技术资料

We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2),with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy,we tested our hypothesis that Sox2,which is a key regulator of the RR phenotype,is under the control of its binding partners. In this study,we focused on DDX17,known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation,we confirmed the binding between DDX17 and Sox2,although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells,it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR,DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2,as well as 3 of its specific gene targets including MUC15,CCND1 and CD133. Correlating with these findings,siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude,DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells,DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes. 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

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

T cells reactive to β cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse,we recently identified the β cell secretory granule protein,chromogranin A (ChgA),as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis,we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast,in NOD.ChgA(+/+) mice,a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice. 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

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

PTEN is a tumour suppressor frequently mutated in many types of cancers. Here we show that targeted disruption of PTEN leads to neoplastic transformation of human neural stem cells (NSCs),but not mesenchymal stem cells. PTEN-deficient NSCs display neoplasm-associated metabolic and gene expression profiles and generate intracranial tumours in immunodeficient mice. PTEN is localized to the nucleus in NSCs,binds to the PAX7 promoter through association with cAMP responsive element binding protein 1 (CREB)/CREB binding protein (CBP) and inhibits PAX7 transcription. PTEN deficiency leads to the upregulation of PAX7,which in turn promotes oncogenic transformation of NSCs and instates 'aggressiveness' in human glioblastoma stem cells. In a large clinical database,we find increased PAX7 levels in PTEN-deficient glioblastoma. Furthermore,we identify that mitomycin C selectively triggers apoptosis in NSCs with PTEN deficiency. Together,we uncover a potential mechanism of how PTEN safeguards NSCs,and establish a cellular platform to identify factors involved in NSC transformation,potentially permitting personalized treatment of glioblastoma. View Publication

We have established a novel cell line,designated as TF-1,from a patient with erythroleukemia,which showed complete growth dependency on granulocyte-macrophage colony-stimulating factor (GM-CSF) or on interleukin-3 (IL-3) and carried a homogeneous chromosomal abnormality (54X). Erythropoietin (EPO) also sustained the short-term growth of TF-1,but did not induce erythroid differentiation. These three hematopoietic growth factors acted on TF-1 synergistically. Transforming growth factor-beta and interferons inhibited the factor-dependent growth of TF-1 cells in a dose-dependent fashion,and monocyte-colony stimulating factor and interkeukin-1 enhanced the GM-CSF-dependent growth of TF-1. Ultrastructural studies revealed some very immature features in this cell line. Although TF-1 cells do not express glycophorin A or carbonyl anhydrase I,the morphological and cytochemical features,and the constitutive expression of globin genes,indicate the commitment of TF-1 to erythroid lineage. When induced to differentiate,TF-1 entered two different pathways. Specifically,hemin and delta-aminolevulinic acid induced hemoglobin synthesis,whereas TPA induced dramatic differentiation of TF-1 into macrophage-like cells. In summary,TF-1 is a cell line of immature erythroid origin that requires GM-CSF,IL-3,or EPO for its growth and that has the ability to undergo differentiation into either more mature erythroid cells or into macrophage-like cells. TF-1 is a useful tool for analyzing the human receptors for IL-3,GM-CSF,and EPO or the signal transduction of these hemopoietic growth factors. View Publication

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here,we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA,12.5% of cell colonies demonstrated CCR5 editing,of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells,we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells,including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication,macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro,and generation of HIV-resistant cells for potential therapeutic applications. View Publication

Limited data are available on the effects of stem cells in non-ischemic dilated cardiomyopathy (DCM). Since the diffuse nature of the disease calls for a broad distribution of cells,this study investigated the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) in a mouse model of DCM. Nanofibrous scaffolds were produced using a clinical grade atelocollagen which was electrospun and cross-linked under different conditions. As assessed by scanning electron microscopy and shearwave elastography,the optimum crosslinking conditions for hiPS-CM colonization proved to be a 10% concentration of citric acid crosslinking agent and 150 min of post-electrospinning baking. Acellular collagen scaffolds were first implanted in both healthy mice and those with induced DCM by a cardiac-specific invalidation of serum response factor (SRF). Seven and fourteen days after implantation,the safety of the scaffold was demonstrated by echocardiography and histological assessments. The subsequent step of implantation of the scaffolds seeded with hiPS-CM in DCM induced mice,using cell-free scaffolds as controls,revealed that after fourteen days heart function decreased in controls while it remained stable in the treated mice. This pattern was associated with an increased number of endothelial cells,in line with the greater vascularity of the scaffold. Moreover,a lesser degree of fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling was observed. These results support the interest of the proposed hiPS-CM seeded electrospun scaffold for the stabilization of the DCM outcome with potential for its clinical use in the future. View Publication

Multi-ciliated airway cells (MCACs) play a role in mucociliary clearance of the lung. However,the efficient induction of functional MCACs from human pluripotent stem cells has not yet been reported. Using carboxypeptidase M (CPM) as a surface marker of NKX2-1(+)-ventralized anterior foregut endoderm cells (VAFECs),we report a three-dimensional differentiation protocol for generating proximal airway epithelial progenitor cell spheroids from CPM(+) VAFECs. These spheroids could be induced to generate MCACs and other airway lineage cells without alveolar epithelial cells. Furthermore,the directed induction of MCACs and of pulmonary neuroendocrine lineage cells was promoted by adding DAPT,a Notch pathway inhibitor. The induced MCACs demonstrated motile cilia with a 9 + 2" microtubule arrangement and dynein arms capable of beating and generating flow for mucociliary transport. This method is expected to be useful for future studies on human airway disease modeling and regenerative medicine." View Publication

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however,which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs,which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion,whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus,the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms,according to self-peptide expression patterns. View Publication