搜索结果: 'ipsc'

Chemotherapy-induced peripheral neuropathy (CIPN) affects up to two-thirds of cancer patients undergoing cytotoxic chemotherapy. Here,we used human iPSC-derived sensory neurons (iPSC-DSN) to model CIPN in vitro. Administration of various chemotherapeutic agents (i.e.,paclitaxel,vincristine,bortezomib and cisplatin) at clinically applicable concentrations resulted in reduced cell viability,axonal degeneration,electrophysiological dysfunction and increased levels of phosphorylated c-Jun in iPSC-DSN. Transcriptomic analyses revealed that the upregulation of c-Jun strongly correlated with the expression of genes of neuronal injury,apoptosis and inflammatory signatures. To test whether c-Jun plays a central role in the development of CIPN,we applied the small molecule inhibitor of the Jun N-terminal kinase,SP600125,to iPSC-DSN treated with neurotoxic chemotherapy. c-Jun inhibition prevented chemotherapy-induced neurotoxicity by preserving cell viability,axonal integrity and electrophysiological function of iPSC-DSN. These findings identify c-Jun as a key mediator of CIPN pathophysiology across multiple drug types and present preclinical evidence that c-Jun inhibition is an attractive therapeutic target to prevent CIPN. View Publication

Pancreatic β cells derived from human induced pluripotent stem cells (hiPSCs) represent a promising therapeutic avenue in regenerative medicine for diabetes treatment. However,current differentiation protocols lack the specificity and efficiency required to reliably produce fully functional β cells,limiting their clinical applicability. Epigenetic barriers,such as histone modifications,may hinder proper differentiation and the acquisition of essential maturation markers in these cells. Methods: hiPSCs were cultured under feeder-free conditions and subjected to lentiviral transduction with shRNA constructs to silence KDM4A. Differentiation into pancreatic β-like cells was performed using stepwise protocols,with or without doxycycline supplementation,to evaluate the effect of KDM4A suppression. Gene expression was quantified by RT-qPCR,protein expression was assessed by western blotting and immunofluorescence,and functional insulin release was determined by glucose-stimulated insulin secretion (GSIS) assays. Statistical analysis was conducted using unpaired two-tailed Student’s t-tests,with significance set at p < 0.05. Results: A reduction in pancreatic development proteins was observed in the different differentiation states evaluated,after blocking KDM4A expression. Knockdown of KDM4A significantly reduced the expression of pancreatic β-cell genes,such as PDX1,Nkx6.1,and Ins,by 50% compared to WT iPSCs differentiated under the same conditions. Similarly,glucose-stimulated insulin secretion was reduced by approximately 80% in KDM4A-deficient β-like cells. Conclusions: These results emphasize the critical role of histone demethylation in hiPSC differentiation toward β cells. Our findings identify KDM4A as a key epigenetic regulator,suggesting that its modulation could enhance the generation of functional β cells for regenerative medicine in diabetes. View Publication

Gastrointestinal (GI) dysfunction,characterized by severe diarrhea and dehydration,is a central contributor to morbidity and mortality in filovirus disease in patients,yet the role of the epithelium in this clinical outcome remains poorly defined. Here,we employ induced pluripotent stem cell (iPSC)-derived human intestinal (HIOs) and colonic organoids (HCOs) to model Ebola virus (EBOV) and Marburg virus (MARV) infection. These organoids are permissive to filovirus infection and support viral replication. Bulk RNA sequencing revealed distinct intestinal and colonic epithelial responses,including apical and junctional disruption and a delayed virus-specific induction of interferon-stimulated genes. Moreover,infection impaired adenylate cyclase signaling and CFTR-mediated ion transport,providing mechanistic insight into virus-induced secretory diarrhea. This platform recapitulates key features of human GI pathology in filoviral disease and serves as a powerful system to dissect host-pathogen interactions and identify therapeutic targets. Author summaryEbola virus (EBOV) and Marburg virus (MARV) are among the most lethal viruses known. Infection with these viruses leads to severe disease and death. One of their most harmful effects is damage to the gastrointestinal tract,causing intense diarrhea and life-threatening dehydration. Yet,how these viruses affect the gut remains poorly understood. In this study,we used human mini-guts—small,three-dimensional tissues grown from stem cells that mimic the human intestinal and colonic epithelium—to investigate how these viruses interact with gut epithelial cells. We found that both EBOV and MARV infect and replicate in these tissues,disrupt key barrier structures,and interfere with the cells’ ability to regulate fluid secretion. These effects mirror the severe symptoms seen in patients. Our study provides new insight into how EBOV and MARV damage the gut and identifies specific cellular pathways that may be targeted for treatment. This research not only improves our understanding of EBOV and MARV infections but also offers new infection platforms for testing therapies aimed at protecting the gastrointestinal system during filovirus outbreaks. View Publication

