技术资料

Patient specific induced pluripotent stem cells (iPSCs) derived ? cells represent an effective means for disease modeling and autologous diabetes cell replacement therapy. In this study,an AG73-5%gelatin methacryloyl (GelMA) /2% alginate methacrylate (AlgMA) hydrogel was employed to generate pancreatic progenitor (PP) organoids and improve stem cell-derived ? (SC-?) cell differentiation protocol. The laminin-derived homolog AG73,which mimics certain cell?matrix interactions,facilitates AKT signaling pathway activation to promote PDX1+/NKX6.1+ PP organoid formation and effectively modulates subsequent epithelial–mesenchymal transition (EMT) in the endocrine lineage. The 5%GelMA/2%AlgMA hydrogel mimics the physiological stiffness of the pancreas,providing the optimal mechanical stress and spatial structure for PP organoid differentiation. The Syndecan-4 (SDC4)-ITGAV complex plays a pivotal role in the early stages of pancreatic development by facilitating the formation of SOX9+/PDX1+ bipotent PPs. Our findings demonstrate that AG73-GelMA/AlgMA hydrogel-derived SC-? cells exhibit enhanced insulin secretion and accelerated hyperglycemia reversal in vivo. This study presents a cost-effective,stable,and efficient alternative for the comprehensive 3D culture of SC-? cells in vitro by mitigating the uncertainties associated with conventional culture methods. View Publication

BackgroundMetastasis,the spread,and growth of malignant cells at secondary sites within a patient’s body,accounts for over 90% of cancer-related mortality. Breast cancer is the most common tumor type diagnosed and the leading cause of cancer lethality in women in the United States. It is estimated that 10–16% breast cancer patients will have brain metastasis. Current therapies to treat patients with breast cancer brain metastasis (BCBM) remain palliative. This is largely due to our limited understanding of the fundamental molecular and cellular mechanisms through which BCBM progresses,which represents a critical barrier for the development of efficient therapies for affected breast cancer patients.MethodsPrevious research in BCBM relied on co-culture assays of tumor cells with rodent neural cells or rodent brain slice ex vivo. Given the need to overcome the obstacle for human-relevant host to study cell-cell communication in BCBM,we generated human embryonic stem cell-derived cerebral organoids to co-culture with human breast cancer cell lines. We used MDA-MB-231 and its brain metastatic derivate MDA-MB-231 Br-EGFP,other cell lines of MCF-7,HCC-1806,and SUM159PT. We leveraged this novel 3D co-culture platform to investigate the crosstalk of human breast cancer cells with neural cells in cerebral organoid.ResultsWe found that MDA-MB-231 and SUM159PT breast cancer cells formed tumor colonies in human cerebral organoids. Moreover,MDA-MB-231 Br-EGFP cells showed increased capacity to invade and expand in human cerebral organoids.ConclusionsOur co-culture model has demonstrated a remarkable capacity to discern the brain metastatic ability of human breast cancer cells in cerebral organoids. The generation of BCBM-like structures in organoid will facilitate the study of human tumor microenvironment in culture. View Publication

Evaluating cell metabolism is crucial during pluripotent stem cell (PSC) differentiation and somatic cell reprogramming as it affects cell fate. As cultured stem cells are heterogeneous,a comparative analysis of relative metabolism using existing metabolic analysis methods is difficult,resulting in inaccuracies. In this study,we measured human PSC basal metabolic levels using a Seahorse analyzer. We used fibroblasts,human induced PSCs,and human embryonic stem cells to monitor changes in basal metabolic levels according to cell number and determine the number of cells suitable for analysis. We evaluated normalization methods using glucose and selected the most suitable for the metabolic analysis of heterogeneous PSCs during the reprogramming stage. The response of fibroblasts to glucose increased with starvation time,with oxygen consumption rate and extracellular acidification rate responding most effectively to glucose 4 hours after starvation and declining after 5 hours of starvation. Fibroblasts and PSCs achieved appropriate responses to glucose without damaging their metabolism 2?4 and 2?3 hours after starvation,respectively. We developed a novel method for comparing basal metabolic rates of fibroblasts and PSCs,focusing on quantitative analysis of glycolysis and oxidative phosphorylation using glucose without enzyme inhibitors. This protocol enables efficient comparison of energy metabolism among cell types,including undifferentiated PSCs,differentiated cells,and cells undergoing cellular reprogramming,and addresses critical issues,such as differences in basal metabolic levels and sensitivity to normalization,providing valuable insights into cellular energetics. View Publication

