技术资料

The recently identified GGGGCC repeat expansion in the noncoding region of C9ORF72 is the most common pathogenic mutation in patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). We generated a human neuronal model and investigated the pathological phenotypes of human neurons containing GGGGCC repeat expansions. Skin biopsies were obtained from two subjects who had textgreater1,000 GGGGCC repeats in C9ORF72 and their respective fibroblasts were used to generate multiple induced pluripotent stem cell (iPSC) lines. After extensive characterization,two iPSC lines from each subject were selected,differentiated into postmitotic neurons,and compared with control neurons to identify disease-relevant phenotypes. Expanded GGGGCC repeats exhibit instability during reprogramming and neuronal differentiation of iPSCs. RNA foci containing GGGGCC repeats were present in some iPSCs,iPSC-derived human neurons and primary fibroblasts. The percentage of cells with foci and the number of foci per cell appeared to be determined not simply by repeat length but also by other factors. These RNA foci do not seem to sequester several major RNA-binding proteins. Moreover,repeat-associated non-ATG (RAN) translation products were detected in human neurons with GGGGCC repeat expansions and these neurons showed significantly elevated p62 levels and increased sensitivity to cellular stress induced by autophagy inhibitors. Our findings demonstrate that key neuropathological features of FTD/ALS with GGGGCC repeat expansions can be recapitulated in iPSC-derived human neurons and also suggest that compromised autophagy function may represent a novel underlying pathogenic mechanism. View Publication

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited,stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently,the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition,the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However,the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation,we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice,hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling,increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. View Publication

Neural stem cells (NSCs) possess immunosuppressive characteristics,but effects of NSCs on human dendritic cells (DCs),the most important antigen presenting cells,are less well studied. We used an in vitro approach to evaluate the effects of human NSCs on differentiation of human blood CD14+ monocytes into DCs. NSCs derived from H1 human embryonic stem cells (hESC-NSCs) and human ReNcell NSC line,as well as human bone marrow derived mesenchymal stem cells (MSCs),were tested. We observed that in response to treatment with interleukin-4 and granulocyte macrophage colony-stimulating factor CD14+ monocytes co-cultured with NSCs were able to down-regulate CD14 and up-regulate the differentiation marker CD1a,whereas MSC co-culture strongly inhibited CD1a expression and supported prolonged expression of CD14. A similar difference between NSCs and MSCs was noted when lipopolysaccharides were included to induce maturation of monocyte-derived DCs. However,when effects on the function of derived DCs were investigated,NSCs suppressed the elevation of the DC maturation marker CD83,although not the up-regulation of costimulatory molecules CD80,CD86 and CD40,and impaired the functional capacity of the derived DCs to stimulate alloreactive T cells. We did not observe any obvious difference between hESC-NSCs and ReNcell NSCs in inhibiting DC maturation and function. Our data suggest that although human NSCs are less effective than human MSCs in suppressing monocyte differentiation into DCs,these stem cells can still affect the function of DCs,ultimately regulating specific immune responses. View Publication

Induced pluripotent stem cells (iPSCs) hold promise for the treatment of motoneuron diseases because of their distinct features including pluripotency,self-derivation and potential ability to differentiate into motoneurons. However,it is still unknown whether human iPSC-derived motoneurons can functionally innervate target muscles in vivo,which is the definitive sign of successful cell therapy for motoneuron diseases. In the present study,we demonstrated that human iPSCs derived from mesenchymal cells of the umbilical cord possessed a high yield in neural differentiation. Using a chemically-defined in vitro system,human iPSCs efficiently differentiated into motoneurons which displayed typical morphology,expressed specific molecules,and generated repetitive trains of action potentials. When transplanted into the injured musculocutaneous nerve of rats,they survived robustly,extended axons along the nerve,and formed functional connections with the target muscle (biceps brachii),thereby protecting the muscle from atrophy. Our study provides evidence for the first time that human iPSC-derived motoneurons are truly functional not only in vitro but also in vivo,and they have potential for stem cell-based therapies for motoneuron diseases. textcopyright 2013 Elsevier B.V. View Publication

