技术资料

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically,4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1),thereby mitigating oxidative stress. This has important relevance for cancer,as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress,thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically,high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells. How cells adapt to glucose starvation is still elusive. Here,Levy et al. show that the mTOR substrate 4EBP1 protects human,mouse,and yeast cells from glucose starvation and is exploited by cancer cells to promote tumorigenesis. View Publication

DNA methylation regulation involves multi-layered chromatin interactions that require remodeling proteins like the helicase,lymphoid-specific (HELLS). Here,we generate HELLS and DNA methyltransferase 3A and B (DNMT3A/B) knockout human pluripotent stem cells and report telomere-to-telomere maps of whole genome bisulfite sequencing data combined with ATAC-sequencing. Disrupting HELLS induces a global loss of DNA methylation that is distinct from the DNMTs,in particular over peri/centromeric satellite repeats as defined in the telomere-to-telomere genome assembly. However,HELLS appears dispensable for local enhancer remodeling and the potential to differentiate into the three embryonic germ layers. Taken together,our results further clarify the genomic targets and role of HELLS in human cells.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-025-03681-9. 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

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

SummaryKnowledge of cell signaling pathways that drive human neural crest differentiation into craniofacial chondrocytes is incomplete,yet essential for using stem cells to regenerate craniomaxillofacial structures. To accelerate translational progress,we developed a differentiation protocol that generated self-organizing craniofacial cartilage organoids from human embryonic stem cell-derived neural crest stem cells. Histological staining of cartilage organoids revealed tissue architecture and staining typical of elastic cartilage. Protein and post-translational modification (PTM) mass spectrometry and snRNA-seq data showed that chondrocyte organoids expressed robust levels of cartilage extracellular matrix (ECM) components: many collagens,aggrecan,perlecan,proteoglycans,and elastic fibers. We identified two populations of chondroprogenitor cells,mesenchyme cells and nascent chondrocytes,and the growth factors involved in paracrine signaling between them. We show that ECM components secreted by chondrocytes not only create a structurally resilient matrix that defines cartilage,but also play a pivotal autocrine cell signaling role in determining chondrocyte fate. Graphical abstract Highlights•Craniofacial cartilage organoids were grown from human neural crest stem cells•These organoids exhibited elastic cartilage architecture and characteristic markers•Paracrine signaling drove chondrogenesis in mesenchyme cells and nascent chondrocytes•ECM components cemented chondrocyte cell fate through autocrine signaling Natural sciences; Biological sciences; Biochemistry; Cell biology; Stem cells research; Specialized functions of cells View Publication

Development requires coordinated interactions between the epiblast,which generates the embryo proper; the trophectoderm,which generates the placenta; and the hypoblast,which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent,as in the mouse. However,while BMP inhibits anterior signalling centre specification in the mouse,it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally,we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies. Weatherbee,Weberling,Gantner et al. find contrasting requirements for BMP in the anterior signalling centre and pre-implantation epiblast between mice and humans. They further find that NOTCH may be indispensable for human epiblast survival. 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

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

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3,SLMAP,and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states. View Publication

AbstractHiBiT 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. Graphical Abstract Graphical Abstract View Publication

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ?0.9 in as little as six-weeks with a mean firing rate of ?13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports,making it an excellent first pass,high-throughput method for studying network properties and neurodegenerative diseases. 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