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Remarkable advances in protocol development have been achieved to manufacture insulin-secreting islets from human pluripotent stem cells (hPSCs). Distinct from current approaches,we devised a tunable strategy to generate islet spheroids enriched for major islet cell types by incorporating PDX1+ cell budding morphogenesis into staged differentiation. In this process that appears to mimic normal islet morphogenesis,the differentiating islet spheroids organize with endocrine cells that are intermingled or arranged in a core-mantle architecture,accompanied with functional heterogeneity. Through in vitro modelling of human pancreas development,we illustrate the importance of PDX1 and the requirement for EphB3/4 signaling in eliciting cell budding morphogenesis. Using this new approach,we model Mitchell-Riley syndrome with RFX6 knockout hPSCs illustrating unexpected morphogenesis defects in the differentiation towards islet cells. The tunable differentiation system and stem cell-derived islet models described in this work may facilitate addressing fundamental questions in islet biology and probing human pancreas diseases. The ability to differentiate human pluripotent stem cells (hPSCs) into insulin producing cells holds potential for diabetes treatments,but many of these approaches lack the complexity needed for in vitro disease modeling. Here they develop an hPSC-derived islet spheroid system,offering an experimental model to study pancreatic budding and islet morphogenesis with human cells. View Publication

SummaryThe human brain microenvironment undergoes dynamic changes during development,which have been incompletely characterized in in vitro models including neural organoids. Here,we used liquid chromatography-mass spectrometry to investigate proteome and secretome changes in human dorsal forebrain organoids derived from three hiPSC lines at days 20,35,and 50 of differentiation. Proteome and immunohistochemical analysis revealed reduced proliferation and increased differentiation of progenitor cells gradually over time. In contrast,secretome analysis showed distinct characteristics at each timepoint — notably,at day 35,the numbers of cell adhesion molecules,synaptic proteins,and proteases were increased. Taken together,we present a resource describing the dynamic features of a neural organoid proteome and secretome across different genetic backgrounds. We describe the unique niche composition of neural organoids during the period of neurogenesis and suggest that synaptic proteins may play a role in guiding neurogenesis. Graphical abstract Highlights•Proteomic analysis of DFOs on three time points shows neural differentiation•Protein secretion increases during peak neurogenesis at D35 and D50•Cell adhesion molecules,synapse proteins,and metalloproteases are mainly secreted at D35•Extracellular matrix proteins are predominantly secreted at D50 Natural sciences; Biological sciences; Neuroscience; Tissue Engineering View Publication

Background and PurposeEsophageal cancer-related gene-4 (ECRG4) participate in inflammation process and can interact with the innate immunity complex TLR4-MD2-CD14 on human granulocytes. In addition,ECRG4 participate in modulation of ion channel function and electrical activity of cardiomyocytes. However,the exact mechanism is unknown. This study aimed to test our hypothesis that ECRG4 contributes to inflammation-induced ion channel dysfunctions in cardiomyocytes.MethodsHuman-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated from three donors were treated with lipopolysaccharide (LPS) to establish an endotoxin-induced inflammatory model. Immunostaining,real-time PCR,and patch-clamp techniques were used for the study.ResultsECRG4 was detected in hiPSC-CMs at different differentiation time. LPS treatment increased ECRG4 expression in hiPSC-CMs. Knockdown of ECRG4 decreased the expression level of Toll-Like-Receptor 4 (TLR4,a LPS receptor) and its associated genes and inflammatory cytokines. Furthermore,ECRG4 knockdown shortened the action potential duration (APD) and intercepted LPS-induced APD prolongation by enhancing ISK (small conductance calcium-activated K channel current) and attenuating INCX (Na/Ca exchanger current). Overexpression of ECRG4 mimicked LPS effects on ISK and INCX,which could be prevented by NF?B signaling blockers.ConclusionThis study demonstrated that LPS effects on cardiac ion channel function were mediated by the upregulation of ECRG4,which affects NF?B signaling. Our findings support the roles of ECRG4 in inflammatory responses and the ion channel dysfunctions induced by LPS challenge. View Publication

