搜索结果: 'ipsc'

The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However,generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)-derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons,which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology,we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure,function,and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders. View Publication

The realization of personalized medicine through human induced pluripotent stem cell (iPSC) technology can be advanced by transcriptomics,epigenomics,and bioinformatics that inform on genetic pathways directing tissue development and function. When possible,population diversity should be included in new studies as resources become available. Previously we derived replicate iPSC lines of African American,Hispanic-Latino and Asian self-designated ethnically diverse (ED) origins with normal karyotype,verified teratoma formation,pluripotency biomarkers,and tri-lineage in vitro commitment. Here we perform bioinformatics of RNA-Seq and ChIP-seq pluripotency data sets for two replicate Asian and Hispanic-Latino ED-iPSC lines that reveal differences in generation of contractile cardiomyocytes but similar and robust differentiation to multiple neural,pancreatic,and smooth muscle cell types. We identify shared and distinct genes and contributing pathways in the replicate ED-iPSC lines to enhance our ability to understand how reprogramming to iPSC impacts genes and pathways contributing to cardiomyocyte contractility potential. View Publication

Although human induced pluripotent stem cells (hiPSCs) hold great potential for the study of human diseases affecting disparate cell types,they have been underutilized in seeking mechanistic insights into the pathogenesis of congenital craniofacial disorders. Craniofrontonasal syndrome (CFNS) is a rare X-linked disorder caused by mutations in EFNB1 and characterized by craniofacial,skeletal,and neurological anomalies. Heterozygous females are more severely affected than hemizygous males,a phenomenon termed cellular interference that involves mosaicism for EPHRIN-B1 function. Although the mechanistic basis for cellular interference in CFNS has been hypothesized to involve Eph/ephrin-mediated cell segregation,no direct evidence for this has been demonstrated. Here,by generating hiPSCs from CFNS patients,we demonstrate that mosaicism for EPHRIN-B1 expression induced by random X inactivation in heterozygous females results in robust cell segregation in human neuroepithelial cells,thus supplying experimental evidence that Eph/ephrin-mediated cell segregation is relevant to pathogenesis in human CFNS patients. View Publication

The heart wall tissue,or the myocardium,is one of the main targets in cardiovascular disease prevention and treatment. Animal models have not been sufficient in mimicking the human myocardium as evident by the very low clinical translation rates of cardiovascular drugs. Additionally,current in vitro models of the human myocardium possess several shortcomings such as lack of physiologically relevant co-culture of myocardial cells,lack of a 3D biomimetic environment,and the use of non-human cells. In this study,we address these shortcomings through the design and manufacture of a myocardium-on-chip (MOC) using 3D cell-laden hydrogel constructs and human induced pluripotent stem cell (hiPSC) derived myocardial cells. The MOC utilizes 3D spatially controlled co-culture of hiPSC derived cardiomyocytes (iCMs) and hiPSC derived endothelial cells (iECs) integrated among iCMs as well as in capillary-like side channels,to better mimic the microvasculature seen in native myocardium. We first fully characterized iCMs using immunostaining,genetic,and electrochemical analysis and iECs through immunostaining and alignment analysis to ensure their functionality,and then seeded these cells sequentially into the MOC device. We showed that iECs could be cultured within the microfluidic device without losing their phenotypic lineage commitment,and align with the flow upon physiological level shear stresses. We were able to incorporate iCMs within the device in a spatially controlled manner with the help of photocrosslinkable polymers. The iCMs were shown to be viable and functional within the device up to 7 days,and were integrated with the iECs. The iCMs and iECs in this study were derived from the same hiPSC cell line,essentially mimicking the myocardium of an individual human patient. Such devices are essential for personalized medicine studies where the individual drug response of patients with different genetic backgrounds can be tested in a physiologically relevant manner. View Publication

Recent successes in deriving human-induced pluripotent stem cells (hiPSCs) allow for the possibility of studying human neurons derived from patients with neurological diseases. Concomitant inhibition of the BMP and TGF-β1 branches of the TGF-β signaling pathways by the endogenous antagonist,Noggin,and the small molecule SB431542,respectively,induces efficient neuralization of hiPSCs,a method known as dual-SMAD inhibition. The use of small molecule inhibitors instead of their endogenous counterparts has several advantages including lower cost,consistent activity,and the maintenance of xeno-free culture conditions. We tested the efficacy of DMH1,a highly selective small molecule BMP-inhibitor for its potential to replace Noggin in the neuralization of hiPSCs. We compare Noggin and DMH1-induced neuralization of hiPSCs by measuring protein and mRNA levels of pluripotency and neural precursor markers over a period of seven days. The regulation of five of the six markers assessed was indistinguishable in the presence of concentrations of Noggin or DMH1 that have been shown to effectively inhibit BMP signaling in other systems. We observed that by varying the DMH1 or Noggin concentration,we could selectively modulate the number of SOX1 expressing cells,whereas PAX6,another neural precursor marker,remained the same. The level and timing of SOX1 expression have been shown to affect neural induction as well as neural lineage. Our observations,therefore,suggest that BMP-inhibitor concentrations need to be carefully monitored to ensure appropriate expression levels of all transcription factors necessary for the induction of a particular neuronal lineage. We further demonstrate that DMH1-induced neural progenitors can be differentiated into β3-tubulin expressing neurons,a subset of which also express tyrosine hydroxylase. Thus,the combined use of DMH1,a highly specific BMP-pathway inhibitor,and SB431542,a TGF-β1-pathway specific inhibitor,provides us with the tools to independently regulate these two pathways through the exclusive use of small molecule inhibitors. View Publication

