技术资料

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

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

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

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

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

A reliable suspension-based platform for scaling engineered cardiac tissue (ECT) production from human induced pluripotent stem cells (hiPSCs) is crucial for regenerative therapies. Here,we compared the production and functionality of ECTs formed using our scaffold-based,engineered tissue microsphere differentiation approach with those formed using the prevalent scaffold-free aggregate platform. We utilized a microfluidic system for the rapid (1 million cells/min),high density (30,40,60 million cells/ml) encapsulation of hiPSCs within PEG-fibrinogen hydrogel microspheres. HiPSC-laden microspheres and aggregates underwent suspension-based cardiac differentiation in chemically defined media. In comparison to aggregates,microspheres maintained consistent size and shape initially,over time,and within and between batches. Initial size and shape coefficients of variation for microspheres were eight and three times lower,respectively,compared to aggregates. On day 10,microsphere cardiomyocyte (CM) content was 27 % higher and the number of CMs per initial hiPSC was 250 % higher than in aggregates. Contraction and relaxation velocities of microspheres were four and nine times higher than those of aggregates,respectively. Microsphere contractile functionality also improved with culture time,whereas aggregate functionality remained unchanged. Additionally,microspheres displayed improved ?-adrenergic signaling responsiveness and uniform calcium transient propagation. Transcriptomic analysis revealed that while both microspheres and aggregates demonstrated similar gene regulation patterns associated with cardiomyocyte differentiation,heart development,cardiac muscle contraction,and sarcomere organization,the microspheres exhibited more pronounced transcriptional changes over time. Taken together,these results highlight the capability of the microsphere platform for scaling up biomanufacturing of ECTs in a suspension-based culture platform. Graphical abstractImage 1 Highlights•Comparison of scaffold-based and scaffold-free 3D hiPSC cardiac differentiation.•Microsphere provided tighter control of size and shape with higher reproducibility.•Microspheres showed higher cardiomyocyte (CM) content and more CMs/initial hiPSC.•Microsphere contracted faster than aggregate with higher cell structural maturity.•Microsphere platform exhibited more pronounced transcriptional changes over time. View Publication

Epithelial tissues sheath organs and electro-mechanically regulate ion and water transport to regulate development,homeostasis,and hydrostatic organ pressure. Here,we demonstrate how external electrical stimulation allows us to control these processes in living tissues. Specifically,we electrically stimulate hollow,3D kidneyoids and gut organoids and find that physiological-strength electrical stimulation of ? 5 - 10 V/cm powerfully inflates hollow tissues; a process we call electro-inflation. Electro-inflation is mediated by increased ion flux through ion channels/transporters and triggers subsequent osmotic water flow into the lumen,generating hydrostatic pressure that competes against cytoskeletal tension. Our computational studies suggest that electro-inflation is strongly driven by field-induced ion crowding on the outer surface of the tissue. Electrically stimulated tissues also break symmetry in 3D resulting from electrotaxis and affecting tissue shape. The ability of electrical cues to regulate tissue size and shape emphasizes the role and importance of the electrical micro-environment for living tissues. Electrical stimulation of hollow,3D kidney tissues causes these tissues to inflate and change shape. The authors call this process electro-inflation and connect it to electricity driving ions into the center of the tissues,causing water to follow by osmosis. View Publication

Diffuse Intrinsic Pontine Glioma (DIPG) is a highly aggressive and fatal pediatric brain cancer. One pre-requisite for tumor cells to infiltrate is adhesion to extracellular matrix (ECM) components. However,it remains largely unknown which ECM proteins are critical in enabling DIPG adhesion and migration and which integrin receptors mediate these processes. Here,we identify laminin as a key ECM protein that supports robust DIPG cell adhesion and migration. To study DIPG infiltration,we developed a DIPG-neural assembloid model,which is composed of a DIPG spheroid fused to a human induced pluripotent stem cell-derived neural organoid. Using this assembloid model,we demonstrate that knockdown of laminin-associated integrins significantly impedes DIPG infiltration. Moreover,laminin-associated integrin knockdown improves DIPG response to radiation and HDAC inhibitor treatment within the DIPG-neural assembloids. These findings reveal the critical role of laminin-associated integrins in mediating DIPG progression and drug response. The results also provide evidence that disrupting integrin receptors may offer a novel therapeutic strategy to enhance DIPG treatment outcomes. Finally,these results establish DIPG-neural assembloid models as a powerful tool to study DIPG disease progression and enable drug discovery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01765-4. View Publication

