mechanisms regulating TH signaling in human hepatocytes. Graphical abstractImage 1 Highlights•THR?1 is essential for T3 effects in human iPSC-derived hepatocytes (iHEPs).•THR?1 knockout reduces iPSC and progenitor cell proliferative capacity.•T3 regulates key genes involved in lipid and carbohydrate metabolism through THR?1.•THR?1 plays a strong ligand-independent role. View Publication

Diabetic retinopathy (DR) is the leading cause of visual impairment,particularly in the proliferative form (proliferative DR [PDR]). The impact of the PDR microenvironment on microglia,which are the resident immune cells in the central nervous system,and the specific pathological changes it may induce remain unclear. This study aimed to investigate the role of microglia in the progression of PDR under hypoxic and inflammatory conditions. We performed a comprehensive gene expression analysis using human-induced pluripotent stem cell-derived microglia under different stimuli (dimethyloxalylglycine (DMOG),lipopolysaccharide (LPS),and DMOG + LPS) to mimic the hypoxic inflammatory environment characteristic of PDR. Principal component analysis revealed distinct gene expression profiles,with 76 genes synergistically upregulated under combined stimulation. Notably,prostaglandin-endoperoxide synthase 2 (encoding cyclooxygenase (COX)-2) exhibited the most pronounced increase,leading to elevated prostaglandin E2 (PGE2) levels and driving pathological angiogenesis and inflammation via the COX-2/PGE2/PGE receptor 2 signaling axis. Additionally,the upregulation of the fibrogenic genes snail family transcriptional repressor 1 and collagen type I alpha 1 chain suggested a role for microglia in fibrosis. These findings underscore the critical involvement of microglia in PDR and suggest that targeting both the angiogenic and fibrotic pathways may present new therapeutic strategies for managing this condition. View Publication

IntroductionEmerging biotechnologies are increasingly being explored for food production,including the development of cell-cultivated meat. Conventional approaches typically rely on satellite cell (SC) biopsies,which present challenges in scalability. Bovine induced pluripotent stem cells (biPSCs) represent a promising alternative due to their capacity for self-renewal and developmental plasticity.MethodsThis study utilized both lentiviral (integrating) and episomal (non-integrating) reprogramming strategies to generate biPSCs suitable for myogenic differentiation. Bovine fetal fibroblasts (bFFs) were reprogrammed using episomal vectors pMaster K and pCXB-EBNA1,leading to the emergence of putative iPSC colonies 13 days post-nucleofection. A clonal line,bFF-iPSCs pMK,was selected for further analysis.ResultsThe bFF-iPSCs pMK line expressed key pluripotency markers including alkaline phosphatase (AP),OCT4,SOX2,and NANOG,and was stably maintained for over 33 passages,although episomal plasmids remained detectable. in vitro myogenic differentiation was assessed by comparing this line to a previously established lentiviral reprogrammed line (bFF-iPSCs mOSKM). Both lines exhibited downregulation of pluripotency markers and upregulation of the early myogenic marker PAX3. By day 30,the bFF-iPSCs pMK line formed elongated,multinucleated cells characteristic of myotubes and displayed a corresponding gene expression profile.DiscussionThese results provide new insights into bovine in vitro myogenesis and its application in cultured meat production. While promising,the study also highlights the difficulty in achieving complete myogenic differentiation,indicating a need for further optimization of differentiation protocols. Graphical abstract View Publication

SummaryNGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study,we present a standardized approach for generating neurons utilizing clonal,targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150 days. Temporal omics,electrophysiological,and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic,imaging,and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally,we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows,profiling data,and functional characterizations enable the development of reproducible human in vitro models of neurological disorders. Graphical abstract Highlights•Optimized NGN2 protocol generates functional postnatal neurons in 28 days•Extensive profiling data provide benchmarks for neuron maturation•Maturation assays reliably assess neuron maturation in single or mixed cell types•Rapid targeted engineering protocol integrates NGN2 into iPSC lines in 3 weeks MotivationUsing induced pluripotent stem cell (iPSC)-derived neurons (iNs) to model diseases requires defined,robust,and reproducible protocols capable of generating predictable neuronal types. In addition,extensive profiling is essential to assess whether iNs are suitable to model specific diseases with desired molecular,functional,and maturation-related features. We sought to establish a standardized protocol for generating iNs at large scales. We also sought to develop systematic profiling data and assays for determining the maturation levels of iN cultures as resources for the community. Shan et al. report methods to generate postnatal-like iPSC-derived neurons at large scale and with long-term stability. They provide extensive characterization data and assays to measure neuronal maturity. They find genes associated with maturation exhibit diverse functions. Their data support the utility of these methods to enable modeling of neurological disorders. View Publication

BackgroundPathological deposition of hyperphosphorylated tau in the brain closely correlates with the course of Alzheimer’s disease (AD). Tau pathology occurs in axons of affected neurons and tau removal from axons might thus be an early intervention strategy.MethodsWe investigated the role of the RNA-binding protein hnRNP R in axonal localization and local translation of Mapt mRNA in neurons cultured from hnRNP R knockout mice. hnRNP R knockout mice were crossed with 5×FAD mice,an AD mouse model,and the effects of hnRNP R loss on the deposition of phospho-tau and amyloid-? plaques were evaluated. We designed antisense oligonucleotides (MAPT-ASOs) to block the binding of hnRNP R to Mapt mRNA. Cultured mouse and human neurons were treated with MAPT-ASOs and axonal Mapt mRNA and tau protein levels were quantified. MAPT-ASO was injected intracerebroventricularly into 5×FAD mice followed by quantification of phospho-tau aggregates and amyloid-? plaques in their brains. Protein changes in brains of 5×FAD mice treated with the MAPT-ASO were measured by mass spectrometry.ResultsMapt mRNA and tau protein were reduced in axons but not cell bodies of primary neurons cultured from hnRNP R knockout mice. Brains of 5×FAD mice deficient for hnRNP R contained less phospho-tau aggregates and amyloid-? plaques in the cortex and hippocampus. Treatment of neurons with MAPT-ASOs to block hnRNP R binding to Mapt similarly reduced axonal tau levels. Intracerebroventricular injection of a MAPT-ASO reduced the phospho-tau and plaque load and prevented neurodegeneration in the brains of 5×FAD mice,accompanied by rescue of proteome alterations.ConclusionLowering of tau selectively in axons thus represents an innovative therapeutic perspective for treatment of AD and other tauopathies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40035-025-00499-0. View Publication

Human hematopoiesis starts at early yolk sac and undergoes site- and stage-specific changes over development. The intrinsic mechanism underlying property changes in hematopoiesis ontogeny remains poorly understood. Here,we analyzed single-cell transcriptome of human primary hematopoietic stem/progenitor cells (HSPCs) at different developmental stages,including yolk-sac (YS),AGM,fetal liver (FL),umbilical cord blood (UCB) and adult peripheral blood (PB) mobilized HSPCs. These stage-specific HSPCs display differential intrinsic properties,such as metabolism,self-renewal,differentiating potentialities etc. We then generated highly co-related gene regulatory network (GRNs) modules underlying the differential HSC key properties. Particularly,we identified GRNs and key regulators controlling lymphoid potentiality,self-renewal as well as aerobic respiration in human HSCs. Introducing selected regulators promotes key HSC functions in HSPCs derived from human pluripotent stem cells. Therefore,GRNs underlying key intrinsic properties of human HSCs provide a valuable guide to generate fully functional HSCs in vitro.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-024-00192-z. View Publication