BackgroundStem-cell-derived therapy is a promising option for tissue regeneration. Human iPSC-derived progenitors of smooth muscle cells (pSMCs) exhibit limited proliferation and differentiation,which minimizes the risk of tumor formation while restoring smooth muscle cells (SMCs). Up to 29% of women suffer from recurrence of vaginal prolapse after prolapse surgery. Therefore,there is a need for therapies that can restore vaginal function. SMCs contribute to vaginal tone and contractility. We sought to examine whether human pSMCs can restore vaginal function in a rat model.MethodsFemale immunocompromised RNU rats were divided into 5 groups: intact controls (n?=?12),VSHAM (surgery?+?saline injection,n?=?35),and three cell-injection groups (surgery?+?cell injection using pSMCs from three patients,n?=?14/cell line). The surgery to induce vaginal injury was analogous to prolapse surgery. Menopause was induced by surgical ovariectomy. The vagina,urethra,bladder were harvested 10 weeks after surgery (5 weeks after cell injection). Organ bath myography was performed to evaluate the contractile function of the vagina,and smooth muscle thickness was examined by tissue immunohistochemistry. Collagen I,collagen III,and elastin mRNA and protein expressions in tissues were assessed.ResultsVaginal smooth muscle contractions induced by carbachol and KCl in the cell-injection groups were significantly greater than those in the VSHAM group. Collagen I protein expression in the vagina of the cell-injections groups was significantly higher than in the VSHAM group. Vaginal elastin protein expression was similar between the cell-injection and VSHAM groups. In the urethra,gene expression levels of collagen I,III,and elastin were all significantly greater in the cell-injection groups than in the VSHAM group. Collagen I,III,and elastin protein expression of the urethra did not show a consistent trend between cell-injection groups and the VSHAM group.ConclusionsHuman iPSC-derived pSMCs transplantation appears to be associated with improved contractile function of the surgically injured vagina in a rat model. This is accompanied by changes in extracellular protein expression the vagina and urethra. These observations support further efforts in the translation of pSMCs into a treatment for regenerating the surgically injured vagina in women who suffer recurrent prolapse after surgery.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03900-3. View Publication

In mammals,early embryonic gastrulation process is high energy demanding. Previous studies showed that,unlike endoderm and mesoderm cells,neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process,which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/?-catenin signaling during neuroectoderm differentiation. Notably,histone lactylation,especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments,we further identified that four genes,including PAX6,were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05510-x. View Publication

BackgroundAbnormalities in T cell activation play an important role in the pathogenesis of myocarditis,and persistent T cell responses can lead to autoimmunity and chronic cardiac inflammation,as well as even dilated cardiomyopathy. Although previous work has examined the role of T cells in myocarditis in animal models,the specific mechanism for human cardiomyocytes has not been investigated.MethodsIn this study,we constructed the human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and established the T cell-mediated cardiac injury model by co-culturing with activated CD4 + T or CD8 + T cells that were isolated from peripheral mononuclear blood to elucidate the pathogenesis of myocardial cell injury caused by inflammation.ResultsBy combination of quantitative proteomics with tissue and cell immunofluorescence examination,we established a proteome profile of inflammatory myocardia from hiPSC-CMs with obvious cardiomyocyte injury and increased levels of lactate dehydrogenase content,creatine kinase isoenzyme MB and cardiac troponin. A series of molecular dysfunctions of hiPSC-CMs was observed and indicated that CD4 + cells could produce direct cardiomyocyte injury by activating the NOD-like receptor signals pathway.ConclusionsThe data presented in our study established a proteome map of inflammatory myocardial based on hiPSC-CMs injury model. These results can provide guidance in the discovery of improved clinical treatments for myocarditis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03791-4. View Publication

IntroductionThe inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states,however,the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype,which is one of the reasons why macrophage transplantation has failed.MethodsIn this study,we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover,we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor,sensory and autonomic functions. Instead of transplanting the cells,we injected the paracrine factors and extracellular vesicles that they secrete,avoiding the loss of the phenotype of the transplanted cells due to local environmental cues.ResultsWe demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival,namely,the alternative activation with IL-10 and TGF-?1 (M(IL-10+TGF-?1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-?1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally,the M(IL-10+TGF-?1) secretome supported the recovery of bladder function and decreased microglial activation,astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-?1)-derived secretome identified clusters of proteins involved in axon extension,dendritic spine maintenance,cell polarity establishment,and regulation of astrocytic activation.DiscussionOverall,our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications. View Publication

Background and purpose: In this study,we applied an induced pluripotent stem cell (iPSC)-based model of inherited erythromelalgia (IEM) to screen a library of 281 small molecules,aiming to identify candidate pain-modulating compounds. Experimental approach: Human iPSC-derived sensory neuron-like cells,which exhibit action potentials in response to noxious stimulation,were evaluated using whole-cell patch-clamp and microelectrode array (MEA) techniques. Key results: Sensory neuron-like cells derived from individuals with IEM showed spontaneous electrical activity characteristic of genetic pain disorders. The drug screen identified four compounds (AZ106,AZ129,AZ037 and AZ237) that significantly decreased spontaneous firing with minimal toxicity. The calculated IC50 values indicate the potential efficacy of these compounds. Electrophysiological analysis confirmed the compounds' ability to reduce action potential generation in IEM patient-specific iPSC-derived sensory neuron-like cells. Conclusions and implications: Our screening approach demonstrates the reproducibility and effectiveness of human neuronal disease modelling offering a promising avenue for discovering new analgesics. These findings address a critical gap in current therapeutic strategies for both general and neuropathic pain,warranting further investigation. This study highlights the innovative use of patient-derived iPSC sensory neuronal models in pain research and emphasises the potential for personalised medicine in developing targeted analgesics. Key points: Utilisation of human iPSCs for efficient differentiation into sensory neuron-like cells offers a novel strategy for studying pain mechanisms. IEM sensory neuron-like cells exhibit key biomarkers and generate action potentials in response to noxious stimulation. IEM sensory neuron-like cells display spontaneous electrical activity,providing a relevant nociceptive model. Screening of 281 compounds identified four candidates that significantly reduced spontaneous firing with low cytotoxicity. Electrophysiological profiling of selected compounds revealed promising insights into their mechanisms of action,specifically modulating the NaV 1.7 channel for targeted analgesia. View Publication

