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Despite progress in the development of drugs that efficiently target cancer cells,treatments for metastatic tumours are often ineffective. The now well-established dependency of cancer cells on their microenvironment suggests that targeting the non-cancer-cell component of the tumour might form a basis for the development of novel therapeutic approaches. However,the as-yet poorly characterized contribution of host responses during tumour growth and metastatic progression represents a limitation to exploiting this approach. Here we identify neutrophils as the main component and driver of metastatic establishment within the (pre-)metastatic lung microenvironment in mouse breast cancer models. Neutrophils have a fundamental role in inflammatory responses and their contribution to tumorigenesis is still controversial. Using various strategies to block neutrophil recruitment to the pre-metastatic site,we demonstrate that neutrophils specifically support metastatic initiation. Importantly,we find that neutrophil-derived leukotrienes aid the colonization of distant tissues by selectively expanding the sub-pool of cancer cells that retain high tumorigenic potential. Genetic or pharmacological inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase (Alox5) abrogates neutrophil pro-metastatic activity and consequently reduces metastasis. Our results reveal the efficacy of using targeted therapy against a specific tumour microenvironment component and indicate that neutrophil Alox5 inhibition may limit metastatic progression. View Publication

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders,highlighted by their successful therapeutic use in inherent immunodeficiencies. However,biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here,we report an RNA-based episomal vector system,amenable for long-term transgene expression in stem cells. Specifically,we used a unique intranuclear RNA virus,Borna disease virus (BDV),as the gene transfer vehicle,capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology,cell surface CD105 expression,or the adipogenicity of MSCs. Similarly,replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs),while maintaining the ability to differentiate into three embryonic germ layers. Thus,the BDV-based vectors offer a genomic modification-free,episomal RNA delivery system for sustained stem cell transduction.Gene Therapy accepted article preview online,03 December 2015. doi:10.1038/gt.2015.108. 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

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

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

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

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

Patient safety is a major concern in the application of induced pluripotent stem cells (iPSCs) in cell-based therapy. Efforts are being made to reprogram,maintain,and differentiate iPSCs in defined conditions to provide a safe source of stem cells for regenerative medicine. Recently,human fibroblasts were successfully reprogrammed into pluripotent stem cells using four recombinant proteins (OCT4,c-Myc,KLF4,and SOX2) fused with a cell-penetrating peptide (9R). These protein-induced pluripotent stem cells (piPSCs) are maintained and propagated on a feeder layer of mouse embryonic fibroblasts. Use of animal-derived products in maintenance and differentiation of iPSCs poses risks of zoonotic disease transmission and immune rejection when transplanted into humans. To avoid potential incorporation of xenogenic products,we cultured piPSCs on recombinant human matrix proteins. We then tested whether recombinant human matrix proteins can support self-renewal and pluripotency of piPSCs. After long-term culture on recombinant human vitronectin in xeno-free conditions,piPSCs retained the expression of pluripotent markers. The pluripotency of these cells was further evaluated by differentiating toward ectoderm,mesoderm,and endoderm lineages in vitro. In conclusion,recombinant human vitronectin can support the long-term culture and maintain the stemness of piPSCs in defined nonxenogenic conditions. 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