技术资料

Induced pluripotent stem cells (iPSCs) can be generated from various adult cells,genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however,their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells,here,we aimed to generate human iPSCs overexpressing interferon-beta (IFNB,IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-?. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation,IFNB-iPSCs formed embryoid bodies and upregulated endoderm,mesoderm,and some ectoderm markers. However,an upregulation of key neuroectoderm markers,PAX6 and LHX2,was compromised. A negative effect of IFN-? on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-?. The study describes new IFN-?-producing iPSC lines suitable for the generation of various types of IFN-?-producing cells for future experimental and clinical applications,and it unravels an inhibitory effect of IFN-? on stem cell neuroectoderm differentiation. View Publication

Introduction: Pathogenic variants in canonical transforming growth factor β (TGFβ) signaling genes predispose patients to thoracic aortic aneurysm and dissection (TAAD),predominantly in aortic root. Although TAAD pathogenesis associated with TGFβ receptor defects is well characterized,distinct and redundant mechanisms of TGFβ isoforms in TAAD incidence and severity remain elusive. Objective: Here we examined the biological role of TGFB2 in smooth muscle cell (SMC) differentiation and investigated how TGFB2 defects can lead to regional TAAD manifestations. Methods: To characterize the role of TGFB2 in SMC differentiation and function,we employed human-induced pluripotent stem cell (hiPSC)-derived SMC differentiation,CRISPR/Cas9 gene editing,three-dimensional SMC constructs,and human aortic tissue samples. Results: Despite the similar effects of different TGFβ isoforms on hiPSC-derived SMC differentiation,siRNA experiments revealed that TGFB2 distinctively displays TGFBR3 dependence for signal transduction,an understudied TGFβ receptor in TAAD. Molecular evaluation of different thoracic aorta regions suggested TGFB2 and TGFBR3 enrichment in the aortic root tunica media. TGFB2 haploinsufficiency (TGFB2KO/+) and TGFB2 neutralization impaired the differentiation of second heart field-derived SMCs. TGFBR3KO/KO prevented the molecular rescue of TGFB2KO/+ by TGFB2 supplementation indicating the involvement of TGFBR3 in TGFB2-mediated SMC differentiation. Lastly,a missense TGFB2 variant (TGFB2G276R/+) caused mechanical defects in SMC tissue ring constructs that were rescued by TGFB2 supplementation or genetic correction. Conclusion: Our data suggests the distinct regulation and action of TGFB2 in SMCs populating the aortic root,while redundant activities of TGFβ isoforms provide implications about the milder TAAD aggressiveness of pathogenic TGFB2 variants. 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

SummaryToxic signaling by extrasynaptic NMDA receptors (eNMDARs) is considered an important promoter of amyotrophic lateral sclerosis (ALS) disease progression. To exploit this therapeutically,we take advantage of TwinF interface (TI) inhibition,a pharmacological principle that,contrary to classical NMDAR pharmacology,allows selective elimination of eNMDAR-mediated toxicity via disruption of the NMDAR/TRPM4 death signaling complex while sparing the vital physiological functions of synaptic NMDARs. Post-disease onset treatment of the SOD1G93A ALS mouse model with FP802,a modified TI inhibitor with a safe pharmacology profile,stops the progressive loss of motor neurons in the spinal cord,resulting in a reduction in the serum biomarker neurofilament light chain,improved motor performance,and an extension of life expectancy. FP802 also effectively blocks NMDA-induced death of neurons in ALS patient-derived forebrain organoids. These results establish eNMDAR toxicity as a key player in ALS pathogenesis. TI inhibitors may provide an effective treatment option for ALS patients. Graphical abstract Highlights•eNMDARs promote ALS disease progression via the NMDAR/TRPM4 death signaling complex•TwinF interface inhibitor FP802 disrupts the NMDAR/TRPM4 death signaling complex•FP802 is therapeutically effective in an ALS mouse model•FP802 protects against NMDA-induced death in brain organoids from ALS patient iPSCs Yan et al. find that FP802,which provides neuroprotection by detoxifying eNMDARs through disruption of the NMDAR/TRPM4 complex,halts motor neuron loss in an ALS mouse model,reduces serum NfL levels,improves motor performance,and extends life expectancy. FP802 is also neuroprotective in brain organoids derived from ALS patients. View Publication

Human induced pluripotent stem cells and their differentiation into cardiac myocytes (hiPSC-CMs) provides a unique and valuable platform for studies of cardiac muscle structure–function. This includes studies centered on disease etiology,drug development,and for potential clinical applications in heart regeneration/repair. Ultimately,for these applications to achieve success,a thorough assessment and physiological advancement of the structure and function of hiPSC-CMs is required. HiPSC-CMs are well noted for their immature and sub-physiological cardiac muscle state,and this represents a major hurdle for the field. To address this roadblock,we have developed a hiPSC-CMs (?-MHC dominant) experimental platform focused on directed physiological enhancement of the sarcomere,the functional unit of cardiac muscle. We focus here on the myosin heavy chain (MyHC) protein isoform profile,the molecular motor of the heart,which is essential to cardiac physiological performance. We hypothesized that inducing increased expression of ?-MyHC in ?-MyHC dominant hiPSC-CMs would enhance contractile performance of hiPSC-CMs. To test this hypothesis,we used gene editing with an inducible ?-MyHC expression cassette into isogeneic hiPSC-CMs,and separately by gene transfer,and then investigated the direct effects of increased ?-MyHC expression on hiPSC-CMs contractility and relaxation function. Data show improved cardiac functional parameters in hiPSC-CMs induced with ?-MyHC. Positive inotropy and relaxation was evident in comparison to ?-MyHC dominant isogenic controls both at baseline and during pacing induced stress. This approach should facilitate studies of hiPSC-CMs disease modeling and drug screening,as well as advancing fundamental aspects of cardiac function parameters for the optimization of future cardiac regeneration,repair and re-muscularization applications. View Publication

The inner ear organs responsible for hearing (cochlea) and balance (vestibular system) are susceptible to oxidative stress due to the high metabolic demands of their sensorineural cells. Oxidative stress-induced damage to these cells can cause hearing loss or vestibular dysfunction,yet the precise mechanisms remain unclear due to the limitations of animal models and challenges of obtaining living human inner ear tissue. Therefore,we developed an in vitro oxidative stress model of the pre-natal human inner ear using otic progenitor cells (OPCs) derived from human-induced pluripotent stem cells (hiPSCs). OPCs,hiPSCs,and HeLa cells were exposed to hydrogen peroxide or ototoxic drugs (gentamicin and cisplatin) that induce oxidative stress to evaluate subsequent cell viability,cell death,reactive oxygen species (ROS) production,mitochondrial activity,and apoptosis (caspase 3/7 activity). Dose-dependent reductions in OPC cell viability were observed post-exposure,demonstrating their vulnerability to oxidative stress. Notably,gentamicin exposure induced ROS production and cell death in OPCs,but not hiPSCs or HeLa cells. This OPC-based human model effectively simulates oxidative stress conditions in the human inner ear and may be useful for modeling the impact of ototoxicity during early pregnancy or evaluating therapies to prevent cytotoxicity. 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

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

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

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

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

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