Human pluripotent stem cells offer a scalable platform to study genetic and signalling mechanisms governing cell lineage decisions during differentiation. Genome-wide and single-cell transcriptomics technologies likewise offer high-throughput analysis of heterogeneous cell differentiation states. While in vivo development has been extensively characterised using these technologies,there remains a need for comprehensive single-cell transcriptomic profiling of stem cell differentiation from pluripotency. Understanding gene expression changes governing differentiation in vitro is key to developing high fidelity differentiation protocols and understanding fundamental mechanisms of development. We generated a single-cell RNA sequencing time course to study the role of developmental signalling pathways on multilineage diversification from pluripotency in vitro. The combined dataset of over 60,000 cells spans cell types from a time course of differentiation across all germ layers,ranging from gastrulation cell states to progenitor and committed cell types. These data provide a diverse benchmarking reference point to compare against in vivo development and advance understanding of signalling regulation of differentiation,providing insights into protocol development,drug screening,and regenerative medicine applications. View Publication

BackgroundPrimary cilia mediate vertebrate development and growth factor signalling. Defects in primary cilia cause inherited developmental conditions termed ciliopathies. Ciliopathies often present with cystic kidney disease,a major cause of early renal failure. Currently,only one drug,Tolvaptan,is licensed to slow the decline of renal function for the ciliopathy polycystic kidney disease. Novel therapeutic interventions are needed.MethodsWe screened clinical development compounds to identify those that reversed cilia loss due to siRNA knockdown. In parallel,we undertook a whole genome siRNA-based reverse genetics phenotypic screen to identify positive modulators of cilia formation.ResultsUsing a clinical development compound screen,we identify fasudil hydrochloride. Fasudil is a generic,off-patent drug that is a potent,broadly selective Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor. In parallel,the siRNA screen identifies ROCK2 and we demonstrate that ROCK2 is a key mediator of cilium formation and function through its possible effects on actin cytoskeleton remodelling.ConclusionsOur results indicate that specific ROCK2 inhibitors (e.g. belumosudil) could be repurposed for cystic kidney disease treatment. We propose that ROCK2 inhibition represents a novel,disease-modifying therapeutic approach for heterogeneous ciliopathies. Plain language summaryPrimary cilia are antennae-like structures on cells that are important for early development and healthy cell function. Defects in primary cilia can cause inherited diseases called ciliopathies. Ciliopathies often cause fluid-filled sacs,called cysts,that are a major cause of kidney disease and failure. There is currently one drug licensed to slow kidney disease progression,but it is poorly tolerated in patients. Therefore,new drugs are needed. In this study,we used screening assays to identify potential drugs and their targets that are effective in promoting the formation of primary cilia. Our results identified ROCK2 (Rho-associated coiled-coil-containing protein kinase 2),an inhibitor of protein signalling,as a key mediator of cilium function. These findings suggest that drugs that specifically target ROCK2 could be a potential treatment option for cystic kidney disease. Smith et al. use clinical development screen and whole genome siRNA-reverse genetics phenotypic screen to identify ROCK2,as a modulator of cilia formation and function via its effects on actin cytoskeleton remodelling. Repurposing ROCK2 is a viable treatment for ciliopathies,for which a limited therapeutic option is available. View Publication

Low Earth Orbit (LEO) has emerged as a unique environment for evaluating altered stem cell properties in microgravity. LEO has become increasingly accessible for research and development due to progress in private spaceflight. Axiom Mission 2 (Ax-2) was launched as the second all-private astronaut mission to the International Space Station (ISS). Frozen human induced pluripotent stem cells (hiPSCs) expressing green fluorescent protein (GFP) under the SOX2 promoter,as well as fibroblasts differentiated from SOX2-GFP hiPSCs,were sent to the ISS. Astronauts then thawed and seeded both cell types into commercially available 96-well plates,which provided surface tension that reduced fluid movement out of individual wells and showed that hiPSCs or hiPSC-derived fibroblasts could survive either in suspension or attached to a Matrigel substrate. Furthermore,both cell types could be transfected with red fluorescent protein (RFP)-expressing plasmid. We demonstrate that hiPSCs and hiPSC-fibroblasts can be thawed in microgravity in off-the-shelf,commercially-available cell culture hardware,can associate into 3D spheroids or grow adherently in Matrigel,and can be transfected with DNA. This lays the groundwork for future biomanufacturing experiments in space. View Publication

