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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

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

SummaryKCNQ1/Kv7,a low-voltage-gated K+ channel,regulates cardiac rhythm and glucose homeostasis. While KCNQ1 mutations are associated with long-QT syndrome and type2 diabetes,its function in human pancreatic cells remains controversial. We identified a homozygous KCNQ1 mutation (R397W) in an individual with permanent neonatal diabetes melitus (PNDM) without cardiovascular symptoms. To decipher the potential mechanism(s),we introduced the mutation into human embryonic stem cells and generated islet-like organoids (SC-islets) using CRISPR-mediated homology-repair. The mutation did not affect pancreatic differentiation,but affected channel function by increasing spike frequency and Ca2+ flux,leading to insulin hypersecretion. With prolonged culturing,the mutant islets decreased their secretion and gradually deteriorated,modeling a diabetic state,which accelerated by high glucose levels. The molecular basis was the downregulated expression of voltage-activated Ca2+ channels and oxidative phosphorylation. Our study provides a better understanding of the role of KCNQ1 in regulating insulin secretion and ?-cell survival in hereditary diabetes pathology. Graphical abstract Highlights•A permanent neonatal diabetes melitus patient carries a homozygous KCNQ1 mutation•KCNQ1R397W is loss of function and shows atypical electrophysiology in hESC-islets•Under high glucose,elevated Ca2+ flux leads to insulin hypersecretion•Mutant cells gradually switch phenotype,deteriorate,accelerated by high glucose Biological sciences; Endocrinology; Endocrinology; Health sciences; Internal medicine; Medical specialty; Medicine; Natural sciences; Physiology View Publication

Stem cell-derived islet cell therapy can effectively treat type 1 diabetes,but its efficacy is hindered by low oxygen supply post-transplantation,particularly in subcutaneous spaces and encapsulation devices,leading to cell dysfunction. The response to hypoxia and effective strategies to alleviate its detrimental effects remain poorly understood. Here,we show that ? cells within stem cell-derived islets gradually undergo a decline in cell identity and metabolic function in hypoxia. This is linked to reduced expression of immediate early genes (EGR1,FOS,and JUN),which downregulates key ? cell transcription factors. We further identified genes important for maintaining ? cell fitness in hypoxia,with EDN3 as a potent player. Elevated EDN3 expression preserves ? cell identity and function in hypoxia by modulating genes involved in ? cell maturation,glucose sensing and regulation. These insights improve the understanding of hypoxia’s impact on stem cell-derived islets,offering a potential intervention for clinical applications. Hypoxia impairs the efficacy of stem cell-derived islet cell therapy,making it a potential barrier for treatment of type 1 diabetes. Wang et al. identify EDN3 as a key factor that preserves ? cell identity and function in hypoxia,offering possible strategies to improve therapeutic outcomes. View Publication

During a heart attack,ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment,thereby shifting focus to cell-free therapies. Consequently,we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature,efficacy in previous studies,and easy obtainability from minimally invasive techniques. Nonetheless,using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome,regardless of donor health,would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors,passaged 6–10 times,underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content,independent of donor health status,had elevated expression of proteins involved in cell survival pathways. In IR conditions,iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05),cell viability (p < 0.001),and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently,only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore,secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences. View Publication

Single-cell technologies can measure the expression of thousands of molecular features in individual cells undergoing dynamic biological processes. While examining cells along a computationally-ordered pseudotime trajectory can reveal how changes in gene or protein expression impact cell fate,identifying such dynamic features is challenging due to the inherent noise in single-cell data. Here,we present DELVE,an unsupervised feature selection method for identifying a representative subset of molecular features which robustly recapitulate cellular trajectories. In contrast to previous work,DELVE uses a bottom-up approach to mitigate the effects of confounding sources of variation,and instead models cell states from dynamic gene or protein modules based on core regulatory complexes. Using simulations,single-cell RNA sequencing,and iterative immunofluorescence imaging data in the context of cell cycle and cellular differentiation,we demonstrate how DELVE selects features that better define cell-types and cell-type transitions. DELVE is available as an open-source python package: https://github.com/jranek/delve. Characteristic genes or proteins driving continuous biological processes are difficult to uncover from noisy single-cell data. Here,authors present DELVE,an unsupervised feature selection method to identify core molecular features driving cell fate decisions. View Publication

Human GWAS have shown that obesogenic FTO polymorphisms correlate with lean mass,but the mechanisms have remained unclear. It is counterintuitive because lean mass is inversely correlated with obesity and metabolic diseases. Here,we use CRISPR to knock-in FTOrs9939609-A into hESC-derived tissue models,to elucidate potentially hidden roles of FTO during development. We find that among human tissues,FTOrs9939609-A most robustly affect human muscle progenitors’ proliferation,differentiation,senescence,thereby accelerating muscle developmental and metabolic aging. An edited FTOrs9939609-A allele over-stimulates insulin/IGF signaling via increased muscle-specific enhancer H3K27ac,FTO expression and m6A demethylation of H19 lncRNA and IGF2 mRNA,with excessive insulin/IGF signaling leading to insulin resistance upon replicative aging or exposure to high fat diet. This FTO-m6A-H19/IGF2 circuit may explain paradoxical GWAS findings linking FTOrs9939609-A to both leanness and obesity. Our results provide a proof-of-principle that CRISPR-hESC-tissue platforms can be harnessed to resolve puzzles in human metabolism. Human GWAS paradoxically linked FTO SNPs to both lean mass and sarcopenia/obesity. Here,Guang et al used CRISPR-edited stem cells to reveal that an obesogenic FTO SNP accelerates both muscle development and aging,by increasing RNA m6A demethylation. View Publication