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia,platelet dysfunction,and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation,the DNA methylation status in FPDMM remains unknown,largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here,using genome editing techniques,we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks),consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs,with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1,an ETS family member,was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation,and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation. 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

SummaryBackgroundMacrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However,the production and application of genetically edited,highly effective,and mass-produced CAR-modified macrophages (CAR-Ms) are challenging.MethodsHere,we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach,together with a simple differentiation protocol,produced stable and highly effective CAR-Ms without heterogeneity.FindingsThese engineered cells phagocytosed cancer cells,leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore,the engineered CARs,which incorporated a combination of CD3? and Megf10 (referred to as FRP5M?),markedly enhanced the antitumour effect of CAR-Ms by promoting M1,but not M2,polarisation. FRP5M? promoted M1 polarisation via nuclear factor kappa B (NF-?B),ERK,and STAT1 signalling,and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling,inducing M1 polarisation of bystander non-CAR macrophages,and enhancing the infiltration of T cells in cancer spheroids.InterpretationOur findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion,the guided insertion of CAR containing CD3? and Megf10 domains is an effective strategy for the immunotherapy of solid tumours.FundingThis work was supported by KRIBB Research Initiative Program Grant (KGM4562431,KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,10.13039/501100003625Ministry of Health and Welfare) (22A0304L1-01). View Publication

Static gene expression programs have been extensively characterized in stem cells and mature human cells. However,the dynamics of RNA isoform changes upon cell-state-transitions during cell differentiation,the determinants and functional consequences have largely remained unclear. Here,we established an improved model for human neurogenesis in vitro that is amenable for systems-wide analyses of gene expression. Our multi-omics analysis reveals that the pronounced alterations in cell morphology correlate strongly with widespread changes in RNA isoform expression. Our approach identifies thousands of new RNA isoforms that are expressed at distinct differentiation stages. RNA isoforms mainly arise from exon skipping and the alternative usage of transcription start and polyadenylation sites during human neurogenesis. The transcript isoform changes can remodel the identity and functions of protein isoforms. Finally,our study identifies a set of RNA binding proteins as a potential determinant of differentiation stage-specific global isoform changes. This work supports the view of regulated isoform changes that underlie state-transitions during neurogenesis. Synopsis Multi-omics analysis of a newly established human neuronal cell differentiation model reveals widespread dynamic changes in RNA isoform expression,their functional consequences and potential determinants,providing evidence that they underlie cell-state-transitions during neurogenesis. Dynamic changes in RNA and protein levels are strongly correlated during all stages of neuronal differentiation.Nanopore sequencing (ONT-seq) during human neurogenesis reveals 12,019 non-annotated RNA isoforms,a large number of which are differentially expressed during differentiation.70% of new RNA isoforms result from the use of alternative transcription start sites (TSSs) or polyadenylation (pA) sites and exon skipping.RNA isoform changes underlie protein isoform changes during human neurogenesis as revealed by integrating ONT-seq,RNA-seq and proteomics time course data.RNA motif enrichment,RNA-seq and available CLIP-seq data uncover a set of RNA binding proteins (RBPs) as potential determinants of differentiation stage-specific global isoform changes. Multi-omics analysis of a newly established human neuronal cell differentiation model reveals widespread dynamic changes in RNA isoform expression,their functional consequences and potential determinants,providing evidence that they underlie cell-state-transitions during neurogenesis. 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

Advancing cardiac tissue engineering requires innovative fabrication techniques,including 3D bioprinting and tissue maturation,to enable the generation of new muscle for repairing or replacing damaged heart tissue. Recent advances in tissue engineering have highlighted the need for rapid,high-resolution bioprinting methods that preserve cell viability and maintain structural fidelity. Traditional collagen-based bioinks gel slowly,limiting their use in bioprinting. Here,we implement TRACE (tunable rapid assembly of collagenous elements),a macromolecular crowding-driven bioprinting technique that enables the immediate gelation of collagen bioinks infused with cells. This overcomes the need for extended incubation,allowing for direct bioprinting of engineered cardiac tissues with high fidelity. Unlike methods that rely on high-concentration acidic collagen or fibrin for gelation,TRACE achieves rapid bioink stabilization without altering the biochemical composition. This ensures greater versatility in bioink selection while maintaining functional tissue outcomes. Additionally,agarose slurry provides stable structural support,preventing tissue collapse while allowing nutrient diffusion. This approach better preserves complex tissue geometries during culture than gelatin-based support baths or polydimethylsiloxane (PDMS) molds. Our results demonstrate that TRACE enables the bioprinting of structurally stable cardiac tissues with high resolution. By supporting the fabrication of biomimetic tissues,TRACE represents a promising advancement in bioprinting cardiac models and other engineered tissues. View Publication

