参考文献

Brohawn DG et al. (AUG 2016) PloS one 11 8 e0160520

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord.

ALS is a rapidly progressive,devastating neurodegenerative illness of adults that produces disabling weakness and spasticity arising from death of lower and upper motor neurons. No meaningful therapies exist to slow ALS progression,and molecular insights into pathogenesis and progression are sorely needed. In that context,we used high-depth,next generation RNA sequencing (RNAseq,Illumina) to define gene network abnormalities in RNA samples depleted of rRNA and isolated from cervical spinal cord sections of 7 ALS and 8 CTL samples. We aligned textgreater50 million 2X150 bp paired-end sequences/sample to the hg19 human genome and applied three different algorithms (Cuffdiff2,DEseq2,EdgeR) for identification of differentially expressed genes (DEG's). Ingenuity Pathways Analysis (IPA) and Weighted Gene Co-expression Network Analysis (WGCNA) identified inflammatory processes as significantly elevated in our ALS samples,with tumor necrosis factor (TNF) found to be a major pathway regulator (IPA) and TNF$$-induced protein 2 (TNFAIP2) as a major network hub" gene (WGCNA). Using the oPOSSUM algorithm View Publication

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mTeSR™1

Stanurova J et al. (AUG 2016) Scientific reports 6 August 30792

Angelman syndrome-derived neurons display late onset of paternal UBE3A silencing.

Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin-specific gene expression that is regulated by a differentially methylated region. Gene mutations or failures in the imprinting process lead to the development of imprinting disorders,such as Angelman syndrome. The symptoms of Angelman syndrome are caused by the absence of functional UBE3A protein in neurons of the brain. To create a human neuronal model for Angelman syndrome,we reprogrammed dermal fibroblasts of a patient carrying a defined three-base pair deletion in UBE3A into induced pluripotent stem cells (iPSCs). In these iPSCs,both parental alleles are present,distinguishable by the mutation,and express UBE3A. Detailed characterization of these iPSCs demonstrated their pluripotency and exceptional stability of the differentially methylated region regulating imprinted UBE3A expression. We observed strong induction of SNHG14 and silencing of paternal UBE3A expression only late during neuronal differentiation,in vitro. This new Angelman syndrome iPSC line allows to study imprinted gene regulation on both parental alleles and to dissect molecular pathways affected by the absence of UBE3A protein. View Publication

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产品名：

mTeSR™1

温和细胞解离试剂

ReLeSR™

Sun Y et al. (JUL 2016) eLife 5

A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

Dravet Syndrome is an intractable form of childhood epilepsy associated with deleterious mutations in SCN1A,the gene encoding neuronal sodium channel Nav1.1. Earlier studies using human induced pluripotent stem cells (iPSCs) have produced mixed results regarding the importance of Nav1.1 in human inhibitory versus excitatory neurons. We studied a Nav1.1 mutation (p.S1328P) identified in a pair of twins with Dravet Syndrome and generated iPSC-derived neurons from these patients. Characterization of the mutant channel revealed a decrease in current amplitude and hypersensitivity to steady-state inactivation. We then differentiated Dravet-Syndrome and control iPSCs into telencephalic excitatory neurons or medial ganglionic eminence (MGE)-like inhibitory neurons. Dravet inhibitory neurons showed deficits in sodium currents and action potential firing,which were rescued by a Nav1.1 transgene,whereas Dravet excitatory neurons were normal. Our study identifies biophysical impairments underlying a deleterious Nav1.1 mutation and supports the hypothesis that Dravet Syndrome arises from defective inhibitory neurons. View Publication

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Barnea-Cramer AO et al. (JUL 2016) Scientific reports 6 29784

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions,resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration,these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals,as evidenced by two visual behavioral tests. Furthermore,the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease. View Publication

