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Differentiation of embryonic stem cells and induced pluripotent stem cells (iPSCs) into endoderm derivatives,including thyroid,thymus,lungs,liver,and pancreas,has broad implications for disease modeling and therapy. We utilize and expand a model development approach previously outlined by the authors to construct a model for the directed differentiation of iPSCs into definitive endoderm (DE). Assuming discrete intermediate stages in the differentiation process with a homogeneous population in each stage,three lineage models with two,three,and four populations and three growth models are constructed. Additionally,three models for error distribution are defined,resulting in a total of 27 models. Experimental data obtained in vitro are used for model calibration,model selection,and final validation. Model selection suggests that no transitory state during differentiation expresses the DE biomarkers CD117 and CD184,a finding corroborated by existing literature. Additionally,space-limited growth models,such as logistic and Gompertz growth,outperform exponential growth. Validation of the inferred model with leave-out data results in prediction errors of 26.4%. Using the inferred model,it is predicted that the optimal differentiation period is between 1.9 and 2.4 days,plating populations closer to 300 000 cells per well result in the highest yield efficiency,and that iPSC differentiation outpaces the DE proliferation as the main driver of the population dynamics. We also demonstrate that the model can predict the effect of growth modulators on cell population dynamics. Our model serves as a valuable tool for optimizing differentiation protocols,providing insights into developmental biology. View Publication

INTRODUCTIONPolygenic risk score (PRS) identifies individuals at high genetic risk for Alzheimer's disease (AD),but its utility in predicting cognitive trajectories and AD pathologies remains unclear. We optimized PRS (optPRS) for AD,investigated its association with cognitive trajectories and AD phenotypes of cerebral organoids.METHODSUsing genome‐wide association study (GWAS) summary statistics from a European population,we developed optPRS to predict AD in Korean individuals (n = 1634). We analyzed the association between optPRS and cognitive trajectories (n = 771). We generated induced pluripotent stem cell–derived cerebral organoids from patients with high (n = 3) and low (n = 4) optPRS to evaluate amyloid beta (Aβ) and phosphorylated tau (p‐tau) levels.RESULTSOptPRS predicted AD dementia and Aβ positivity,independent of apolipoprotein E (APOE). Higher optPRSs correlated with rapid cognitive decline. Cerebral organoids from the high optPRS group exhibited increased Aβ insolubility and p‐tau levels.CONCLUSIONOptPRS predicted cognitive decline and AD phenotypes of cerebral organoids,supporting its use in risk assessments and drug‐screening platform. View Publication

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects communication,social interaction,and behavior. Calcium (Ca2+) signaling dysregulation has been frequently highlighted in genetic studies as a contributing factor to aberrant developmental processes in ASD. Herein,we used ASD and control induced pluripotent stem cells (iPSCs) to investigate transcriptomic and functional Ca2+ dynamics at various stages of differentiation to cortical neurons. Idiopathic ASD and control iPSC lines underwent the dual SMAD inhibition differentiation protocol to direct their fate toward cortical neurons. Samples from multiple time points along the course of differentiation were processed for bulk RNA sequencing,spanning the following sequential stages: the iPSC stage,neural induction (NI) stage,neurosphere (NSP) stage,and differentiated cortical neuron (Diff) stage. Our transcriptomic analyses suggested that the numbers of Ca2+ signaling-relevant differentially expressed genes between ASD and control samples were higher in the iPSC and Diff stages. Accordingly,samples from the iPSC and Diff stages were processed for Ca2+ imaging studies. Results revealed that iPSC-stage ASD samples displayed elevated maximum Ca2+ levels in response to ATP compared to controls. By contrast,in the Diff stage,ASD neurons showed reduced maximum Ca2+ levels in response to ATP but increased maximum Ca2+ levels in response to KCl and DHPG relative to controls. Considering the distinct functional signaling contexts of these stimuli,this differential profile of receptor- and ionophore-mediated Ca2+ response suggests that aberrant calcium homeostasis underlies the pathophysiology of ASD neurons. Our data provides functional evidence for Ca2+ signaling dysregulation during neurogenesis in idiopathic ASD. View Publication

BackgroundDiabetic peripheral neuropathy (DPN) is one of the most prevalent and debilitating complications of diabetes,marked by chronic neuroinflammation,immune dysregulation,and progressive neuronal degeneration. Current treatments offer limited efficacy,largely focusing on symptomatic relief rather than addressing the underlying disease mechanisms. There is a critical need for disease-modifying therapies that target the molecular basis of DPN.ResultsIn this study,we developed a novel targeted nanotherapeutic system—ZH-1c-EVs@SIN—composed of neural stem cell-derived extracellular vesicles (NSC-EVs) modified with the ZH-1c aptamer and loaded with the anti-inflammatory compound sinomenine (SIN). This system was specifically designed to target microglia and inhibit the WNT5a/TRPV1 signaling pathway. Transcriptomic profiling of microglia revealed key gene networks implicated in DPN pathology and responsive to SIN treatment. Functional assays demonstrated that ZH-1c-EVs@SIN facilitated a shift in microglial phenotype from pro-inflammatory M1 to anti-inflammatory M2,significantly reduced inflammatory cytokine expression,and restored levels of neuronal regulatory proteins. Nanoparticle tracking analysis and transmission electron microscopy confirmed optimal vesicle size and morphology,while fluorescence imaging showed efficient uptake by microglia. In vivo studies in a murine model of DPN revealed marked improvements in pain-related behavior and histopathological signs of nerve damage.ConclusionZH-1c-EVs@SIN represents a promising therapeutic strategy for DPN,offering targeted immunomodulation and enhanced neural repair via regulation of the WNT5a/TRPV1 signaling axis. This nano-delivery platform introduces a novel and precise approach to intervening in diabetic neuropathy and may be applicable to other neuroinflammatory conditions.Graphical abstractMechanism of ZH-1c-EVs@SIN Mediating the WNT5a/TRPV1 Pathway to Improve Immune-Inflammatory Homeostasis in the Treatment of DPN in Mice. View Publication

Craniosynostosis is a multigenic congenital condition in which one or more calvarial sutures have prematurely fused during the development of the fetus. Pathogenic variants in FGFR2 are associated with the development of syndromic craniosynostosis,such as Crouzon,Apert and Pfeifer syndromes. Investigation of FGFR2 -linked craniosynostosis is hindered by the lack of appropriate in vitro models. Patient-derived human induced pluripotent stem cell (hiPSC) in vitro disease models provide the opportunity to investigate the disease,identify molecular targets for pharmaceutical treatments,and enable the generation of autologous pluripotent stem cell catalogues. Here,we report three patient-derived hiPSC lines carrying the C342Y,S252W or E565G FGFR2 pathogenic variant. The patient hiPSC lines express characteristic pluripotency markers and display distinct phosphorylation profiles under unstimulated conditions. FGFR2 C342Y showed autophosphorylation in the absence of bFGF ligand,although downstream docking proteins PLCγ and FRS2α were not phosphorylated. FGFR2 S252W and FGFR2 E565G hiPSCs showed increased phosphorylation of docking proteins PLCγ and FRS2α,whereas FGFR2 was not phosphorylated. These patient hiPSC lines provide molecular and cellular options to investigate FGFR2 -linked craniosynostosis in the patient-specific genomic context and develop therapeutic modalities. View Publication