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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

Glioblastoma multiforme (GBM),the most common and lethal tumor of the adult brain,generally shows chemo- and radioresistance. MicroRNAs (miRs) regulate physiological processes,such as resistance of GBM cells to temozolomide (TMZ). Although miRs are attractive targets for cancer therapeutics,the effectiveness of this approach requires targeted delivery. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers,including GBM. We report on an increase in miR-9 in TMZ-resistant GBM cells. miR-9 was involved in the expression of the drug efflux transporter,P-glycoprotein. To block miR-9,methods were developed with Cy5-tagged anti-miR-9. Dye-transfer studies indicated intracellular communication between GBM cells and MSCs. This occurred by gap junctional intercellular communication and the release of microvesicles. In both cases,anti-miR-9 was transferred from MSCs to GBM cells. However,the major form of transfer occurred with the microvesicles. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to TMZ,as shown by increased cell death and caspase activity. The data showed a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse the chemoresistance of GBM cells.Molecular Therapy-Nucleic Acids (2013) 2,e126; doi:10.1038/mtna.2013.60; published online 1 October 2013. View Publication

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro,and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously,we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this NIA Mouse ESC Bank we generated and characterized 48 additional mouse ESC lines,in which single TFs in each line could be induced in a doxycycline-controllable manner. Together,with the previous ESC lines,the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g.,neural lineages by Myt1 Isl1,and St18; mesodermal lineages by Pitx1,Pitx2,Barhl2,and Lmx1a; white blood cells by Myb,Etv2,and Tbx6,and ovary by Pitx1,Pitx2,and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs. View Publication

The mammalian central nervous system (CNS) undergoes significant expansion postnatally,producing astrocytes,oligodendrocytes and inhibitory neurons to modulate the activity of neural circuits. This is coincident in humans with the emergence of pediatric epilepsy,a condition commonly treated with valproate/valproic acid (VPA),a potent inhibitor of histone deacetylases (HDACs). The sequential activity of specific HDACs,however,may be essential for the differentiation of distinct subpopulations of neurons and glia. Here,we show that different subsets of CNS neural stem cells (NSCs) and progenitors switch expression of HDAC1 and HDAC2 as they commit to a neurogenic lineage in the subventricular zone (SVZ) and dentate gyrus (DG). The administration of VPA for only one week from P7-P14,combined with sequential injections of thymidine analogs reveals that VPA stimulates a significant and differential decrease in the production and differentiation of progeny of NSCs in the DG,rostral migratory stream (RMS),and olfactory bulb (OB). Cross-fostering VPA-treated mice revealed,however,that a postnatal failure to thrive induced by VPA treatment had a greater effect on DG neurogenesis than VPA action directly. By one month after VPA,OB interneuron genesis was significantly and differentially reduced in both periglomerular and granule neurons. Using neurosphere assays to test if VPA directly regulates NSC activity,we found that short term treatment with VPA in vivo reduced neurosphere numbers and size,a phenotype that was also obtained in neurospheres from control mice treated with VPA and an alternative HDAC inhibitor,Trichostatin A (TSA) at 0 and 3 days in vitro (DIV). Collectively,these data show that clinically used HDAC inhibitors like VPA and TSA can perturb postnatal neurogenesis; and their use should be carefully considered,especially in individuals whose brains are actively undergoing key postnatal time windows of development. View Publication

The aim of this investigation was to characterize the proliferative precursor cells in the adult mouse hippocampal region. Given that a very large number of new hippocampal cells are generated over the lifetime of an animal,it is predicted that a neural stem cell is ultimately responsible for maintaining this genesis. Although it is generally accepted that a proliferative precursor resides within the hippocampus,contradictory reports exist regarding the classification of this cell. Is it a true stem cell or a more limited progenitor? Using a strict functional definition of a neural stem cell and a number of in vitro assays,we report that the resident hippocampal precursor is a progenitor capable of proliferation and multipotential differentiation but is unable to self-renew and thus proliferate indefinitely. Furthermore,the mitogen FGF-2 stimulates proliferation of these cells to a greater extent than epidermal growth factor (EGF). In addition,we found that BDNF was essential for the production of neurons from the hippocampal progenitor cells,being required during proliferation to trigger neuronal fate. In contrast,a bona fide neural stem cell was identified in the lateral wall of the lateral ventricle surrounding the hippocampus. Interestingly,EGF proved to be the stronger mitogenic factor for this cell,which was clearly a different precursor from the resident hippocampal progenitor. These results suggest that the stem cell ultimately responsible for adult hippocampal neurogenesis resides outside the hippocampus,producing progenitor cells that migrate into the neurogenic zones and proliferate to produce new neurons and glia. View Publication