An estimated 65 million people globally suffer from post-acute sequelae of COVID-19 (PASC),with many experiencing cardiovascular symptoms (PASC-CVS) like chest pain and heart palpitations. This study examines the role of chronic inflammation in PASC-CVS,particularly in individuals with symptoms persisting over a year after infection. Blood samples from three groups—recovered individuals,those with prolonged PASC-CVS and SARS-CoV-2-negative individuals—revealed that those with PASC-CVS had a blood signature linked to inflammation. Trace-level pro-inflammatory cytokines were detected in the plasma from donors with PASC-CVS 18?months post infection using nanotechnology. Importantly,these trace-level cytokines affected the function of primary human cardiomyocytes. Plasma proteomics also demonstrated higher levels of complement and coagulation proteins in the plasma from patients with PASC-CVS. This study highlights chronic inflammation’s role in the symptoms of PASC-CVS. Sinclair et al. explore the contribution of chronic inflammation to cardiovascular symptoms associated with post-acute sequelae of SARS-CoV-2 infection (PASC-CVS). The authors identify trace levels of inflammatory cytokines in individuals with PASC-CVS that impair the function of cardiomyocytes derived from induced pluripotent stem cells. 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

Parkinson’s disease (PD) is a neurodegenerative illness characterized by motor and non-motor features. Hallmarks of the disease include an extensive loss of dopaminergic neurons in the substantia nigra pars compacta,evidence of neuroinflammation,and the accumulation of misfolded proteins leading to the formation of Lewy bodies. While PD etiology is complex and identifying a single disease trigger has been a challenge,accumulating evidence indicates that non-neuronal and peripheral factors may likely contribute to disease onset and progression. The brain is shielded from peripheral factors by the blood-brain barrier (BBB),which tightly controls the entry of systemic molecules and cells from the blood to the brain. The BBB integrates molecular signals originating from the luminal (blood) and abluminal (brain) sides of the endothelial wall,regulating these exchanges. Of particular interest are erythrocytes,which are not only the most abundant cell type in the blood,but they also secrete extracellular vesicles (EVs) that display disease-specific signatures over the course of PD. Erythrocyte-derived EVs (EEVs) could provide a route by which pathological molecular signals travel from the periphery to the central nervous system. The primary objective of this study was to evaluate,in a human-based platform,mechanisms of EEV transport from the blood to the brain under physiological conditions. The secondary objective was to determine the ability of EEVs,generated by erythrocytes of healthy donors or patients,to induce PD-like features. We leveraged two in vitro models of the BBB,the transwell chambers and a microfluidic BBB chip generated using human induced pluripotent stem cells. Our findings suggest that EEVs transcytose from the vascular to the brain compartment of the human BBB model via a caveolin-dependant mechanism. Furthermore,EEVs derived from individuals with PD altered BBB integrity compared to healthy EEV controls,and clinical severity aggravated the loss of barrier integrity and increased EEV extravasation into the brain compartment. PD-derived EEVs reduced ZO-1 and Claudin 5 tight junction levels in BMEC-like cells and induced the selective atrophy of dopaminergic neurons. In contrast,non-dopaminergic neurons were not affected by treatment with PD EEVs. In summary,our data suggest that EEV interactions at the human BBB can be studied using a highly translational human-based brain chip model,and EEV toxicity at the neurovascular unit is exacerbated by disease severity.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00646-9. HighlightsErythrocytes secrete extracellular vesicles that can transcytose into the brain via a caveolin-dependant mechanism.A microfluidic brain chip can be used to evaluate mechanisms of transcytosis across the blood-brain barrier.The clinical severity of Parkinson’s disease affects how erythrocyte-derived extracellular vesicles interact with cerebral endothelial cells.Erythrocyte-derived extracellular vesicles generated from donors with Parkinson’s disease alter the blood-brain barrier and induce atrophy of dopaminergic neurons.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00646-9. View Publication