With the advent of increasingly sophisticated organoids,there is growing demand for technology to replicate the interactions between multiple tissues or organs. This is challenging to achieve,however,due to the varying culture conditions of the different cell types that make up each tissue. Current methods often require complicated microfluidic setups,but fragile tissue samples tend not to fare well with rough handling. Furthermore,the more complicated the human system to be replicated,the more difficult the model becomes to operate. Here,we present the development of a multi-tissue chip platform that takes advantage of the modularity and convenient handling ability of a CUBE device. We first developed a blood-brain barrier-in-a-CUBE by layering astrocytes,pericytes,and brain microvascular endothelial cells in the CUBE,and confirmed the expression and function of important tight junction and transporter proteins in the blood-brain barrier model. Then,we demonstrated the application of integrating Tissue-in-a-CUBE with a chip in simulating the in vitro testing of the permeability of a drug through the blood-brain barrier to the brain and its effect on treating the glioblastoma brain cancer model. We anticipate that this platform can be adapted for use with organoids to build complex human systems in vitro by the combination of multiple simple CUBE units. Development of platform to integrate multiple Tissue-in-a-CUBEs in a chip for tissue-tissue interaction,demonstrated by simulating the testing of the permeability and effect of a cancer drug in a BBB-Brain cancer model. View Publication

Peripheral nerve and large-scale muscle injuries result in significant disability,necessitating the development of biomaterials that can restore functional deficits by promoting tissue regrowth in an electroactive environment. Among these materials,graphene is favored for its high conductivity,but its low bioactivity requires enhancement through biomimetic components. In this study,we extrusion printed graphene-poly(lactide-co-glycolide) (graphene) lattice scaffolds,aiming to increase bioactivity by incorporating decellularized extracellular matrix (dECM) derived from mouse pup skeletal muscle. We first evaluated these scaffolds using human-induced pluripotent stem cell (hiPSC)-derived motor neurons co-cultured with supportive glia,observing significant improvements in axon outgrowth. Next,we tested the scaffolds with C2C12 mouse and human primary myoblasts,finding no significant differences in myotube formation between dECM-graphene and graphene scaffolds. Finally,using a more complex hiPSC-derived 3D motor neuron spheroid model co-cultured with human myoblasts,we demonstrated that dECM-graphene scaffolds significantly improved axonal expansion towards peripheral myoblasts and increased axonal network density compared to graphene-only scaffolds. Features of early neuromuscular junction formation were identified near neuromuscular interfaces in both scaffold types. These findings suggest that dECM-graphene scaffolds are promising candidates for enhancing neuromuscular regeneration,offering robust support for the growth and development of diverse neuromuscular tissues. View Publication

Febrile seizures (FS) are a common childhood neurological condition triggered by fever in children without prior neurological disorders. While generally benign,some individuals,particularly those with complex FS or genetic predispositions,may develop epilepsy or other neurological comorbidities. The mechanisms underlying this transition remain unclear. Mutations in SCN1A,encoding the NaV1.1 sodium channel ?-subunit,have been linked to several epilepsy syndromes associated with FS. This study examines phenotypic variability in individuals carrying the same SCN1A c.434T?>?C mutation,using induced pluripotent stem cell (iPSC)-derived neurons from two siblings with FS. Despite sharing the mutation,only the older sibling developed temporal lobe epilepsy (TLE). Transcriptomic analysis revealed downregulation of GABAergic pathway genes in both siblings’ neurons,aligning with SCN1A-associated epilepsy. However,neurons from the sibling with TLE exhibited additional abnormalities,including altered AMPA receptor subunit composition,changes in GABAA receptor subunits and chloride cotransporters expression,and reduced brain-derived neurotrophic factor (BDNF) levels,indicative of developmental immaturity. Voltage-clamp recordings confirmed impaired GABAergic and AMPA receptor-mediated synaptic activity. These findings suggest that combined GABAergic dysfunction,aberrant AMPA receptor composition,and reduced BDNF signaling contribute to the more severe phenotype and increased epilepsy susceptibility.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-09208-3. View Publication

Backgroundc-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers,c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development,its impact on nervous system development in higher mammals,especially for some deep structures,for example,thalamus in diencephalon,remains unclear.MethodsTo investigate the influence of c-JUN on early nervous system development,c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs),cerebral organoids (COs),and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining,omics analysis,and loss/gain of function analysis.ResultsAt embryonic day 14.5,c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6+/NESTIN+ exterior layer but lacked a fibers-connected core. Moreover,the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably,in guided ThOs,c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2,WNT8B,MXRA5,HSPG2 and PLAGL1 while 24 genes including MSX1,CYP1B1,LMX1B,NQO1 and COL2A1 were significantly up-regulated.ConclusionOur findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01303-8. View Publication