技术资料

In this study,we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy,qPCR and flow cytometry,we classified the treatment outcome as inducing pluripotency (hESC,flurbiprofen and gatifloxacin),mesendoderm (sinomenine),differentiation (cyamarin,digoxin,digitoxin,selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions,the above classification was reassorted. Hyperacetylation at H3K4,9,14,18,56 and 122 as well as H4K5,8,12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9,K20,K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to differentiation initiators"� View Publication

The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics,force generation,and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness,we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly,our approach has the potential for broad application in the study of cardiac disease,drug discovery,and cardiotoxicity screening. View Publication

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts,an apical domain,marked by EZRIN and atypical PKC$\$,is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly,actin polymerization,regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA),promotes lumen formation,whereas actin contraction,mediated by MYOSIN-II,inhibits this process. Finally,we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development,this system can provide a powerful model for investigation of this process in a controlled environment. Overall,our data establish that lumenogenesis is a fundamental cell biological property of human PSCs. View Publication

BACKGROUND Friedreich's ataxia (FRDA),a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy,is caused by silencing of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. METHODS Application of our previously established FRDA human induced pluripotent stem cell (hiPSC) derived cardiomyocytes model as a platform to assess the efficacy of treatment with either the antioxidant coenzyme Q10 analog,idebenone (IDE) or the iron chelator,deferiprone (DFP),which are both under clinical trial. RESULTS DFP was able to more significantly suppress synthesis of reactive oxygen species (ROS) than IDE at the dosages of 25 $\$ and 10nM respectively which agreed with the reduced rate of intracellular accumulation of iron by DFP treatment from 25 to 50 $\$ With regard to cardiac electrical-contraction (EC) coupling function,decay velocity of calcium handling kinetics in FRDA-hiPSC-cardiomyocytes was significantly improved by DFP treatment but not by IDE. Further mechanistic studies revealed that DFP also modulated iron induced mitochondrial stress as reflected by mitochondria network disorganization and decline level of respiratory chain protein,succinate dehydrogenase (CxII) and cytochrome c oxidase (COXIV). In addition,iron-response protein (IRP-1) regulatory loop was overridden by DFP as reflected by resumed level of ferritin (FTH) back to basal level and the attenuated transferrin receptor (TSFR) mRNA level suppression thereby reducing further iron uptake. CONCLUSIONS DFP modulated iron homeostasis in FRDA-hiPSC-cardiomyocytes and effectively relieved stress-stimulation related to cardiomyopathy. The resuming of redox condition led to the significantly improved cardiac prime events,cardiac electrical-coupling during contraction. View Publication

Pig induced pluripotent stem cells (piPSCs) offer a great opportunity and a number of advantages in the generation of transgenic animals. These immortalized cells can undergo multiple rounds of genetic modifications (e.g.,gene knock-in,knockout) and selection leading to animals that have optimized traits of biomedical or agricultural interests. In this chapter we describe the production and characterization of piPSCs,microinjection of piPSCs into embryos,embryo transfer and production of chimeric animals based on successful protocols. View Publication

Induced pluripotent stem cells hold great potential in regenerative medicine as it enables to generate pluripotent stem cells from any available cell types. Ectopic expression of four transcription factors (Oct4,Sox2,Klf4,and c-Myc) can reprogram fibroblasts directly to pluripotency as shown in multiple species. Here,we describe detailed protocols for generation of iPSCs from adult canine fibroblasts. Robust canine iPSCs will provide powerful tools not only to study human diseases,but also for the development of therapeutic approaches. View Publication

Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation,but they often clump in culture,which has always represented a challenge for neurodifferentiation. In this study,we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM) with 10% CO2,which doubled the expression of the NESTIN,PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore,an additional step (AdSTEP) was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the neurosphederm". The large neural tube-type rosette (NTTR) structure formed from the neurosphederm� View Publication

