Scientific Resources

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

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations,gene expression profiles and response to treatment: WNT,Sonic Hedgehog (SHH),Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example,expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however,its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs),but not their normal counterparts (hENs),resemble Groups 3 and 4 MB in vitro and in vivo. Here,we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs,respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth,self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes,such as SOX2,and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast,OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression. View Publication

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup),carrying different duplication sizes,to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition,using multi-electrodes arrays,we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level,we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor,NCH-51,was validated as a potential clinical candidate. Interestingly,this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders. View Publication

GTF2IRD1 is one of the three members of the GTF2I gene family,clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities,mental retardation,visuospatial deficits and hypersociability of WBS. However,the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here,for the first time,we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner,yeast two-hybrid libraries were screened,isolating 38 novel interaction partners,which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function,as the isolated partners are mostly involved in chromatin modification and transcriptional regulation,whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain,behaviour and human disease. View Publication

Induced pluripotent stem cells (iPSC) and their differentiated derivatives offer a unique source of human primary cells for toxicity screens. Here,we report on the comparative cytotoxicity of 80 compounds (neurotoxicants,developmental neurotoxicants,and environmental compounds) in iPSC as well as isogenic iPSC-derived neural stem cells (NSC),neurons,and astrocytes. All compounds were tested over a 24-h period at 10 and 100$\$,in duplicate,with cytotoxicity measured using the MTT assay. Of the 80 compounds tested,50 induced significant cytotoxicity in at least one cell type; per cell type,32,38,46,and 41 induced significant cytotoxicity in iPSC,NSC,neurons,and astrocytes,respectively. Four compounds (valinomycin,3,3',5,5'-tetrabromobisphenol,deltamethrin,and triphenyl phosphate) were cytotoxic in all four cell types. Retesting these compounds at 1,10,and 100$\$ using the same exposure protocol yielded consistent results as compared with the primary screen. Using rotenone,we extended the testing to seven additional iPSC lines of both genders; no substantial difference in the extent of cytotoxicity was detected among the cell lines. Finally,the cytotoxicity assay was simplified by measuring luciferase activity using lineage-specific luciferase reporter iPSC lines which were generated from the parental iPSC line. This article is part of a Special Issue entitled SI: PSC and the brain. View Publication

Insulin-like growth factor 1 (IGF-1) enhances cellular proliferation and reduces apoptosis during the early differentiation of bone marrow derived mesenchymal stem cells (BMSCs) into neural progenitor-like cells (NPCs) in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). BMSCs were differentiated in three groups of growth factors: (A) EGF + bFGF,(B) EGF + bFGF + IGF-1,and (C) without growth factor. To unravel the molecular mechanisms of the NPCs derivation,microarray analysis using GeneChip miRNA arrays was performed. The profiles were compared among the groups. Annotated microRNA fingerprints (GSE60060) delineated 46 microRNAs temporally up-regulated or down-regulated compared to group C. The expressions of selected microRNAs were validated by real-time PCR. Among the 46 microRNAs,30 were consistently expressed for minimum of two consecutive time intervals. In Group B,only miR-496 was up-regulated and 12 microRNAs,including the let-7 family,miR-1224,miR-125a-3p,miR-214,miR-22,miR-320,miR-708,and miR-93,were down-regulated. Bioinformatics analysis reveals that some of these microRNAs (miR-22,miR-214,miR-125a-3p,miR-320 and let-7 family) are associated with reduction of apoptosis. Here,we summarize the roles of key microRNAs associated with IGF-1 in the differentiation of BMSCs into NPCs. These findings may provide clues to further our understanding of the mechanisms and roles of microRNAs as key regulators of BMSC-derived NPC maintenance. View Publication

Characterizing clinically relevant brain metastasis models and assessing the therapeutic efficacy in such models are fundamental for the development of novel therapies for metastatic brain cancers. In this study,we have developed an in vivo imageable breast-to-brain metastasis mouse model. Using real time in vivo imaging and subsequent composite fluorescence imaging,we show a widespread distribution of micro- and macro-metastasis in different stages of metastatic progression. We also show extravasation of tumour cells and the close association of tumour cells with blood vessels in the brain thus mimicking the multi-foci metastases observed in the clinics. Next,we explored the ability of engineered adult stem cells to track metastatic deposits in this model and show that engineered stem cells either implanted or injected via circulation efficiently home to metastatic tumour deposits in the brain. Based on the recent findings that metastatic tumour cells adopt unique mechanisms of evading apoptosis to successfully colonize in the brain,we reasoned that TNF receptor superfamily member 10A/10B apoptosis-inducing ligand (TRAIL) based pro-apoptotic therapies that induce death receptor signalling within the metastatic tumour cells might be a favourable therapeutic approach. We engineered stem cells to express a tumour selective,potent and secretable variant of a TRAIL,S-TRAIL,and show that these cells significantly suppressed metastatic tumour growth and prolonged the survival of mice bearing metastatic breast tumours. Furthermore,the incorporation of pro-drug converting enzyme,herpes simplex virus thymidine kinase,into therapeutic S-TRAIL secreting stem cells allowed their eradication post-tumour treatment. These studies are the first of their kind that provide insight into targeting brain metastasis with stem-cell mediated delivery of pro-apoptotic ligands and have important clinical implications. View Publication