Neutrophils are critical for host defense against fungi. However,the short life span and lack of genetic tractability of primary human neutrophils has limited in vitro analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils. Here,we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against Aspergillus fumigatus. GATA1 knockout (KO) iNeutrophils have increased maturation,antifungal pattern recognition receptor expression and more readily execute neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal ?-glucan,including an upregulation of the pentose phosphate pathway (PPP),similar to primary human neutrophils in vitro. Furthermore,we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill A. fumigatus but is not necessary for the upregulation of PPP. Collectively,these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response. Author SummaryNeutrophils are important first responders to fungal infections,and understanding their antifungal functions is essential to better elucidating disease dynamics. Primary human neutrophils are short lived and do not permit genetic manipulation,limiting their use to study neutrophil-fungal interactions in vitro. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) are a genetically tractable alternative to primary human neutrophils for in vitro analyses. In this report we show that GATA1-deficient iPSCs generate neutrophils (iNeutrophils) that are more mature than wild-type iNeutrophils and display increased antifungal activity against the human fungal pathogen Aspergillus fumigatus. We also show that GATA1-deficient iNeutrophils have increased expression of antifungal receptors than wild-type cells and shift their metabolism and execute neutrophil antifungal functions at levels comparable to primary human neutrophils. Deletion of the integrin CD18 blocks the ability of GATA1-deficient iNeutrophils to kill and control the growth of A. fumigatus,demonstrating an important role for this integrin in iNeutrophil antifungal activity. Collectively,these findings support the use of iNeutrophils as a model to study neutrophil antifungal immunity. View Publication

Diabetes mellitus,a chronic and non-transmissible disease,triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ?-like cells (P?LCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways,directing the differentiation of iPSCs into pancreatic ? cells. Despite significant progress and improved protocols,the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here,we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into P?LCs. Our protocol has shown to be effective,with the resulting cells exhibiting key functional properties of pancreatic ? cells,including the expression of crucial molecular markers (pdx1,nkx6.1,ngn3) and the capability to secrete insulin in response to glucose. Furthermore,the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ? cell genes. View Publication

Disease modeling with isogenic Induced Pluripotent Stem Cell (iPSC)-differentiated organoids serves as a powerful technique for studying disease mechanisms. Multiplexed coculture is crucial to mitigate batch effects when studying the genetic effects of disease-causing variants in differentiated iPSCs or organoids,and demultiplexing at the single-cell level can be conveniently achieved by assessing natural genetic barcodes. Here,to enable cost-efficient time-series experimental designs via multiplexed bulk and single-cell RNA-seq of hybrids,we introduce a computational method in our Vireo Suite,Vireo-bulk,to effectively deconvolve pooled bulk RNA-seq data by genotype reference,and thereby quantify donor abundance over the course of differentiation and identify differentially expressed genes among donors. Furthermore,with multiplexed scRNA-seq and bulk RNA-seq,we demonstrate the usefulness and necessity of a pooled design to reveal donor iPSC line heterogeneity during macrophage cell differentiation and to model rare WT1 mutation-driven kidney disease with chimeric organoids. Our work provides an experimental and analytic pipeline for dissecting disease mechanisms with chimeric organoids. IPSC-derived organoids model diseases. Multiplexed coculture and demultiplexing natural genetic barcodes aid in studying genetic effects. Here,authors introduce Vireo-bulk to deconvolve bulk RNA-seq data,quantify donor abundance and identify differentially expressed genes. View Publication

Oxidative phosphorylation (OXPHOS) in the mitochondrial inner membrane is a therapeutic target in many diseases. Neural stem cells (NSCs) show progress in improving mitochondrial dysfunction in the central nervous system (CNS). However,translating neural stem cell-based therapies to the clinic is challenged by uncontrollable biological variability or heterogeneity,hindering uniform clinical safety and efficacy evaluations. We propose a systematic top-down design based on membrane self-assembly to develop neural stem cell-derived oxidative phosphorylating artificial organelles (SAOs) for targeting the central nervous system as an alternative to NSCs. We construct human conditionally immortal clone neural stem cells (iNSCs) as parent cells and use a streamlined closed operation system to prepare neural stem cell-derived highly homogenous oxidative phosphorylating artificial organelles. These artificial organelles act as biomimetic organelles to mimic respiration chain function and perform oxidative phosphorylation,thus improving ATP synthesis deficiency and rectifying excessive mitochondrial reactive oxygen species production. Conclusively,we provide a framework for a generalizable manufacturing procedure that opens promising prospects for disease treatment. Regulating oxidative phosphorylation and restoring redox homeostasis are crucial in neurological disorders. Here,the authors develop a top-down membrane self-assembly strategy to develop stem cell-derived artificial organelles (SAOs) that mimic mitochondrial oxidative phosphorylation without the risks associated with stem cell therapy. View Publication

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents,and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue,in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas,but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here,we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells,and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method,new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity,suitable stability and biodistribution,with negligible toxicity to islets,qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-84574-y. View Publication