搜索结果: 'gmp t cell activator'

BackgroundUnderstanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models,lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate,and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule.MethodsTo establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells,we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways,and construct cell lines carrying an inducible NKX3-1 expressing cassette,together with three-dimensional culture system. Unpaired t test was applied for statistical analyses.ResultsWe first successfully generate the definitive endoderm,hindgut,and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1,but fail to express NKX3-1. Therefore,we construct NKX3-1-inducible cell line by homologous recombination,which is eventually able to yield AR,FOXA1,and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally,combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations.ConclusionsThis study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line,as well as provides a stepwise differentiation protocol to generate human prostate-like organoids,which should facilitate the studies on prostate development and disease pathogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03886-y. View Publication

Cell fate progression of pluripotent progenitors is strictly regulated,resulting in high human cell diversity. Epigenetic modifications also orchestrate cell fate restriction. Unveiling the epigenetic mechanisms underlying human cell diversity has been difficult. In this study,we use human brain and retina organoid models and present single-cell profiling of H3K27ac,H3K27me3 and H3K4me3 histone modifications from progenitor to differentiated neural fates to reconstruct the epigenomic trajectories regulating cell identity acquisition. We capture transitions from pluripotency through neuroepithelium to retinal and brain region and cell type specification. Switching of repressive and activating epigenetic modifications can precede and predict cell fate decisions at each stage,providing a temporal census of gene regulatory elements and transcription factors. Removing H3K27me3 at the neuroectoderm stage disrupts fate restriction,resulting in aberrant cell identity acquisition. Our single-cell epigenome-wide map of human neural organoid development serves as a blueprint to explore human cell fate determination. The mechanisms underlying human cell diversity are unclear. Here the authors provide a single-cell epigenome map of human neural organoid development and dissect how epigenetic changes control cell fate specification from pluripotency to distinct cerebral and retina neural types. View Publication

BackgroundLiver disease imposes a significant medical burden that persists due to a shortage of liver donors and an incomplete understanding of liver disease progression. Hepatobiliary organoids (HBOs) could provide an in vitro mini-organ model to increase the understanding of the liver and may benefit the development of regenerative medicine.MethodsIn this study,we aimed to establish HBOs with bile duct (BD) structures and mature hepatocytes (MHs) using human chemically induced liver progenitor cells (hCLiPs). hCLiPs were induced in mature cryo-hepatocytes using a small-molecule cocktail of TGF-? inhibitor (A-83-01,A),GSK3 inhibitor (CHIR99021,C),and 10% FBS (FAC). HBOs were then formed by seeding hCLiPs into ultralow attachment plates and culturing them with a combination of small molecules of Rock-inhibitor (Y-27632) and AC (YAC).ResultsThese HBOs exhibited bile canaliculi of MHs connected to BD structures,mimicking bile secretion and transportation functions of the liver. The organoids showed gene expression patterns consistent with both MHs and BD structures,and functional assays confirmed their ability to transport the bile analogs of rhodamine-123 and CLF. Functional patient-specific HBOs were also successfully created from hCLiPs sourced from cirrhotic liver tissues.ConclusionsThis study demonstrated the potential of human HBOs as an efficient model for studying hepatobiliary diseases,drug discovery,and personalized medicine.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03877-z. View Publication

Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal genetic disease,with most patients carrying mutations in PKD1. The main feature is the formation of bilateral renal cysts,leading to end stage renal failure in a significant proportion of those affected. Despite recent advances made in understanding ADPKD,there are currently no effective curative therapies. The emergence of human induced pluripotent stem cell (hiPSC)-derived kidney disease models has led to renewed hope that more physiological systems will allow for the development of novel treatments. hiPSC-derived organoid models have been used to recapitulate ADPKD,however they present numerous limitations which remain to be addressed. In the present study,we report an efficient method for generating organoids containing a network of polarised and ciliated epithelial tubules. PKD1 null (PKD1?/?) organoids spontaneously develop dilated tubules,recapitulating early ADPKD cystogenesis. Furthermore,PKD1?/? tubules present primary cilia defects when dilated. Our model could therefore serve as a valuable tool to study early ADPKD cystogenesis and to develop novel therapies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-94855-9. View Publication