3D suspension culture is generally considered a promising method to achieve efficient expansion and controlled differentiation of human pluripotent stem cells (hPSCs). In this work,we focused on developing an integrated culture platform for expansion and neural commitment of hPSCs into neural precursors using 3D suspension conditions and chemically-defined culture media. We evaluated different inoculation methodologies for hPSC expansion as 3D aggregates and characterized the resulting cultures in terms of aggregate size distribution. It was demonstrated that upon single-cell inoculation,after four days of culture,3D aggregates were composed of homogenous populations of hPSC and were characterized by an average diameter of 139 ± 26 μm,which was determined to be the optimal size to initiate neural commitment. Temporal analysis revealed that upon neural specification it is possible to maximize the percentage of neural precursor cells expressing the neural markers Sox1 and Pax6 after nine days of culture. These results highlight our ability to define a robust method for production of hPSC-derived neural precursors that minimizes processing steps and that constitutes a promising alternative to the traditional planar adherent culture system due to a high potential for scaling-up. View Publication

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability,with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia,coarsened facial features and intellectual disability,due to truncating mutations in the sorting nexin gene SNX14,encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate,a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma,accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction. View Publication

For diseases of the brain,the pig (Sus scrofa) is increasingly being used as a model organism that shares many anatomical and biological similarities with humans. We report that pig induced pluripotent stem cells (iPSC) can recapitulate events in early mammalian neural development. Pig iPSC line (POU5F1(high)/SSEA4(low)) had a higher potential to form neural rosettes (NR) containing neuroepithelial cells than either POU5F1(low)/SSEA4(low) or POU5F1(low)/SSEA4(high) lines. Thus,POU5F1 and SSEA4 pluripotency marker profiles in starting porcine iPSC populations can predict their propensity to form more robust NR populations in culture. The NR were isolated and expanded in vitro,retaining their NR morphology and neuroepithelial molecular properties. These cells expressed anterior central nervous system fate markers OTX2 and GBX2 through at least seven passages,and responded to retinoic acid,promoting a more posterior fate (HOXB4+,OTX2-,and GBX2-). These findings offer insight into pig iPSC development,which parallels the human iPSC in both anterior and posterior neural cell fates. These in vitro similarities in early neural differentiation processes support the use of pig iPSC and differentiated neural cells as a cell therapy in allogeneic porcine neural injury and degeneration models,providing relevant translational data for eventual human neural cell therapies. View Publication

Emerging evidence shows that glioblastoma multiforme (GBM) originates from cancer stem cells (CSCs). Characterization of CSC-specific signalling pathways would help identify new therapeutic targets and perhaps lead to the development of more efficient therapies selectively targeting CSCs. Here; we successfully dedifferentiated two patient-derived GBM cell lines into CSC-like cells (induced glioma stem cells,iGSCs) through expression of Oct4,Sox2 and Nanog transcription factors. Transformed cells exhibited significant suppression of epidermal growth factor receptor and its downstream pathways. Compared with parental GBM cells,iGSCs formed large neurospheres even in the absence of exogenous mitogens; they exhibited significant sensitivity to salinomycin and chemoresistance to temozolomide. Further characterization of iGSCs revealed induction of NOTCH1 and Wnt/β-catenin signalling and expression of CD133,CD44 and ALDH1A1. Our results indicate that iGSCs may help us understand CSC physiology and lead to development of potential therapeutic interventions aimed at differentiating tumour cells to render them more sensitive to chemotherapy or other standard agents. View Publication

Induced pluripotent stem cells (iPSC) are important tools for drug discovery assays and toxicology screens. In this manuscript,we design high efficiency TALEN and ZFN to target two safe harbor sites on chromosome 13 and 19 in a widely available and well-characterized integration-free iPSC line. We show that these sites can be targeted in multiple iPSC lines to generate reporter systems while retaining pluripotent characteristics. We extend this concept to making lineage reporters using a C-terminal targeting strategy to endogenous genes that express in a lineage-specific fashion. Furthermore,we demonstrate that we can develop a master cell line strategy and then use a Cre-recombinase induced cassette exchange strategy to rapidly exchange reporter cassettes to develop new reporter lines in the same isogenic background at high efficiency. Equally important we show that this recombination strategy allows targeting at progenitor cell stages,further increasing the utility of the platform system. The results in concert provide a novel platform for rapidly developing custom single or dual reporter systems for screening assays. View Publication