Control of cellular (de)differentiation in a temporal,cell-specific,and exchangeable manner is of paramount importance in the field of reprogramming. Here,we have generated and characterized a mouse strain that allows iPSC generation through the Cre/loxP conditional and doxycycline/rtTA-controlled inducible expression of the OSKM reprogramming factors entirely from within the ROSA26 locus. After reprogramming,these factors can be replaced by genes of interest-for example,to enhance lineage-directed differentiation-with the use of a trap-coupled RMCE reaction. We show that,similar to ESCs,Dox-controlled expression of the cardiac transcriptional regulator Mesp1 together with Wnt inhibition enhances the generation of functional cardiomyocytes upon in vitro differentiation of such RMCE-retargeted iPSCs. This ROSA26-iPSC mouse model is therefore an excellent tool for studying both cellular reprogramming and lineage-directed differentiation factors from the same locus and will greatly facilitate the identification and ease of functional characterization of the genetic/epigenetic determinants involved in these complex processes. View Publication

The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results,NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2,LMX1A,TH,GIRK2,PITX3,EN1,NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue,with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation,the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future. 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

AbstractA basic assumption underlying induced pluripotent stem cell (iPSC) models of neurodegeneration is that disease-relevant pathologies present in brain tissue are also represented in donor-matched cells differentiated from iPSCs. However,few studies have tested this hypothesis in matched iPSCs and neuropathologically characterized donated brain tissues. To address this,we assessed iPSC-neuron production of ?-amyloid (A?) A?40,A?42,and A?43 in 24 iPSC lines matched to donor brains with primary neuropathologic diagnoses of sporadic AD (sAD),familial AD (fAD),control,and other neurodegenerative disorders. Our results demonstrate a positive correlation between A?43 production by fAD iPSC-neurons and A?43 accumulation in matched brain tissues but do not reveal a substantial correlation in soluble A? species between control or sAD iPSC-neurons and matched brains. However,we found that the ApoE4 genotype is associated with increased A? production by AD iPSC-neurons. Pathologic tau phosphorylation was found to be increased in AD and fAD iPSC-neurons compared to controls and positively correlated with the relative abundance of longer-length A? species produced by these cells. Taken together,our results demonstrate that sAD-predisposing genetic factors influence iPSC-neuron phenotypes and that these cells are capturing disease-relevant and patient-specific components of the amyloid cascade. View Publication

SummaryCytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore,while mutations in TARDBP (encoding TDP-43) have been associated with ALS,the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9,we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C,two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization,phosphorylation,solubility,or aggregation compared with isogenic control MNs. However,our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures,as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively,our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease. Graphical abstract Highlights•Mutant MNs maintain viability but are more vulnerable to cellular stress•Mutant MNs do not show TDP-43 pathology•TDP-43 variants lead to a progressive decline in spontaneous neuronal activity•Functional impairments are accompanied by abnormal synaptic marker expression Molecular neuroscience; Cellular neuroscience View Publication

SUMMARY Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance,there are currently no stem-cell-derived models of human engineered cardiac tissues (hECTs) that include resident macrophages. In this study,we made an induced pluripotent stem cell (iPSC)-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. The inclusion of iM0s significantly impacted hECT function,increasing contractile force production. A potential mechanism underlying these changes was revealed by the interrogation of calcium signaling,which demonstrated the modulation of ?-adrenergic signaling in +iM0 hECTs. Collectively,these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models,emphasizing the need to further explore their contributions not only in healthy hECT models but also in the contexts of disease and injury. In brief Lock and Graney et al. develop a human engineered cardiac tissue with an incorporated iPSC-derived macrophage population to better mimic the complex cell landscape of the native myocardium. Macrophage inclusion leads to increased contractile function of the tissue,which is attributed to macrophage stimulation of the cardiomyocyte ?-adrenergic signaling pathway. Graphical Abstract View Publication

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking,which has impeded their broader application. In this study,we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics,immunogenicity,immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs),iMSCs demonstrated an enhanced proliferative capacity,a higher level of regenerative gene expression,and lower immunogenicity,strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition,iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models,iMSCs engrafted in the lungs,liver,and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity,low immunogenicity and unique immunomodulatory properties,and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects. View Publication