技术资料

Human embryonic stem cells (hESCs) serve as a valuable in vitro model for studying early human developmental processes due to their ability to differentiate into all three germ layers. Here,we present a comprehensive multi-omics dataset generated by differentiating hESCs into cardiomyocytes via the mesodermal lineage,collecting samples at 10 distinct time points. We measured mRNA levels by mRNA sequencing (mRNA-seq),translation levels by ribosome profiling (Ribo-seq),and protein levels by quantitative mass spectrometry-based proteomics. Technical validation confirmed high quality and reproducibility across all datasets,with strong correlations between replicates. This extensive dataset provides critical insights into the complex regulatory mechanisms of cardiomyocyte differentiation and serves as a valuable resource for the research community,aiding in the exploration of mammalian development and gene regulation. View Publication

Human induced pluripotent stem cell-derived sensory neuron (iPSC-dSN) models are a valuable resource for the study of neurotoxicity but are affected by poor replicability and reproducibility,often due to a lack of optimization. Here,we identify experimental factors related to culture conditions that substantially impact cellular drug response in vitro and determine optimal conditions for improved replicability and reproducibility. Treatment duration and cell seeding density were both found to be significant factors,while cell line differences also contributed to variation. A replicable dose–response in viability was demonstrated after 48-h exposure to docetaxel or paclitaxel. Additionally,a replicable dose-dependent reduction in neurite outgrowth was demonstrated,demonstrating the applicability of the model for the examination of additional phenotypes. Overall,we have established an optimized iPSC-dSN model for the study of taxane-induced neurotoxicity. View Publication

The rare,accelerated aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) is commonly caused by a de novo c.1824 C?>?T point mutation of the LMNA gene that results in the protein progerin. The primary cause of death is a heart attack or stroke arising from atherosclerosis. A characteristic feature of HGPS arteries is loss of smooth muscle cells. An adenine base editor (ABE7.10max) corrected the point mutation and produced significant improvement in HGPS mouse lifespan,vascular smooth muscle cell density,and adventitial fibrosis. To assess whether base editing correction of human HGPS tissue engineered blood vessels (TEBVs) prevents the HGPS vascular phenotype and to identify the minimum fraction of edited smooth muscle cells needed to effect such changes,we transduced HGPS iPSCs with lentivirus containing ABE7.10max. Endothelial cells (viECs) and smooth muscle cells (viSMCs) obtained by differentiation of edited HGPS iPSCs did not express progerin and had double-stranded DNA breaks and reactive oxygen species at the same levels as healthy viSMCs and viECs. Editing HGPSviECs restored a normal response to shear stress. Normal vasodilation and viSMC density were restored in TEBVs made with edited cells. When TEBVs were prepared with at least 50% edited smooth muscle cells,viSMC proliferation and myosin heavy chain levels significantly improved. Sequencing of TEBV cells after perfusion indicated an enrichment of edited cells after 5?weeks of perfusion when they comprised 50% of the initial number of cells in the TEBVs. Thus,base editing correction of a fraction of HGPS vascular cells improves human TEBV phenotype. View Publication

The cytoplasmic RIG-I-like receptors (RLRs) recognize viral RNA and initiate innate antiviral immunity. RLR signaling also triggers glycolytic reprogramming through glucose transporters (GLUTs),whose role in antiviral immunity is elusive. Here,we unveil that insulin-responsive GLUT4 inhibits RLR signaling independently of glucose uptake in adipose and muscle tissues. At steady state,GLUT4 is docked at the Golgi matrix by ubiquitin regulatory X domain 9 (UBXN9,TUG). Following RNA virus infection,GLUT4 is released and translocated to the cell surface where it spatially segregates a significant pool of cytosolic RLRs,preventing them from activating IFN-? responses. UBXN9 deletion prompts constitutive GLUT4 trafficking,sequestration of RLRs,and attenuation of antiviral immunity,whereas GLUT4 deletion heightens RLR signaling. Notably,reduced GLUT4 expression is uniquely associated with human inflammatory myopathies characterized by hyperactive interferon responses. Overall,our results demonstrate a noncanonical UBXN9-GLUT4 axis that controls antiviral immunity via plasma membrane tethering of cytosolic RLRs. View Publication