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mTeSR™1

Robinson M et al. (AUG 2016) Stem Cell Reviews and Reports 12 4 476--483

Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells

Pluripotent stem cells can become any cell type found in the body. Accordingly,one of the major challenges when working with pluripotent stem cells is producing a highly homogenous population of differentiated cells,which can then be used for downstream applications such as cell therapies or drug screening. The transcription factor Ascl1 plays a key role in neural development and previous work has shown that Ascl1 overexpression using viral vectors can reprogram fibroblasts directly into neurons. Here we report on how a recombinant version of the Ascl1 protein functionalized with intracellular protein delivery technology (Ascl1-IPTD) can be used to rapidly differentiate human induced pluripotent stem cells (hiPSCs) into neurons. We first evaluated a range of Ascl1-IPTD concentrations to determine the most effective amount for generating neurons from hiPSCs cultured in serum free media. Next,we looked at the frequency of Ascl1-IPTD supplementation in the media on differentiation and found that one time supplementation is sufficient enough to trigger the neural differentiation process. Ascl1-IPTD was efficiently taken up by the hiPSCs and enabled rapid differentiation into TUJ1-positive and NeuN-positive populations with neuronal morphology after 8 days. After 12 days of culture,hiPSC-derived neurons produced by Ascl1-IPTD treatment exhibited greater neurite length and higher numbers of branch points compared to neurons derived using a standard neural progenitor differentiation protocol. This work validates Ascl1-IPTD as a powerful tool for engineering neural tissue from pluripotent stem cells. View Publication

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100-0483

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100-1061

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100-1077

产品名：

STEMdiff™ 神经花环选择试剂

STEMdiff™神经前体细胞培养基

STEMdiff™神经祖细胞冻存液

Vitronectin XF™

CellAdhere™ 稀释缓冲液

DMEM/F-12 with 15 mM HEPES

CryoStor® CS10

STEMdiff™ 神经诱导培养基

STEMdiff™SMADi神经诱导试剂盒

STEMdiff™SMADi神经诱导试剂盒，2套

Hausser Scientificᵀᴹ 明线血球计数板

ReLeSR™

CryoStor® CS10

Vitronectin XF™

温和细胞解离试剂

ReLeSR™

Dafinca R et al. (APR 2016) Stem cells (Dayton,Ohio) 34 8 2016

C9orf72 Hexanucleotide Expansions are Associated with Altered ER Calcium Homeostasis and Stress Granule Formation in iPSC-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS),accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions,differentiated these to functional motor and cortical neurons and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons,decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis,reduced levels of the anti-apoptotic protein Bcl-2,increased endoplasmic reticulum (ER) stress and reduced mitochondrial membrane potential. Furthermore,C9orf72 motor neurons,and also cortical neurons,show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats,which describes a novel pathogenic link between C9orf72 mutations,dysregulation of calcium signalling and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia (FTD). This article is protected by copyright. All rights reserved. View Publication

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mTeSR™1

Nekrasov ED et al. (DEC 2016) Molecular Neurodegeneration 11 1 1--15

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons.

Background: Huntington's disease (HD) is an incurable hereditary neurodegenerative disorder,which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD,existing pharmaceutical can only relieve its symptoms. Results: Here,induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene,and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro,as evidenced by mutant huntingtin protein aggregation,increased number of lysosomes/autophagosomes,nuclear indentations,and enhanced neuronal death during cell aging. Moreover,store-operated channel (SOC) currents were detected in the differentiated neurons,and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally,the quinazoline derivative,EVP4593,reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging. Conclusions: Our data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks,providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug. [ABSTRACT FROM AUTHOR] View Publication

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mFreSR™

mTeSR™1

Francis KR et al. (APR 2016) Nature medicine 22 4 388--396

Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/&dollar;&dollar;-catenin defects in neuronal cholesterol synthesis phenotypes.