We investigate the effects of myocardial transplantation of human induced pluripotent stem cell (iPSC)-derived progenitors and cardiomyocytes into acutely infarcted myocardium in severe combined immune deficiency mice. A total of 2 × 10(5) progenitors,cardiomyocytes or cell-free saline were injected into peri-infarcted anterior free wall. Sham-operated animals received no injection. Myocardial function was assessed at 2-week and 4-week post-infarction by using echocardiography and pressure-volume catheterization. Early myocardial remodelling was observed at 2-week with echocardiography derived stroke volume (SV) in saline (20.45 ± 7.36 $\$,P textless 0.05) and cardiomyocyte (19.52 ± 3.97 $\$,P textless 0.05) groups,but not in progenitor group (25.65 ± 3.61 $\$),significantly deteriorated as compared to sham control group (28.41 ± 4.41 $\$). Consistently,pressure-volume haemodynamic measurements showed worsening chamber dilation in saline (EDV: 23.24 ± 5.01 $\$,P textless 0.05; ESV: 17.08 ± 5.82 $\$,P textless 0.05) and cardiomyocyte (EDV: 26.45 ± 5.69 $\$,P textless 0.05; ESV: 18.03 ± 6.58 $\$,P textless 0.05) groups by 4-week post-infarction as compared to control (EDV: 15.26 ± 2.96 $\$; ESV: 8.41 ± 2.94 $\$). In contrast,cardiac progenitors (EDV: 20.09 ± 7.76 $\$; ESV: 13.98 ± 6.74 $\$) persistently protected chamber geometry against negative cardiac remodelling. Similarly,as compared to sham control (54.64 ± 11.37%),LV ejection fraction was preserved in progenitor group from 2-(38.68 ± 7.34%) to 4-week (39.56 ± 13.26%) while cardiomyocyte (36.52 ± 11.39%,P textless 0.05) and saline (35.34 ± 11.86%,P textless 0.05) groups deteriorated early at 2-week. Improvements of myocardial function in the progenitor group corresponded to increased vascularization (16.12 ± 1.49/mm(2) to 25.48 ± 2.08/mm(2) myocardial tissue,P textless 0.05) and coincided with augmented networking of cardiac telocytes in the interstitial space of infarcted zone. View Publication

Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency,little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs,human pluripotency-associated transcripts 2,3 and 5 (HPAT2,HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2,HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells,followed by whole-transcriptome analysis,identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate. View Publication

X-ALD is an inherited neurodegenerative disorder where mutations in the ABCD1 gene result in clinically diverse phenotypes: the fatal disorder of cerebral childhood ALD (cALD) or a milder disorder of adrenomyeloneuropathy (AMN). The various models used to study the pathobiology of X-ALD disease lack the appropriate presentation for different phenotypes of cALD vs AMN. This study demonstrates that induced pluripotent stem cells (IPSC) derived brain cells astrocytes (Ast),neurons and oligodendrocytes (OLs) express morphological and functional activities of the respective brain cell types. The excessive accumulation of saturated VLCFA,a hallmark" of X-ALD� View Publication

Hyaluronic acid (HA) has been cross-linked to form hydrogel for potential applications in the self-renewal and differentiation of human pluripotent stem cells (hPSCs) for years. However,HA hydrogel with improved residence time and mechanical integrity that allows the survival of hPSCs under defined conditions is still much needed for clinical applications. In this study,HA was modified with methacrylate functional groups (MeHA) and cross-linked by photo-crosslinking method. After subsequent conjugation with adhesive peptide,these MeHA surfaces demonstrated performance in facilitating human induced pluripotent stem cells (hiPSCs) proliferation,and good pluripotency maintenance of hiPSCs under defined conditions. Moreover,MeHA films on glass-slides exhibited long residence time and mechanical stability throughout hiPSC culture. Our photo-crosslinkable MeHA possesses great value in accelerating the application of HA hydrogel in hiPSCs proliferation and differentiation with the conjugation of adhesive peptides. View Publication

The prevalence and specificity of unique fusion oncogenes are high in a number of soft tissue sarcomas (STSs). The close relationship between fusion genes and clinicopathological features suggests that a correlation may exist between the function of fusion proteins and cellular context of the cell-of-origin of each tumor. However,most STSs are origin-unknown tumors and this issue has not yet been investigated in detail. In the present study,we examined the effects of the cellular context on the function of the synovial sarcoma (SS)-specific fusion protein,SS18-SSX,using human pluripotent stem cells (hPSCs) containing the drug-inducible SS18-SSX gene. We selected the neural crest cell (NCC) lineage for the first trial of this system,induced SS18-SSX at various differentiation stages from PSCs to NCC-derived mesenchymal stromal cells (MSCs),and compared its biological effects on each cell type. We found that the expression of FZD10,identified as an SS-specific gene,was induced by SS18-SSX at the PSC and NCC stages,but not at the MSC stage. This stage-specific induction of FZD10 correlated with stage-specific changes in histone marks associated with the FZD10 locus and also with the loss of the BAF47 protein,a member of the SWI/SNF chromatin-remodeling complex. Furthermore,the global gene expression profile of hPSC-derived NCCs was the closest to that of SS cell lines after the induction of SS18-SSX. These results clearly demonstrated that the cellular context is an important factor in the function of SS18-SSX as an epigenetic modifier. View Publication