Introduction: The lack of functional hepatocytes poses a significant challenge for drug safety testing and therapeutic applications due to the inability of mature hepatocytes to expand and their tendency to lose functionality in vitro. Previous studies have demonstrated the potential of Human Liver Stem Cells (HLSCs) to differentiate into hepatocyte-like cells within an in vitro rotary cell culture system,guided by a combination of growth factors and molecules known to regulate hepatocyte maturation. In this study,we employed a matrix multi-assay approach to comprehensively characterize HLSC differentiation. Methods: We evaluated the expression of hepatic markers using qRT-PCR,immunofluorescence,and Western blot analysis. Additionally,we measured urea and FVIII secretion into the supernatant and developed an updated indocyanine green in vitro assay to assess hepatocyte functionality. Results: Molecular analyses of differentiated HLSC aggregates revealed significant upregulation of hepatic genes,including CYP450,urea cycle enzymes,and uptake transporters exclusively expressed on the sinusoidal side of mature hepatocytes,evident as early as 1 day post-differentiation. Interestingly,HLSCs transiently upregulated stem cell markers during differentiation,followed by downregulation after 7 days. Furthermore,differentiated aggregates demonstrated the ability to release urea and FVIII into the supernatant as early as the first 24 h,with accumulation over time. Discussion: These findings suggest that a 3D rotation culture system may facilitate rapid hepatic differentiation of HLSCs. Despite the limitations of this rotary culture system,its unique advantages hold promise for characterizing HLSC GMP batches for clinical applications. View Publication

ABSTRACTThe ultimate aim of nuclear reprogramming is to provide stem cells or differentiated cells from unrelated cell types as a cell source for regenerative medicine. A popular route towards this is transcription factor induction,and an alternative way is an original procedure of transplanting a single somatic cell nucleus to an unfertilized egg. A third route is to transplant hundreds of cell nuclei into the germinal vesicle (GV) of a non-dividing Amphibian meiotic oocyte,which leads to the activation of silent genes in 24 h and robustly induces a totipotency-like state in almost all transplanted cells. We apply this third route for potential therapeutic use and describe a procedure by which the differentiated states of cells can be reversed so that totipotency and pluripotency gene expression are regained. Differentiated cells are exposed to GV extracts and are reprogrammed to form embryoid bodies,which shows the maintenance of stemness and could be induced to follow new directions of differentiation. We conclude that much of the reprogramming effect of eggs is already present in meiotic oocytes and does not require cell division or selection of dividing cells. Reprogrammed cells by oocytes could serve as replacements for defective adult cells in humans. Summary: Stem cell therapy has shed light on incurable diseases. We describe a novel method for cell reprogramming and provide personalized stem cell sources for stem cell therapies. View Publication

