技术资料

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation,migration,and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme,a highly aggressive brain cancer,suggesting that ion channel expression may be perturbed in this population. However,little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing,we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance,expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally,genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes,gene mutations,survival outcomes,regional tumor expression,and experimental responses to loss-of-function. Together,the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. View Publication

Neural stem cells (NSCs) have the ability to self-renew and to differentiate into various cell types of the central nervous system. This potential can be recapitulated by human induced pluripotent stem cells (hiPSCs) in vitro. The differentiation capacity of hiPSCs is characterized by several stages with distinct morphologies and the expression of various marker molecules. We used the monoclonal antibodies (mAbs) 487(LeX),5750(LeX) and 473HD to analyze the expression pattern of particular carbohydrate motifs as potential markers at six differentiation stages of hiPSCs. Mouse ESCs were used as a comparison. At the pluripotent stage,487(LeX)-,5750(LeX)- and 473HD-related glycans were differently expressed. Later,cells of the three germ layers in embryoid bodies (hEBs) and,even after neuralization of hEBs,subpopulations of cells were labeled with these surface antibodies. At the human rosette-stage of NSCs (hR-NSC),LeX- and 473HD-related epitopes showed antibody-specific expression patterns. We also found evidence that these surface antibodies could be used to distinguish the hR-NSCs from the hSR-NSCs stages. Characterization of hNSCs(FGF-2/EGF) derived from hSR-NSCs revealed that both LeX antibodies and the 473HD antibody labeled subpopulations of hNSCs(FGF-2/EGF). Finally,we identified potential LeX carrier molecules that were spatiotemporally regulated in early and late stages of differentiation. Our study provides new insights into the regulation of glycoconjugates during early human stem cell development. The mAbs 487(LeX),5750(LeX) and 473HD are promising tools for identifying distinct stages during neural differentiation. View Publication

Mouse embryonic stem (ES) cells cultured as aggregates and exposed to retinoic acid are induced to express multiple phenotypes normally associated with neurons. A large percentage of treated aggregates produce a rich neuritic outgrowth. Dissociating the induced aggregates with trypsin and plating the cells as a monolayer results in cultures in which a sizable percentage of the cells have a neuronal appearance. These neuron-like cells express class III beta-tubulin and the neurofilament M subunit. Induced cultures express transcripts for neural-associated genes including the neurofilament L subunit,glutamate receptor subunits,the transcription factor Brn-3,and GFAP. Levels of neurofilament L and GAD67 and GAD65 transcripts rise dramatically upon induction. Physiological studies show that the neuron-like cells generate action potentials and express TTX-sensitive sodium channels,as well as voltage-gated potassium channels and calcium channels. We conclude that a complex system of neuronal gene expression can be activated in cultured ES cells. This system should be favorable for investigating some of the mechanisms that regulate neuronal differentiation. View Publication

BACKGROUND Congenital human cytomegalovirus (HCMV) infection,a leading cause of birth defects,is most often manifested as neurological disorders. The pathogenesis of HCMV-induced neurological disorders is,however,largely unresolved,primarily because of limited availability of model systems to analyze the effects of HCMV infection on neural cells. METHODS An induced pluripotent stem cell (iPSC) line was established from the human fibroblast line MRC5 by introducing the Yamanaka's four factors and then induced to differentiate into neural stem/progenitor cells (NSPCs) by dual inhibition of the SMAD signaling pathway using Noggin and SB-431542. RESULTS iPSC-derived NSPCs (NSPC/iPSCs) were susceptible to HCMV infection and allowed the expression of both early and late viral gene products. HCMV-infected NSPC/iPSCs underwent apoptosis with the activation of caspase-3 and -9 as well as positive staining by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Cytochrome c release from mitochondria to cytosol was observed in these cells,indicating the involvement of mitochondrial dysfunction in their apoptosis. In addition,phosphorylation of proteins involved in the unfolded protein response (UPR),such as PKR-like eukaryotic initiation factor 2a kinase (PERK),c-Jun NH2-terminal kinase (JNK),inositol-requiring enzyme 1 (IRE1),and the alpha subunit of eukaryotic initiation factor 2 (eIF2$$) was observed in HCMV-infected NSPC/iPSCs. These results,coupled with the finding of increased expression of mRNA encoding the C/EBP-homologous protein (CHOP) and the detection of a spliced form of X-box binding protein 1 (XBP1) mRNA,suggest that endoplasmic reticulum (ER) stress is also involved in HCMV-induced apoptosis of these cells. CONCLUSIONS iPSC-derived NSPCs are thought to be a useful model to study HCMV neuropathogenesis and to analyze the mechanisms of HCMV-induced apoptosis in neural cells. View Publication

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS),as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear,with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed,and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased,leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α,suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6,and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. View Publication

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells,modulate the injury environment,or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible,although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417–427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics,although they were not similar when grown as adherent cells. In addition,iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases. J. Comp. Neurol. 522:2707–2728,2014. textcopyright 2014 Wiley Periodicals,Inc. View Publication