Copy number variations of the human CNTN6 gene,resulting from megabase-scale microdeletions or microduplications in the 3p26.3 region,are frequently implicated in neurodevelopmental disorders such as intellectual disability and developmental delay. However,duplication of the full-length human CNTN6 gene presents with variable penetrance,resulting in phenotypes that range from neurodevelopmental disorders to no visible pathologies,even within the same family. Previously,we obtained a set of induced pluripotent stem cell lines derived from a patient with a CNTN6 gene duplication and from two healthy donors. Our findings demonstrated that CNTN6 expression in neurons carrying the duplication was significantly reduced. Additionally,the expression from the CNTN6 duplicated allele was markedly lower compared to the wild-type allele. Here,we first introduce a system for correcting megabase-scale duplications in induced pluripotent stem cells and secondly analyze the impact of this correction on CNTN6 gene expression. We showed that the deletion of one copy of the CNTN6 duplication did not affect the expression levels of the remaining allele in the neuronal cells. View Publication

Naive human embryonic stem cells (hESCs) that resemble the pre-implantation epiblasts are fueled by a combination of aerobic glycolysis and oxidative phosphorylation,but their mitochondrial regulators are poorly understood. Here we report that,proline dehydrogenase (PRODH),a mitochondria-localized proline metabolism enzyme,is dramatically upregulated in naive hESCs compared to their primed counterparts. The upregulation of PRODH is induced by a reduction in c-Myc expression that is dependent on PD0325901,a MEK inhibitor routinely present in naive hESC culture media. PRODH knockdown in naive hESCs significantly promoted mitochondrial oxidative phosphorylation (mtOXPHOS) and reactive oxygen species (ROS) production that triggered autophagy,DNA damage,and apoptosis. Remarkably,MitoQ,a mitochondria-targeted antioxidant,effectively restored the pluripotency and proliferation of PRODH-knockdown naive hESCs,indicating that PRODH maintains naive pluripotency by preventing excessive ROS production. Concomitantly,PRODH knockdown significantly slowed down the proteolytic degradation of multiple key mitochondrial electron transport chain complex proteins. Thus,we revealed a crucial role of PRODH in limiting mtOXPHOS and ROS production,and thereby safeguarding naive pluripotency of hESCs. Synopsis Downregulation of PRODH promotes oxidative phosphorylation and ROS production,which in turn impair pluripotency and proliferation of naive but not primed hESCs,revealing a crucial role of PRODH in safeguarding human naive pluripotency. PRODH is expressed in naive hESCs at a higher level compared to their primed counterparts.MEK inhibitor present in naive culture media upregulates PRODH by suppressing MYC.PRODH depletion boosts mtOXPHOS and ROS production in naive hESCs.PRODH promotes proteolytic degradation of the ETC complex components. Downregulation of PRODH promotes oxidative phosphorylation and ROS production,which in turn impair pluripotency and proliferation of naive but not primed hESCs,revealing a crucial role of PRODH in safeguarding human naive pluripotency. View Publication

SummaryGenome-wide association studies (GWAS) of Alzheimer’s disease (AD) have identified a plethora of risk loci. However,the disease variants/genes and the underlying mechanisms remain largely unknown. For a strong AD-associated locus near Clusterin (CLU),we tied an AD protective allele to a role of neuronal CLU in promoting neuron excitability through lipid-mediated neuron-glia communication. We identified a putative causal SNP of CLU that impacts neuron-specific chromatin accessibility to transcription-factor(s),with the AD protective allele upregulating neuronal CLU and promoting neuron excitability. Transcriptomic analysis and functional studies in induced pluripotent stem cell (iPSC)-derived neurons co-cultured with mouse astrocytes show that neuronal CLU facilitates neuron-to-glia lipid transfer and astrocytic lipid droplet formation coupled with reactive oxygen species (ROS) accumulation. These changes cause astrocytes to uptake less glutamate thereby altering neuron excitability. Our study provides insights into how CLU confers resilience to AD through neuron-glia interactions. View Publication