Inclusion body myositis (IBM) is an inflammatory myopathy that displays proximal and distal muscle weakness. At the histopathological level,the muscles of IBM patients show inflammatory infiltrates,rimmed vacuoles and mitochondrial changes. The etiology of IBM remains unknown,and there is a lack of validated disease models,biomarkers and effective treatments. To contribute to unveil disease underpins we developed a cell model based on myotubes derived from induced pluripotent stem cells (iPSC-myotubes) from IBM patients and compared the molecular phenotype vs. age and sex-paired controls (n?=?3 IBM and 4 CTL). We evaluated protein histological findings and the gene expression profile by mRNA-seq,alongside functional analysis of inflammation,degeneration and mitochondrial function. Briefly,IBM iPSC-myotubes replicated relevant muscle histopathology features of IBM,including aberrant expression of HLA,TDP-43 and COX markers. mRNA seq analysis identified 1007 differentially expressed genes (DEGs) (p-value adj? View Publication

Cell-autonomous barriers to reprogramming somatic cells into induced pluripotent stem cells (iPSCs) remain poorly understood. Using a focused CRISPR-Cas9 screen,we identified Ubiquitin-specific peptidase 22 (USP22) as a key chromatin-based barrier to human iPSC derivation. Suppression of USP22 significantly enhances reprogramming efficiency. Surprisingly,this effect is likely to be independent of USP22’s deubiquitinase activity or its association with the SAGA complex,as shown through module-specific knockouts,and genetic rescue experiments. USP22 is not required for iPSC derivation or maintenance. Mechanistically,USP22 loss during reprogramming downregulates fibroblast-specific genes while activating pluripotency-associated genes,including DNMT3L,LIN28A,SOX2,and GDF3. Additionally,USP22 loss enhances reprogramming efficiency under naïve stem cell conditions. These findings reveal an unrecognized role for USP22 in maintaining somatic cell identity and repressing pluripotency genes,highlighting its potential as a target to improve reprogramming efficiency. Ubiquitin-specific peptidase 22 (USP22) is identified as a key chromatin-based barrier to human iPSC derivation through a chromatin-focused CRISPR-Cas9 screen. View Publication

SUMMARYHuman neurogenesis is disproportionately protracted,lasting >10 times longer than in mouse,allowing neural progenitors to undergo more rounds of self-renewing cell divisions and generate larger neuronal populations. In the human spinal cord,expansion of the motor neuron lineage is achieved through a newly evolved progenitor domain called vpMN (ventral motor neuron progenitor) that uniquely extends and expands motor neurogenesis. This behavior of vpMNs is controlled by transcription factor NKX2-2,which in vpMNs is co-expressed with classical motor neuron progenitor (pMN) marker OLIG2. In this study,we sought to determine the molecular basis of NKX2-2-mediated extension and expansion of motor neurogenesis. We found that NKX2-2 represses proneural gene NEUROG2 by two distinct,Notch-independent mechanisms that are respectively apparent in rodent and human spinal progenitors: in rodents (and chick),NKX2-2 represses Olig2 and the motor neuron lineage through its tinman domain,leading to loss of Neurog2 expression. In human vpMNs,however,NKX2-2 represses NEUROG2 but not OLIG2,thereby allowing motor neurogenesis to proceed,albeit in a delayed and protracted manner. Interestingly,we found that ectopic expression of tinman-mutant Nkx2-2 in mouse pMNs phenocopies human vpMNs,repressing Neurog2 but not Olig2,and leading to delayed and protracted motor neurogenesis. Our studies identify a Notch- and tinman-independent mode of Nkx2-2-mediated Neurog2 repression that is observed in human spinal progenitors,but is normally masked in rodents and chicks due to Nkx2-2’s tinman-dependent repression of Olig2. View Publication