The genetic reprogramming technology allows one to generate pluripotent stem cells for individual patients. These cells,called induced pluripotent stem cells (iPSCs),can be an unlimited source of specialized cell types for the body. Thus,autologous somatic cell replacement therapy becomes possible,as well as the generation of in vitro cell models for studying the mechanisms of disease pathogenesis and drug discovery. Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that leads to a loss of upper and lower motor neurons. About 10% of cases are genetically inherited,and the most common familial form of ALS is associated with mutations in the SOD1 gene. We used the reprogramming technology to generate induced pluripotent stem cells with patients with familial ALS. Patient-specific iPS cells were obtained by both integration and transgene-free delivery methods of reprogramming transcription factors. These iPS cells have the properties of pluripotent cells and are capable of direct differentiation into motor neurons. View Publication

Adaptive immunity to self-antigens causes autoimmune disorders,such as multiple sclerosis,psoriasis and type 1 diabetes; paradoxically,T- and B-cell responses to amyloid-$\$(A$\$) reduce Alzheimer's disease (AD)-associated pathology and cognitive impairment in mouse models of the disease. The manipulation of adaptive immunity has been a promising therapeutic approach for the treatment of AD,although vaccine and anti-A$\$ approaches have proven difficult in patients,thus far. CD4(+) T cells have a central role in regulating adaptive immune responses to antigens,and A$\$-specific CD4(+) T cells have been shown to reduce AD pathology in mouse models. As these cells may facilitate endogenous mechanisms that counter AD,an evaluation of their abundance before and during AD could provide important insights. A$\$-CD4see is a new assay developed to quantify A$\$-specific CD4(+) T cells in human blood,using dendritic cells derived from human pluripotent stem cells. In tests of textgreater50 human subjects A$\$-CD4see showed an age-dependent decline of A$\$-specific CD4(+) T cells,which occurs earlier in women than men. In aggregate,men showed a 50% decline in these cells by the age of 70 years,but women reached the same level before the age of 60 years. Notably,women who carried the AD risk marker apolipoproteinE-ɛ4 (ApoE4) showed the earliest decline,with a precipitous drop between 45 and 52 years,when menopause typically begins. A$\$-CD4see requires a standard blood draw and provides a minimally invasive approach for assessing changes in A$\$ that may reveal AD-related changes in physiology by a decade. Furthermore,CD4see probes can be modified to target any peptide,providing a powerful new tool to isolate antigen-specific CD4(+) T cells from human subjects. View Publication

AbstractBack to TopbackslashnNeurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However,it remains unclear to what extent these new mechanistic insights will translate to animal models,an important step in the validation of new targets. Previously,we found that glia from mice carrying the SOD1G93A mutation,a model of ALS,were toxic to stem cell–derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore,we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span,decreased microglial activation,and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo. View Publication

The enteric nervous system (ENS) is composed of neural crest-derived neurons (also known as ganglion cells) the cell bodies of which are located in the submucosal and myenteric plexuses of the intestinal wall. Intramucosal ganglion cells are known to exist but are rare and often considered ectopic. Also derived from the neural crest are enteric glial cells that populate the ganglia and the associated nerves,as well as the lamina propria of the intestinal mucosa. In Hirschsprung disease (HSCR),ganglion cells are absent from the distal gut because of a failure of neural crest-derived progenitor cells to complete their rostrocaudal migration during embryogenesis. The fate of intramucosal glial cells in human HSCR is essentially unknown. We demonstrate a network of intramucosal cells that exhibit dendritic morphology typical of neurons and glial cells. These dendritic cells are present throughout the human gut and express Tuj1,S100,glial fibrillary acidic protein,CD56,synaptophysin,and calretinin,consistent with mixed or overlapping neuroglial differentiation. The cells are present in aganglionic colon from patients with HSCR,but with an altered immunophenotype. Coexpression of Tuj1 and HNK1 in this cell population supports a neural crest origin. These findings extend and challenge the current understanding of ENS microanatomy and suggest the existence of an intramucosal population of neural crest-derived cells,present in HSCR,with overlapping immunophenotype of neurons and glia. Intramucosal neuroglial cells have not been previously recognized,and their presence in HSCR poses new questions about ENS development and the pathobiology of HSCR that merit further investigation. View Publication

OBJECTIVE Charcot-Marie-Tooth (CMT) disease is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here,we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. METHODS iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. RESULTS iPSC-derived motor neurons exhibited gene and protein expression,ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient,but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities,including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. INTERPRETATION Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations,including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies. View Publication

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor,with dismal survival outcomes. Recently,cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here,we have investigated the expression of TNC in tissue microarrays including 17 GBMs,18 WHO grade III astrocytomas,15 WHO grade II astrocytomas,4 WHO grade I astrocytomas,and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas,whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs,where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population. View Publication