Smith-Lemli-Opitz syndrome (SLOS) is a malformation disorder caused by mutations in DHCR7,which impair the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. SLOS results in cognitive impairment,behavioral abnormalities and nervous system defects,though neither affected cell types nor impaired signaling pathways are fully understood. Whether 7DHC accumulation or cholesterol loss is primarily responsible for disease pathogenesis is also unclear. Using induced pluripotent stem cells (iPSCs) from subjects with SLOS,we identified cellular defects that lead to precocious neuronal specification within SLOS derived neural progenitors. We also demonstrated that 7DHC accumulation,not cholesterol deficiency,is critical for SLOS-associated defects. We further identified downregulation of Wnt/$$-catenin signaling as a key initiator of aberrant SLOS iPSC differentiation through the direct inhibitory effects of 7DHC on the formation of an active Wnt receptor complex. Activation of canonical Wnt signaling prevented the neural phenotypes observed in SLOS iPSCs,suggesting that Wnt signaling may be a promising therapeutic target for SLOS. View Publication

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Dispase (1 U/mL)

mTeSR™1

Chamma I et al. (MAR 2016) Nature Communications 7 10773

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short,enzymatically biotinylated tag,compatible with SRI techniques including uPAINT,STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues,with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β,neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody,and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore,Nlg1 is dynamic,disperse and sensitive to synaptic stimulation,whereas LRRTM2 is organized in compact and stable nanodomains. Thus,mSA is a versatile tool to image membrane proteins at high resolution in complex live environments,providing novel information about the nano-organization of biological structures. View Publication

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05711

100-1281

产品名：

NeuroCult™ SM1 神经添加物

Takayama Y and Kida YS (FEB 2016) PloS one 11 2 e0148559

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices.

Morphology and function of the nervous system is maintained via well-coordinated processes both in central and peripheral nervous tissues,which govern the homeostasis of organs/tissues. Impairments of the nervous system induce neuronal disorders such as peripheral neuropathy or cardiac arrhythmia. Although further investigation is warranted to reveal the molecular mechanisms of progression in such diseases,appropriate model systems mimicking the patient-specific communication between neurons and organs are not established yet. In this study,we reconstructed the neuronal network in vitro either between neurons of the human induced pluripotent stem (iPS) cell derived peripheral nervous system (PNS) and central nervous system (CNS),or between PNS neurons and cardiac cells in a morphologically and functionally compartmentalized manner. Networks were constructed in photolithographically microfabricated devices with two culture compartments connected by 20 microtunnels. We confirmed that PNS and CNS neurons connected via synapses and formed a network. Additionally,calcium-imaging experiments showed that the bundles originating from the PNS neurons were functionally active and responded reproducibly to external stimuli. Next,we confirmed that CNS neurons showed an increase in calcium activity during electrical stimulation of networked bundles from PNS neurons in order to demonstrate the formation of functional cell-cell interactions. We also confirmed the formation of synapses between PNS neurons and mature cardiac cells. These results indicate that compartmentalized culture devices are promising tools for reconstructing network-wide connections between PNS neurons and various organs,and might help to understand patient-specific molecular and functional mechanisms under normal and pathological conditions. View Publication

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mTeSR™1

RNAseq Analyses Identify Tumor Necrosis Factor-Mediated Inflammation as a Major Abnormality in ALS Spinal Cord.

Angelman syndrome-derived neurons display late onset of paternal UBE3A silencing.

A deleterious Nav1.1 mutation selectively impairs telencephalic inhibitory neurons derived from Dravet Syndrome patients.

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Functionalizing Ascl1 with Novel Intracellular Protein Delivery Technology for Promoting Neuronal Differentiation of Human Induced Pluripotent Stem Cells

C9orf72 Hexanucleotide Expansions are Associated with Altered ER Calcium Homeostasis and Stress Granule Formation in iPSC-Derived Neurons from Patients with Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons.

Modeling Smith-Lemli-Opitz syndrome with induced pluripotent stem cells reveals a causal role for Wnt/&dollar;&dollar;-catenin defects in neuronal cholesterol synthesis phenotypes.

Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

In Vitro Reconstruction of Neuronal Networks Derived from Human iPS Cells Using Microfabricated Devices.

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