Huntington's disease is caused by expanded CAG repeats in HTT,conferring toxic gain of function on mutant HTT (mHTT) protein. Reducing mHTT amounts is postulated as a strategy for therapeutic intervention. We conducted genome-wide RNA interference screens for genes modifying mHTT abundance and identified 13 hits. We tested 10 in vivo in a Drosophila melanogaster Huntington's disease model,and 6 exhibited activity consistent with the in vitro screening results. Among these,negative regulator of ubiquitin-like protein 1 (NUB1) overexpression lowered mHTT in neuronal models and rescued mHTT-induced death. NUB1 reduces mHTT amounts by enhancing polyubiquitination and proteasomal degradation of mHTT protein. The process requires CUL3 and the ubiquitin-like protein NEDD8 necessary for CUL3 activation. As a potential approach to modulating NUB1 for treatment,interferon-β lowered mHTT and rescued neuronal toxicity through induction of NUB1. Thus,we have identified genes modifying endogenous mHTT using high-throughput screening and demonstrate NUB1 as an exemplar entry point for therapeutic intervention of Huntington's disease. View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Most neurodegenerative diseases are linked to aberrant accumulation of aggregation-prone proteins. Among them,Huntington's disease (HD) is caused by an expanded polyglutamine repeat stretch in the N terminus of the mutant huntingtin protein (mHTT),which gets cleaved and aggregates in the brain. Recently established human induced pluripotent stem cell-derived HD neurons exhibit some disease-relevant phenotypes and provide tools for HD research. However,they have limitations such as genetic heterogeneity and an absence of mHTT aggregates and lack a robust neurodegeneration phenotype. In addition,the relationship between the phenotype and mHTT levels has not been elucidated. Herein,we present a human embryonic stem cell (hESC)-derived HD neuronal model expressing HTTexon1 fragments,which addresses the deficiencies enumerated above. The wild-type and HD lines are derived from an isogenic background and exhibit insoluble mHTT aggregates and neurodegeneration. We also demonstrate a quantitative relationship between neurodegeneration and soluble monomeric (but not oligomeric or aggregated) mHTT levels. Reduction of ∼10% of mHTT is sufficient to prevent toxicity,whereas ∼90% reduction of wild-type HTT is safe and well-tolerated in these cells. A known HD toxicity modifier (Rhes) showed expected rescue of neurodegeneration. Therefore,the hESC-derived neuronal models complement existing induced pluripotent stem cell-derived neuronal models and provide valuable tools for HD research.—Lu,B.,Palacino,J. A novel human embryonic stem cell-derived Huntington's disease neuronal model exhibits mutant huntingtin (mHTT) aggregates and soluble mHTT-dependent neurodegeneration. View Publication

Stochastic processes and imprinting,along with genetic factors,lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system,and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition,allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates,such as A2BP1 (RBFOX1),ERBB4,NLGN4X,NRG1,NRG3,NRXN1,and NLGN1. Overall,there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square,p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance,the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications. View Publication

Efficient derivation of neural cells from human embryonic stem cells (hESCs) remains an unmet need for the treatment of neurological disorders. The limiting factors for current methods include being labor-intensive,time-consuming and expensive. In this study,we hypothesize that the substrate topography,with optimal geometry and dimension,can modulate the neural fate of hESCs and enhance the efficiency of differentiation. A multi-architectural chip (MARC) containing fields of topographies varying in geometry and dimension was developed to facilitate high-throughput analysis of topography-induced neural differentiation in vitro. The hESCs were subjected to direct differentiation"� View Publication

Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types,including neurons. However,differentiation of hPSCs with extrinsic factors is a slow,step-wise process,mimicking the protracted timing of human development. Using a small-molecule screen,we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at textgreater75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors,including tetrodotoxin (TTX)-resistant,SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development,suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain. View Publication

Cockayne syndrome (CS) is a human premature aging disorder associated with neurological and developmental abnormalities,caused by mutations mainly in the CS group B gene (ERCC6). At the molecular level,CS is characterized by a deficiency in the transcription-couple DNA repair pathway. To understand the role of this molecular pathway in a pluripotent cell and the impact of CSB mutation during human cellular development,we generated induced pluripotent stem cells (iPSCs) from CSB skin fibroblasts (CSB-iPSC). Here,we showed that the lack of functional CSB does not represent a barrier to genetic reprogramming. However,iPSCs derived from CSB patient's fibroblasts exhibited elevated cell death rate and higher reactive oxygen species (ROS) production. Moreover,these cellular phenotypes were accompanied by an up-regulation of TXNIP and TP53 transcriptional expression. Our findings suggest that CSB modulates cell viability in pluripotent stem cells,regulating the expression of TP53 and TXNIP and ROS production. View Publication

Transactive response DNA-binding (TDP-43) protein is the dominant disease protein in amyotrophic lateral sclerosis (ALS) and a subgroup of frontotemporal lobar degeneration (FTLD-TDP). Identification of mutations in the gene encoding TDP-43 (TARDBP) in familial ALS confirms a mechanistic link between misaccumulation of TDP-43 and neurodegeneration and provides an opportunity to study TDP-43 proteinopathies in human neurons generated from patient fibroblasts by using induced pluripotent stem cells (iPSCs). Here,we report the generation of iPSCs that carry the TDP-43 M337V mutation and their differentiation into neurons and functional motor neurons. Mutant neurons had elevated levels of soluble and detergent-resistant TDP-43 protein,decreased survival in longitudinal studies,and increased vulnerability to antagonism of the PI3K pathway. We conclude that expression of physiological levels of TDP-43 in human neurons is sufficient to reveal a mutation-specific cell-autonomous phenotype and strongly supports this approach for the study of disease mechanisms and for drug screening. View Publication