技术资料

Although hepatocyte-like cells derived from human pluripotent stem cells (hPSC-HLCs) are considered a promising model for predicting hepatotoxicity,their application has been restricted because of the low activity of drug metabolizing enzymes (DMEs). Here we found that the low expression of xenobiotic receptors (constitutive androstane receptor,CAR; and pregnane X receptor,PXR) contributes to the low activity of DMEs in hPSC-HLCs. Most CAR- and PXR-regulated DMEs and transporters were transcriptionally down-regulated in hPSC-HLC. Transcriptional expression of CAR and PXR was highly repressed in hPSC-HLCs,whereas mRNA levels of aryl hydrocarbon receptor (AHR) were comparable to those of adult liver. Furthermore,ligand-induced transcriptional activation was observed only at AHR in hPSC-HLCs. Bisulfite sequencing analysis demonstrated that promoter hypermethylation of CAR and PXR was associated with diminished transcriptional activity in hPSC-HLCs. Treatment with AHR-selective ligands increased the transcription of AHR-dependent target genes by direct AHR-DNA binding at the xenobiotic response element. In addition,an antagonist of AHR significantly inhibited AHR-dependent target gene expression. Thus,AHR may function intrinsically as a xenosensor as well as a ligand-dependent transcription factor in hPSC-HLCs. Our results indicate that hPSC-HLCs can be used to screen toxic substances related to AHR signaling and to identify potential AHR-targeted therapeutics. View Publication

Epigenomic regulation is likely to be important in the maintenance of genomic integrity of human pluripotent stem cells,however,the mechanisms are unknown. We explored the epigenomes and transcriptomes of human pluripotent stem cells before and after spontaneous transformation to abnormal karyotypes and in correlation to cancer cells. Our results reveal epigenetic silencing of Catalase,a key regulator of oxidative stress and DNA damage control in abnormal cells. Our findings provide novel insight into the mechanisms associated with spontaneous transformation of human pluripotent stem cells towards malignant fate. The same mechanisms may control the genomic stability of cells in somatic tissues. View Publication

BACKGROUND: Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart. METHODS AND RESULTS: Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide-cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore,nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart. CONCLUSION: Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy. View Publication

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays,as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here,we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders,such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs,yielding electrophysiologically active neurons within just 3 wk. Using this platform,we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus,this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types. View Publication

Cockayne syndrome (CS) is a severe neurodevelopmental disorder characterized by growth abnormalities,premature aging,and photosensitivity. Mutation of Cockayne syndrome B (CSB) affects neuronal gene expression and differentiation,so we attempted to bypass its function by expressing downstream target genes. Intriguingly,ectopic expression of Synaptotagmin 9 (SYT9),a key component of the machinery controlling neurotrophin release,bypasses the need for CSB in neuritogenesis. Importantly,brain-derived neurotrophic factor (BDNF),a neurotrophin implicated in neuronal differentiation and synaptic modulation,and pharmacological mimics such as 7,8-dihydroxyflavone and amitriptyline can compensate for CSB deficiency in cell models of neuronal differentiation as well. SYT9 and BDNF are downregulated in CS patient brain tissue,further indicating that sub-optimal neurotrophin signaling underlies neurological defects in CS. In addition to shedding light on cellular mechanisms underlying CS and pointing to future avenues for pharmacological intervention,these data suggest an important role for SYT9 in neuronal differentiation. View Publication

Summary Human pluripotent stem cells (hPSCs) are uniquely dependent on aerobic glycolysis to generate ATP. However,the importance of oxidative phosphorylation (OXPHOS) has not been elucidated. Detailed amino acid profiling has revealed that glutamine is indispensable for the survival of hPSCs. Under glucose- and glutamine-depleted conditions,hPSCs quickly died due to the loss of ATP. Metabolome analyses showed that hPSCs oxidized pyruvate poorly and that glutamine was the main energy source for OXPHOS. hPSCs were unable to utilize pyruvate-derived citrate due to negligible expression of aconitase 2 (ACO2) and isocitrate dehydrogenase 2/3 (IDH2/3) and high expression of ATP-citrate lyase. Cardiomyocytes with mature mitochondria were not able to survive without glucose and glutamine,although they were able to use lactate to synthesize pyruvate and glutamate. This distinguishing feature of hPSC metabolism allows preparation of clinical-grade cell sources free of undifferentiated hPSCs,which prevents tumor formation during stem cell therapy. View Publication

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism,genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs,the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells,which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSC-based differentiation technology and facilitates screening for molecules in modulating liver differentiation,metabolism,genetic network,and response to infection or other external stimuli. View Publication

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid,we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3,Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells,whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine. View Publication

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP),we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels,IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes,including a reduction in cell adhesion and increase in cell death. For cell adhesion,we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally,we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus,transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. View Publication

BACKGROUND Patient-derived induced pluripotent stem cells (iPSCs) are an innovative source as an in vitro model for neurological diseases. Recent studies have demonstrated the differentiation of brain microvascular endothelial cells (BMECs) from various stem cell sources,including iPSC lines. However,the impact of the culturing conditions used to maintain such stem cell pluripotency on their ability to differentiate into BMECs remains undocumented. In this study,we investigated the effect of different sources of Matrigel and stem cell maintenance medium on BMEC differentiation efficiency. METHODS The IMR90-c4 iPSC line was maintained on mTeSR1 or in essential-8 (E-8) medium on growth factor-reduced (GFR) Matrigel from three different manufacturers. Cells were differentiated into BMECs following published protocols. The phenotype of BMEC monolayers was assessed by immunocytochemistry. Barrier function was assessed by transendothelial electrical resistance (TEER) and permeability to sodium fluorescein,whereas the presence of drug efflux pumps was assessed by uptake assay using fluorescent substrates. RESULTS Stem cell maintenance medium had little effect on the yield and barrier phenotype of IMR90-derived BMECs. The source of GFR-Matrigel used for the differentiation process significantly impacted the ability of IMR90-derived BMECs to form tight monolayers,as measured by TEER and fluorescein permeability. However,the Matrigel source had minimal effect on BMEC phenotype and drug efflux pump activity. CONCLUSION This study supports the ability to differentiate BMECs from iPSCs grown in mTeSR1 or E-8 medium and also suggests that the origin of GFR-Matrigel has a marked inpact on BMEC barrier properties. View Publication

Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways,however,it remains unclear as to what extent these pathways can be exploited,either individually or in combination,in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro,but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways,we developed a high-density microbioreactor array (HDMA) - a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt,Hedgehog,IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation,inducing cell cycle activity marked by Ki67,and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration. View Publication

Generation of induced pluripotent stem cells (iPSCs) from human urine-derived cells (hUCs) provides a convenient and non-invasive way to obtain patient-specific iPSCs. However,many isolated hUCs exhibit very poor proliferation and are difficult to reprogram. In this study,we optimized reprogramming approaches for hUCs with very poor proliferation. We report here that a compound cocktail containing cyclic pifithrin-a (a P53 inhibitor),A-83-01,CHIR99021,thiazovivin,NaB,and PD0325901 significantly improves the reprogramming efficiency (170-fold more) for hUCs. In addition,we showed that replacement of Matrigel with autologous hUC feeders can overcome the reprogramming failure due to the massive cell death that occurs during delivery of reprogramming factors. In summary,we describe improved approaches to enable iPSC generation from hUCs that were otherwise difficult to reprogram,a valuable asset for banking patient-specific iPSCs. View Publication