Better understanding of the earliest molecular pathologies of all neurodegenerative diseases is expected to improve human therapeutics. We investigated the earliest molecular pathology of spinocerebellar ataxia type 1 (SCA1),a rare familial neurodegenerative disease that primarily induces death and dysfunction of cerebellum Purkinje cells. Extensive prior studies have identified involvement of transcription or RNA-splicing factors in the molecular pathology of SCA1. However,the regulatory network of SCA1 pathology,especially central regulators of the earliest developmental stages and inflammatory events,remains incompletely understood. Here,we elucidated the earliest developmental pathology of SCA1 using originally developed dynamic molecular network analyses of sequentially acquired RNA-seq data during differentiation of SCA1 patient-derived induced pluripotent stem cells (iPSCs) to Purkinje cells. Dynamic molecular network analysis implicated histone genes and cytokine-relevant immune response genes at the earliest stages of development,and revealed relevance of ISG15 to the following degradation and accumulation of mutant ataxin-1 in Purkinje cells of SCA1 model mice and human patients. Molecular changes in neurodegeneration occur much earlier than previously expected. In this study,dynamic molecular network analysis of iPSC differentiation uncovers a temporal pathway from histone to ISG15 with the earliest molecular changes of SCA1. View Publication

SummaryGABAergic interneurons are inhibitory neurons of the CNS,playing a fundamental role in neural circuitry and activity. Here,we provide a robust protocol for the successful enrichment of human cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs; generating embryoid bodies; and differentiating and enriching for cerebellar GABAergic neurons (cGNs),with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators,followed by intracellular calcium imaging and analyses.For complete details on the use and execution of this protocol,please refer to Pilotto et al.1 Graphical abstract Highlights•Steps described for generating GABAergic neurons from human iPSCs•Instructions for the enrichment of cerebellar GABAergic interneurons (cGNs)•Guide to calcium imaging of cGNs using genetically encoded calcium indicators Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. GABAergic interneurons are inhibitory neurons of the CNS,playing a fundamental role in neural circuitry and activity. Here,we provide a robust protocol for the successful enrichment of human-cerebellar GABAergic interneurons from human induced pluripotent stem cells (iPSCs) and measuring intracellular calcium transients. We describe in detail steps for culturing iPSCs,and generating embryoid bodies,differentiating and enriching for cerebellar GABAergic neurons (cGNs),with precise steps for their molecular characterization. We then detail the procedure for adeno-associated virus-mediated transduction of cGNs with genetically encoded calcium indicators,followed by intracellular calcium imaging and analyses. View Publication

Epicardium,the most outer mesothelium,exerts crucial functions in fetal heart development and adult heart regeneration. Here we use a three-step manipulation of WNT signalling entwined with BMP and RA signalling for generating a self-organized epicardial organoid that highly express with epicardium makers WT1 and TCF21 from human embryonic stem cells. After 8-days treatment of TGF-beta following by bFGF,cells enter into epithelium-mesenchymal transition and give rise to smooth muscle cells. Epicardium could also integrate and invade into mouse heart with SNAI1 expression,and give birth to numerous cardiomyocyte-like cells. Single-cell RNA seq unveils the heterogeneity and multipotency exhibited by epicardium-derived-cells and fetal-like epicardium. Meanwhile,extracellular matrix and growth factors secreted by epicardial organoid mimics the ecology of subepicardial space between the epicardium and cardiomyocytes. As such,this epicardial organoid offers a unique ground for investigating and exploring the potential of epicardium in heart development and regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01339-w. View Publication

Haloperidol is a typical antipsychotic used to treat schizophrenia and induces dopamine D2 receptor antagonism. Long-term use of haloperidol can reduce brain size in animals and humans; however,the underlying mechanism of this effect remains unclear. Notch1 signaling regulates the development and function of the nervous system by balancing stem cell proliferation and differentiation. Therefore,we investigated the effects of long-term exposure to haloperidol on human-derived brain organoids,which served as sophisticated in vitro models of human brain development. Long-term exposure to haloperidol reduced the size of brain organoids and decreased the ventricular zone and Notch1 signaling. When propionate,which protects against haloperidol-induced toxicity,was combined with haloperidol,it rescued both the overall size of brain organoids and Notch1 expression levels. Additionally,treatment with valproic acid,a Notch1 activator,partially restored the size of brain organoids and the thickness of the ventricular layer. Taken together,these data suggest that long-term exposure to haloperidol impairs neurodevelopment via Notch1 signaling in brain organoids. These findings contribute to our understanding of antipsychotic drug safety and provide information for new neurodevelopmental toxicity assessments.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-08855-w. View Publication