Mohr-Tranebjaerg syndrome (MTS) is a rare X-linked recessive neurodegenerative disorder caused by mutations in the Translocase of Inner Mitochondrial Membrane 8A (TIMM8A) gene,which encodes TIMM8a,a protein localized to the mitochondrial intermembrane space (IMS). The pathophysiology of MTS remains poorly understood. To investigate the molecular mechanisms underlying MTS,we established induced pluripotent stem cells (iPSCs) from a male MTS patient carrying a novel TIMM8A mutation (c.225-229del,p.Q75fs95*),referred to as MTS-iPSCs. To generate an isogenic control,we introduced the same mutation into healthy control iPSCs (CTRL-iPSCs) using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9),resulting in mutant iPSCs (MUT-iPSCs). We differentiated the three iPSC lines into neurons and evaluated their mitochondrial function and neuronal development. Both MTS- and MUT-iPSCs exhibited impaired neuronal differentiation,characterized by smaller somata,fewer branches,and shorter neurites in iPSC-derived neurons. Additionally,these neurons showed increased susceptibility to apoptosis under stress conditions,as indicated by elevated levels of cytochrome c and cleaved caspase-3. Mitochondrial function analysis revealed reduced protein levels and activity of complex IV,diminished ATP synthesis,and increased reactive oxygen species (ROS) generation in MTS- and MUT-neurons. Furthermore,transmission electron microscopy revealed mitochondrial fragmentation in MTS-neurons. RNA sequencing identified differentially expressed genes (DEGs) involved in axonogenesis,synaptic activity,and apoptosis-related pathways. Among these DEGs,coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2),which encodes a mitochondrial IMS protein essential for mitochondrial homeostasis,was significantly downregulated in MTS-neurons. Western blot analysis confirmed decreased CHCHD2 protein levels in both MTS- and MUT-neurons. Overexpression of CHCHD2 rescued mitochondrial dysfunction and promoted neurite elongation in MTS-neurons,suggesting that CHCHD2 acts as a downstream effector of TIMM8a in the pathogenesis of MTS. In summary,loss-of-function of TIMM8a leads to a downstream reduction in CHCHD2 levels,collectively impairing neurogenesis by disrupting mitochondrial homeostasis. TIMM8a mutation (p.Q75fs95*) leads to mitochondrial dysfunction and neuronal defects in iPSC-derived neurons from patient with Mohr-Tranebjaerg syndrome,which are rescued by overexpression of CHCHD2. TIMM8a translocase of inner mitochondrial membrane 8a,CHCHD2 coiled-coil-helix-coiled-coil-helix domain-containing protein 2,MTS Mohr-Tranebjaerg syndrome,I mitochondrial complex I,II mitochondrial complex II,III mitochondrial complex III,IV mitochondrial complex IV,Q coenzyme Q10,Cyt c cytochrome c. View Publication

HiBiT is an engineered luciferase’s 11-amino-acid component that can be introduced as a tag at either terminus of a protein of interest. When the LgBiT component and a substrate are present,HiBiT and LgBiT dimerize forming a functional luciferase. The HiBiT technology has been extensively used for high-throughput protein turnover studies in cells. Here,we have adapted the use of the HiBiT technology to quantify messenger RNA (mRNA) translation temporally in vitro in the rabbit reticulocyte system and in cellulo in HEK293 cells constitutively expressing LgBiT. The assay system can uniquely detect differences in cap,5?UTR,modified nucleotide composition,coding sequence optimization and poly(A) length,and their effects on mRNA translation over time. Importantly,using these assays we established the optimal mRNA composition varied depending on the encoded protein of interest,highlighting the importance of screening methods tailored to the protein of interest,and not reliant on reporter proteins. Our findings demonstrated that HiBiT can be easily and readily adapted to monitor real-time mRNA translation in live cells and offers a novel and highly favourable method for the development of mRNA-based therapeutics. View Publication

Stem cell fate decisions,including proliferation,differentiation,morphological changes,and viability,are impacted by microenvironmental cues such as physical and biochemical signals. However,the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study,we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion,differentiation,and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically,hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering,disease modeling,and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses. View Publication