BACKGROUND: Although both the alkylating agent temozolomide (TMZ) and oncolytic viruses hold promise for treating glioblastoma,which remains uniformly lethal,the effectiveness of combining the two treatments and the mechanism of their interaction on cancer stem cells are unknown. METHODS: We investigated the efficacy of combining TMZ and the oncolytic herpes simplex virus (oHSV) G47Δ in killing glioblastoma stem cells (GSCs),using Chou-Talalay combination index analysis,immunocytochemistry and fluorescence microscopy,and neutral comet assay. The role of treatment-induced DNA double-strand breaks,activation of DNA damage responses,and virus replication in the cytotoxic interaction between G47Δ and TMZ was examined with a panel of pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of DNA repair pathways. Comparisons of cell survival and virus replication were performed using a two-sided t test (unpaired). The survival of athymic mice (n = 6-8 mice per group) bearing GSC-derived glioblastoma tumors treated with the combination of G47Δ and TMZ was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test. RESULTS: The combination of G47Δ and TMZ acted synergistically in killing GSCs but not neurons,with associated robust induction of DNA damage. Pharmacological and shRNA-mediated knockdown studies suggested that activated ataxia telangiectasia mutated (ATM) is a crucial mediator of synergy. Activated ATM relocalized to HSV DNA replication compartments where it likely enhanced oHSV replication and could not participate in repairing TMZ-induced DNA damage. Sensitivity to TMZ and synergy with G47Δ decreased with O(6)-methylguanine-DNA-methyltransferase (MGMT) expression and MSH6 knockdown. Combined G47Δ and TMZ treatment extended survival of mice bearing GSC-derived intracranial tumors,achieving long-term remission in four of eight mice (median survival = 228 days; G47Δ alone vs G47Δ + TMZ,hazard ratio of survival = 7.1,95% confidence interval = 1.9 to 26.1,P = .003) at TMZ doses attainable in patients. CONCLUSIONS: The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation. View Publication

Zika virus (ZIKV) infection is associated with microcephaly in fetuses,but the pathogenesis of ZIKV-related microcephaly is not well understood. Here we show that ZIKV infects the subventricular zone in human fetal brain tissues and that the tissue tropism broadens with the progression of gestation. Our research demonstrates also that intermediate progenitor cells (IPCs) are the main target cells for ZIKV. Post-mitotic committed neurons become susceptible to ZIKV infection as well at later stages of gestation. Furthermore,activation of microglial cells,DNA fragmentation,and apoptosis of infected or uninfected cells could be found in ZIKV-infected brain tissues. Our studies identify IPCs as the main target cells for ZIKV. They also suggest that immune activation after ZIKV infection may play an important role in the pathogenesis of ZIKV-related microcephaly. View Publication

Huntington disease (HD) is a dominant neurodegenerative disorder caused by a CAG repeat expansion in HTT. Here we report correction of HD human induced pluripotent stem cells (hiPSCs) using a CRISPR-Cas9 and piggyBac transposon-based approach. We show that both HD and corrected isogenic hiPSCs can be differentiated into excitable,synaptically active forebrain neurons. We further demonstrate that phenotypic abnormalities in HD hiPSC-derived neural cells,including impaired neural rosette formation,increased susceptibility to growth factor withdrawal,and deficits in mitochondrial respiration,are rescued in isogenic controls. Importantly,using genome-wide expression analysis,we show that a number of apparent gene expression differences detected between HD and non-related healthy control lines are absent between HD and corrected lines,suggesting that these differences are likely related to genetic background rather than HD-specific effects. Our study demonstrates correction of HD hiPSCs and associated phenotypic abnormalities,and the importance of isogenic controls for disease modeling using hiPSCs. 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

We have previously demonstrated that lineage negative cells (Lin(neg)) from umbilical cord blood (UCB) develop into multipotent cells capable of differentiation into bone,muscle,endothelial and neural cells. The objective of this study was to determine the optimal conditions required for Lin(neg) UCB cells to differentiate into neuronal cells and oligodendrocytes. We demonstrate that early neural stage markers (nestin,neurofilament,A2B5 and Sox2) are expressed in Lin(neg) cells cultured in FGF4,SCF,Flt3-ligand reprogramming culture media followed by the early macroglial cell marker O4. Early stage oligodendrocyte markers CNPase,GalC,Olig2 and the late-stage marker MOSP are observed,as is the Schwann cell marker PMP22. In summary,Lin(neg) UCB cells,when appropriately cultured,are able to exhibit characteristics of neuronal and macroglial cells that can specifically differentiate into oligodendrocytes and Schwann cells and express proteins associated with myelin production after in vitro differentiation. 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