The potential of the immune system to decrease cancer progression is widely recognized and has led to the development of innovative anti-cancer immunotherapies. Here,we studied human macrophages derived from genetically engineered iPSCs (iMac) with angiotensin-converting enzyme (ACE) expression regulatable by a doxycycline (dox)-inducible promoter as a novel anti-cancer immunotherapy. Increased ACE expression in iMac (cells now termed ACE-iMac) augments polarization towards an M1 macrophage phenotype characterized by increased production of proinflammatory cytokines,reactive oxygen species,nitric oxide,and an RNA profile indicating an aggressive immune response. ACE-iMac kills tumor cells in vitro significantly better than iMac. In vivo,studies using tumor xenografts for melanoma,breast cancer,and head and neck squamous cell carcinoma (HNSCC) showed a highly significant 3.4- to 7.2-fold reduction in solid tumor size following ACE-expressing ACE-iMac immunotherapy as compared to results with iMac. To further investigate the impact of ACE on human anti-tumor responses,we developed a humanized BLT-NSG mouse model with a fully functional adaptive immune system. Here,ACE-iMac treatment significantly reduced the growth of human melanoma xenografts by enhancing the activation of human T cells and NK cells. In conclusion,enhancing ACE expression in human-derived macrophages (ACE-iMac) greatly amplifies their anti-cancer phenotype,offering a compelling new therapeutic strategy with the potential to improve clinical outcomes for cancer patients. View Publication

Microvascular dysfunction due to hypoxia is a key contributor in the pathogenesis of many disorders including cancer and retinal and cardiovascular diseases,but relevant human models are missing. Here,we present a robust 3D in vitro method with the use of human induced pluripotent stem cell–derived blood vessel organoids to analyze in vitro microvascular remodeling. We present a detailed practical pipeline combining optical tissue clearing,high-resolution immunofluorescence,and surface marker analysis to quantitatively assess hypoxia-driven changes in endothelial cells,pericytes,and the basal lamina. Exposure of these blood vessel organoids to chronic hypoxia (1% O2) for 1 week recapitulated key pathological features,including structural remodeling and a dysregulated secretome with altered vascular endothelial growth factor signaling. This approach establishes a versatile and human-relevant platform to study microvascular remodeling induced by chronic hypoxia and other pathological stimuli and their contribution to microvascular-related diseases. View Publication

Progressive supranuclear palsy-Richardson’s syndrome (PSP-RS) is a primary 4R tauopathy in which early axonal dysfunction may precede overt neurodegeneration; however,the mechanisms linking Tau dysregulation to cytoskeletal vulnerability remain poorly defined. Here,we generated induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons from individuals with sporadic PSP-RS and matched healthy controls and performed integrated transcriptomic and proteomic analyses. PSP-RS neurons exhibited coordinated suppression of dopaminergic and synaptic programs alongside activation of cytoskeletal remodeling and stress-related pathways. These changes were accompanied by increased Tau phosphorylation,neurofilament accumulation,and structural alterations of the axonal compartment,consistent with an early axonopathic phenotype. Notably,mechanistic target of rapamycin (mTOR) signaling significantly increased. Pharmacological inhibition of mTOR reduced Tau phosphorylation and neurofilament levels,indicating that mTOR activity contributes to the maintenance of cytoskeletal imbalance. In conclusion,our findings support a model in which early cytoskeletal dysfunction in PSP-RS arises from the convergence of Tau dysregulation,impaired structural homeostasis,and altered signaling pathways. Rather than acting as a primary driver,mTOR appears to function as a pathogenic amplifier that sustains axonal stress. This study provides a human cellular framework to investigate early axonopathic mechanisms in sporadic PSP-RS. View Publication

Human induced pluripotent stem cells (hiPSCs) provide new possibilities for regenerative therapies. In order for this potential to be achieved,it is critical to efficiently monitor the differentiation of these hiPSCs into specific lineages. Here,we describe a lentiviral reporter vector sensitive to specific microRNAs (miRNA) to show that a single vector bearing multiple miRNA target sequences conjugated to different reporters can be used to monitor hiPSC formation and subsequent differentiation from human fetal fibroblasts (HFFs). The reporter vector encodes EGFP conjugated to the targets of human embryonic stem cell (hESC) specific miRNAs (miR-302a and miR-302d) and mCherry conjugated to the targets of differentiated cells specific miRNAs (miR-142-3p,miR-155,and miR-223). The vector was used to track reprogramming of HFF to iPSC. HFFs co-transduced with this reporter vector and vectors encoding 4 reprogramming factors (OCT4,SOX2,KLF4 and cMYC) were mostly positive for EGFP (67%) at an early stage of hiPSC formation. EGFP expression gradually disappeared and mCherry expression increased indicating less miRNAs specific to differentiated cells and expression of miRNAs specific to hESCs. Upon differentiation of the hiPSC into embryoid bodies,a large fraction of these hiPSCs regained EGFP expression and some of those cells became single positive for EGFP. Further differentiation into neural lineages showed distinct structures demarcated by either EGFP or mCherry expression. These findings demonstrate that a miRNA dependent reporter vector can be a useful tool to monitor living cells during reprogramming of hiPSC and subsequent differentiation to lineage specific cells. View Publication