Abnormal trophoblast self-renewal and differentiation during early gestation is the major cause of miscarriage,yet the underlying regulatory mechanisms remain elusive. Here,we show that trophoblast specific deletion of Kat8,a MYST family histone acetyltransferase,leads to extraembryonic ectoderm abnormalities and embryonic lethality. Employing RNA-seq and CUT&Tag analyses on trophoblast stem cells (TSCs),we further discover that KAT8 regulates the transcriptional activation of the trophoblast stemness marker,CDX2,via acetylating H4K16. Remarkably,CDX2 overexpression partially rescues the defects arising from Kat8 knockout. Moreover,increasing H4K16ac via using deacetylase SIRT1 inhibitor,EX527,restores CDX2 levels and promoted placental development. Clinical analysis shows reduced KAT8,CDX2 and H4K16ac expression are associated with recurrent pregnancy loss (RPL). Trophoblast organoids derived from these patients exhibit impaired TSC self-renewal and growth,which are significantly ameliorated with EX527 treatment. These findings suggest the therapeutic potential of targeting the KAT8-H4K16ac-CDX2 axis for mitigating RPL,shedding light on early gestational abnormalities. Embryo implantation failure is a leading cause of miscarriage,though the mechanisms underlying trophoblast defects are not well understood. Here they show that the histone acetyltransferase KAT8 is essential for proper activation of the trophoblast stemness gene CDX2,and that placental development can be partially rescued by inhibiting histone deacetylase activity. View Publication

ABSTRACTMedium chain acyl?CoA dehydrogenase deficiency (MCADD) is an inherited metabolic disease,characterized by biallelic variants in the ACADM gene. Interestingly,even with the same genotype,patients often present with very heterogeneous symptoms,ranging from fully asymptomatic to life?threatening hypoketotic hypoglycemia. The mechanisms underlying this heterogeneity remain unclear. Therefore,there is a need for in vitro models of MCADD that recapitulate the clinical phenotype as a tool to study the pathophysiology of the disease. Fibroblasts of control and symptomatic MCADD patients with the c.985A>G (p.K329E) were reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs were then differentiated into hepatic expandable organoids (EHOs),further matured to Mat?EHOs,and functionally characterized. EHOs and Mat?EHOs performed typical hepatic metabolic functions,such as albumin and urea production. The organoids metabolized fatty acids,as confirmed by acyl?carnitine profiling and high?resolution respirometry. MCAD protein was fully ablated in MCADD organoids,in agreement with the instability of the mutated MCAD protein. MCADD organoids accumulated medium?chain acyl?carnitines,with a strongly elevated C8/C10 ratio,characteristic of the biochemical phenotype of the disease. Notably,C2 and C14 acyl?carnitines were found decreased in MCADD Mat?EHOs. Finally,MCADD organoids exhibited differential expression of genes involved in ??oxidation,mitochondrial ??oxidation,TCA cycle,and peroxisomal coenzyme A metabolism,particularly upregulation of NUDT7. iPSC?derived organoids of MCADD patients recapitulated the major biochemical phenotype of the disease. Mat?EHOs expressed relevant pathways involved in putative compensatory mechanisms,notably CoA metabolism and the TCA cycle. The upregulation of NUDT7 expression may play a role in preventing excessive accumulation of dicarboxylic acids in MCADD. This patient?specific hepatic organoid system is a promising platform to study the phenotypic heterogeneity between MCADD patients. View Publication

SummaryThe medial ganglionic eminence (MGE) gives rise to parvalbumin (PV)- and somatostatin (SST)-expressing cortical interneurons essential for regulating cortical excitability. Although PV interneurons are linked to various neurodevelopmental and neurodegenerative disorders,reliably generating them from human pluripotent stem cells (hPSCs) has been extremely challenging. We present a robust,reproducible protocol for generating single-rosette MGE organoids (MGEOs) from hPSCs. Transcriptomic analyses reveal that MGEOs exhibit MGE regional identity and faithfully model the developing human fetal MGE. As MGEOs mature,they generate abundant PV-expressing cortical interneurons,including putative basket and axoaxonic cells,at a scale not previously achieved in vitro. When fused with hPSC-derived cortical organoids,these interneurons rapidly migrate into cortical regions,integrate into excitatory networks,and contribute to complex electrophysiological patterns and the emergence of large numbers of fast-spiking neurons. MGEOs thus offer a powerful in vitro approach for probing human MGE-lineage cortical and subcortical GABAergic neuron development,modeling various neuropsychiatric disorders,and advancing cell-based therapies for neurodevelopmental and neurodegenerative disorders. Graphical abstract View Publication