The advent of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing has marked a significant advancement in genetic engineering technology. However,the editing of induced pluripotent stem cells (iPSCs) with CRISPR presents notable challenges in ensuring cell survival and achieving high editing efficiency. These challenges become even more complex when considering the specific target site. P53 activation as a result of traditional CRISPR editing can lead to apoptosis,potentially worsening cell health or even resulting in cell death. Mitigating this apoptotic response can enhance cell survival post-CRISPR editing,which will ultimately increase editing efficiency. In our study,we observed that combining p53 inhibition with pro-survival small molecules yields a homologous recombination rate of over 90% when using CRISPR in human iPSCs. This protocol significantly streamlines the editing process and reduces the time and resources necessary for creating isogenic lines. View Publication

In the present study,we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely,in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons,while TH-positive catecholamine neurons were significantly underrepresented. Furthermore,high NMDAR1 expression was also detected in the KS spheroid,similarly to other patients-derived neuronal cultures,denoting high NMDAR1 expression as a general,KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat,rotenone,bardoxolone,and doxorubicin),and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions,drug development,and screening towards individual therapeutic strategies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-72791-4. View Publication

Krabbe disease (Kd) is a lysosomal storage disorder (LSD) caused by the deficiency of the lysosomal galactosylceramidase (GALC) which cleaves the myelin enriched lipid galactosylceramide (GalCer). Accumulated GalCer is catabolized into the cytotoxic lipid psychosine that causes myelinating cells death and demyelination which recruits microglia/macrophages that fail to digest myelin debris and become globoid cells. Here,to understand the pathological mechanisms of Kd,we used induced pluripotent stem cells (iPSCs) from Kd patients to produce myelinating organoids and microglia. We show that Kd organoids have no obvious defects in neurogenesis,astrogenesis,and oligodendrogenesis but manifest early myelination defects. Specifically,Kd organoids showed shorter but a similar number of myelin internodes than Controls at the peak of myelination and a reduced number and shorter internodes at a later time point. Interestingly,myelin is affected in the absence of autophagy and mTOR pathway dysregulation,suggesting lack of lysosomal dysfunction which makes this organoid model a very valuable tool to study the early events that drive demyelination in Kd. Kd iPSC-derived microglia show a marginal rate of globoid cell formation under normal culture conditions that is drastically increased upon GalCer feeding. Under normal culture conditions,Kd microglia show a minor LAMP1 content decrease and a slight increase in the autophagy protein LC3B. Upon GalCer feeding,Kd cells show accumulation of autophagy proteins and strong LAMP1 reduction that at a later time point are reverted showing the compensatory capabilities of globoid cells. Altogether,this supports the value of our cultures as tools to study the mechanisms that drive globoid cell formation and the compensatory mechanism in play to overcome GalCer accumulation in Kd. View Publication

Background: Phenotypically unstable Schwann cell-like cells (SCLCs),derived from mesenchymal stem cells (MSCs) require intercellular contact-mediated cues for Schwann cell (SCs)-fate commitment. Although rat dorsal root ganglion (DRG) neurons provide contact-mediated signals for the conversion of SCLCs into fate-committed SCs,the use of animal cells is clinically unacceptable. To overcome this problem,we previously acquired human induced pluripotent stem cell-derived sensory neurons (hiPSC-dSNs) as surrogates of rat DRG neurons that committed rat bone marrow SCLCs to the SC fate. In this study,we explored whether hiPSC-dSNs could mimic rat DRG neuron effects to obtain fate-committed SCs from hBMSC-derived SCLCs. Methods: hiPSCs were induced into hiPSC-dSNs using a specific chemical small molecule combination. hBMSCs were induced into hBMSC-derived SCLCs in a specific culture medium and then co-cultured with hiPSC-dSNs to generate SCs. The identity of hBMSC-derived SCs (hBMSC-dSCs) was examined by immunofluorescence,western bolt,electronic microscopy,and RNA-seq. Immunofluorescence was also used to detect the myelination capacity. Enzyme-linked immunosorbent assay and neurite outgrowth analysis were used to test the secretion of neurotrophic factors. Results: The hBMSC-dSCs exhibited bi-/tri-polar morphology of SCs and maintained the expression of the SC markers S100,p75NTR,p0,GFAP,and Sox10,even after withdrawing the glia-inducing factors or hiPSC-dSNs. Electronic microscopy and RNA-seq analysis provided evidence that hBMSC-dSCs were similar to the original human SCs in terms of their function and a variety of characteristics. Furthermore,these cells formed MBP-positive segments and secreted neurotrophic factors to facilitate the neurite outgrowth of Neuro2A. Conclusions: These results demonstrated that phenotypically stable and functionally mature hBMSC-dSCs were generated efficiently via the co-culture of hiPSC-dSNs and hBMSC-derived SCLCs. Our findings may provide a promising protocol through which stable and fully developed hBMSC-dSCs can be used for transplantation to regenerate myelin sheath. View Publication