UNLABELLED Among the known genetic risk factors for Parkinson disease,mutations in GBA1,the gene responsible for the lysosomal disorder Gaucher disease,are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics,we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease,two with and two without parkinsonism,and one patient with Type 2 (acute neuronopathic) Gaucher disease,and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine,demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein,a protein present as aggregates in Parkinson disease and related synucleinopathies,were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607,a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme,restored glucocerebrosidase activity and protein levels,and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons,indicating its potential for treating neuronopathic Gaucher disease. In addition,NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism,suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease,dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity,reduced lysosomal glucocerebrosidase levels,and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype,the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone,which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition,the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons,indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease. View Publication

Accurate modeling of human neuronal cell biology has been a long-standing challenge. However,methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately,these methods entail numerous challenges,including poor efficiency,variable cell type heterogeneity,and lengthy,expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol,these lines can be inducibly differentiated into either cortical (i3 Neurons) or lower motor neurons (i3 LMN) in a rapid,efficient,and scalable manner (Wang et al.,2017). In this manuscript,we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i3 Neurons or i3 LMNs,and we present neuronal culture conditions for various experimental applications. {\textcopyright} 2018 by John Wiley & Sons,Inc. View Publication

Nonhuman primate (NHP) models are more predictive than rodent models for developing induced pluripotent stem cell (iPSC)-based cell therapy,but robust and reproducible NHP iPSC-cardiomyocyte differentiation protocols are lacking for cardiomyopathies research. We developed a method to differentiate integration-free rhesus macaque iPSCs (RhiPSCs) into cardiomyocytes with {\textgreater}85{\%} purity in 10 days,using fully chemically defined conditions. To enable visualization of intracellular calcium flux in beating cardiomyocytes,we used CRISPR/Cas9 to stably knock-in genetically encoded calcium indicators at the rhesus AAVS1 safe harbor locus. Rhesus cardiomyocytes derived by our stepwise differentiation method express signature cardiac markers and show normal electrochemical coupling. They are responsive to cardiorelevant drugs and can be successfully engrafted in a mouse myocardial infarction model. Our approach provides a powerful tool for generation of NHP iPSC-derived cardiomyocytes amenable to utilization in basic research and preclinical studies,including in vivo tissue regeneration models and drug screening. View Publication

Neural rosettes derived from human induced pluripotent stem cells (iPSCs) have been claimed to be a highly robust in vitro cellular model for biomedical application. They are able to propagate in vitro in the presence of mitogens,including basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). However,these two mitogens are also involved in anterior-posterior patterning in a gradient dependent manner along the neural tube axis. Here,we compared the regional identity of neural rosette cells and specific neural subtypes of their progeny propagated with low and high concentrations of bFGF and EGF. We observed that low concentrations of bFGF and EGF in the culturing system were able to induce forebrain identity of the neural rosettes and promote subsequent cortical neuronal differentiation. On the contrary,high concentrations of these mitogens stimulate a mid-hindbrain fate of the neural rosettes,resulting in subsequent cholinergic neuron differentiation. Thus,our results indicate that different concentrations of bFGF and EGF supplemented during propagation of neural rosettes are involved in altering the identity of the resultant neural cells. View Publication

Vascularization of engineered human skin constructs is crucial for recapitulation of systemic drug delivery and for their long-term survival,functionality,and viable engraftment. In this study,the latest microfabrication techniques are used and a novel bioengineering approach is established to micropattern spatially controlled and perfusable vascular networks in 3D human skin equivalents using both primary and induced pluripotent stem cell (iPSC)-derived endothelial cells. Using 3D printing technology makes it possible to control the geometry of the micropatterned vascular networks. It is verified that vascularized human skin equivalents (vHSEs) can form a robust epidermis and establish an endothelial barrier function,which allows for the recapitulation of both topical and systemic delivery of drugs. In addition,the therapeutic potential of vHSEs for cutaneous wounds on immunodeficient mice is examined and it is demonstrated that vHSEs can both promote and guide neovascularization during wound healing. Overall,this innovative bioengineering approach can enable in vitro evaluation of topical and systemic drug delivery as well as improve the potential of engineered skin constructs to be used as a potential therapeutic option for the treatment of cutaneous wounds. View Publication