技术资料

AbstractBackgroundParkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra,which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed,a decrease in Treg number and function has been reported in PD. In this context,pramipexole,a dopaminergic receptor agonist used to treat PD symptoms,has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells.MethodsTreg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28?activated and non?activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co?cultured with in vitro?differentiated human dopaminergic neurons in a cytotoxicity assay with 6?hydroxydopamine (6?OHDA). The role of interleukin?10 (IL?10) was investigated by co?culturing activated IL?10?producing Treg cells with neurons. To further investigate the effect of treatment on Tregs,gene expression in pramipexole?treated,CD3/CD28?activated Treg cells was determined by Fluidigm analysis.ResultsPramipexole?treated CD3/CD28?activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6?OHDA. Pramipexole?treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL?10 release and the activation of genes associated with regulation and neuroprotection.ConclusionAnti?CD3/CD28?activated Treg cells protect dopaminergic neurons against 6?OHDA?induced damage. In addition,activated,IL?10?producing,pramipexole?treated Tregs also induced a neuroprotective effect,and the supernatants of these co?cultures promoted axonal growth. Pramipexole?treated,activated Tregs altered their gene expression in a concentration?dependent manner,and enhanced TGF??related dopamine receptor regulation and immune?related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD. Pramipexole?treated,activated regulatory T cells protect dopaminergic neurons against 6 OHDA damage and promote primary neurite length. This could be due to the production of the regulatory cytokine IL?10 and an increased expression of genes related to regulation and neuroprotection. 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

The immune-modulatory effects of mesenchymal stromal cells (MSCs) are widely used to treat inflammatory disorders,with indoleamine 2,4-dioxygenase-1 (IDO-1) playing a pivotal role in suppressing stimulated T-cell proliferation. Taking that three-dimensional (3D) cultures enhance MSCs’ anti-inflammatory properties compared with two-dimensional (2D) cultures,the differentially expressed miRNAs were examined. Thus,we identified hsa-miR-4662a-5p (miR-4662a) as a key inducer of IDO-1 via its suppression of bridging integrator-1 (BIN-1),a negative regulator of the IDO-1 gene. The IDO-1-inducing potential of miR-4662a was conserved across primary MSCs from various donors and sources but exhibited variability. Notably,iPSC-derived MSCs (iMSCs) demonstrated superior IDO-1 induction and immune-modulatory efficacy compared with their donor-matched primary MSCs. Accordingly,iMSCs expressing miR-4662a (4662a/iMSC) exhibited stronger suppressive effects on T-cell proliferation and more potent suppressive effects on graft-versus-host disease (GVHD),improving survival rates and reducing tissue damage in the liver and gut. Our results point to the therapeutic potential of standardized,off-the-shelf 4662a/iMSC as a robust immune-modulating cell therapy for GVHD. View Publication

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data,while reducing turnover time and costs,and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay,a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes,we sought to introduce standardization steps to the protocol,improving the overall robustness of the PluriLum assay,as well as a shortening of the assay protocol. First,we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm,robust differentiation can be anticipated. In terms of reproducibility,exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data,compared to more reliable concentration–response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol,resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid,both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally,we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter,which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion,we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing. View Publication

Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function,DYSF exon 44,founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing,rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation,hEx44mut,recapitulates the patients’ phenotype and an identical re-framing outcome in primary muscle stem cells. Finally,gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility,not only in treating dysferlin deficiency syndromes,but also perhaps other forms of muscular dystrophy. Dysferlin-deficient muscular dystrophy is a devastating and untreatable disease. Using Cas9,the authors restored dysferlin in muscle stem cells from patients ex vivo and show proof-of-concept for autologous cell replacement therapies in a new humanized mouse model. View Publication

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