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Alessandra Giorgetti,
Maria C. N. Marchetto,
Mo Li,
Diana Yu,
Raffaella Fazzina, Yangling Mu,
Antonio Adamo,
Ida Paramonov,
Julio Castaño Cardoso,
Montserrat Barragan Monasterio,
Cedric Bardy,
Riccardo Cassiani-Ingoni,
Guang-Hui Liu,
Fred H. Gage,
Juan Carlos Izpisua Belmonte
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ABSTRACT: The finding that certain somatic cells can be directly converted into cells of other lineages by the delivery of specific
sets of transcription factors paves the way to novel therapeutic applications. Here we show that human cord blood (CB) CD133+ cells lose their hematopoietic signature and are converted into CB-induced neuronal-like cells (CB-iNCs) by the ectopic expression
of the transcription factor Sox2, a process that is further augmented by the combination of Sox2 and c-Myc. Gene-expression
analysis, immunophenotyping, and electrophysiological analysis show that CB-iNCs acquire a distinct neuronal phenotype characterized
by the expression of multiple neuronal markers. CB-iNCs show the ability to fire action potentials after in vitro maturation
as well as after in vivo transplantation into the mouse hippocampus. This system highlights the potential of CB cells and
offers an alternative means to the study of cellular plasticity, possibly in the context of drug screening research and of
future cell-replacement therapies.
Proceedings of the National Academy of Sciences 07/2012; 109(31):12556-12561. · 9.68 Impact Factor
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ABSTRACT: In the adult brain, neural stem cells have been found in two major niches: the dentate gyrus and the subventricular zone [corrected]. Neurons derived from these stem cells contribute to learning, memory, and the autonomous repair of the brain under pathological conditions. Hence, the physiology of adult neural stem cells has become a significant component of research on synaptic plasticity and neuronal disorders. In addition, the recently developed induced pluripotent stem cell technique provides a powerful tool for researchers engaged in the pathological and pharmacological study of neuronal disorders. In this review, we briefly summarize the research progress in neural stem cells in the adult brain and in the neuropathological application of the induced pluripotent stem cell technique.
Protein & Cell 04/2012; 3(4):251-61.
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Mason A Israel,
Shauna H Yuan,
Cedric Bardy,
Sol M Reyna, Yangling Mu,
Cheryl Herrera,
Michael P Hefferan,
Sebastiaan Van Gorp,
Kristopher L Nazor,
Francesca S Boscolo,
Christian T Carson,
Louise C Laurent,
Martin Marsala,
Fred H Gage,
Anne M Remes,
Edward H Koo,
Lawrence S B Goldstein
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ABSTRACT: Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-β precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-β(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3β (aGSK-3β). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with β-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3β levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-β, in GSK-3β activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.
Nature 02/2012; 482(7384):216-20. · 36.28 Impact Factor
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ABSTRACT: The hippocampus, a brain area critical for learning and memory, is especially vulnerable to damage at early stages of Alzheimer's disease (AD). Emerging evidence has indicated that altered neurogenesis in the adult hippocampus represents an early critical event in the course of AD. Although causal links have not been established, a variety of key molecules involved in AD pathogenesis have been shown to impact new neuron generation, either positively or negatively. From a functional point of view, hippocampal neurogenesis plays an important role in structural plasticity and network maintenance. Therefore, dysfunctional neurogenesis resulting from early subtle disease manifestations may in turn exacerbate neuronal vulnerability to AD and contribute to memory impairment, whereas enhanced neurogenesis may be a compensatory response and represent an endogenous brain repair mechanism. Here we review recent findings on alterations of neurogenesis associated with pathogenesis of AD, and we discuss the potential of neurogenesis-based diagnostics and therapeutic strategies for AD.
Molecular Neurodegeneration 12/2011; 6:85. · 4.28 Impact Factor
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ABSTRACT: Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition.
Proceedings of the National Academy of Sciences 12/2011; 108(51):20382-7. · 9.68 Impact Factor
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Kristen J Brennand,
Anthony Simone,
Jessica Jou,
Chelsea Gelboin-Burkhart,
Ngoc Tran,
Sarah Sangar,
Yan Li, Yangling Mu,
Gong Chen,
Diana Yu,
Shane McCarthy,
Jonathan Sebat,
Fred H Gage
Nature 10/2011; · 36.28 Impact Factor
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Kristen J Brennand,
Anthony Simone,
Jessica Jou,
Chelsea Gelboin-Burkhart,
Ngoc Tran,
Sarah Sangar,
Yan Li, Yangling Mu,
Gong Chen,
Diana Yu,
Shane McCarthy,
Jonathan Sebat,
Fred H Gage
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ABSTRACT: Schizophrenia (SCZD) is a debilitating neurological disorder with a world-wide prevalence of 1%; there is a strong genetic component, with an estimated heritability of 80-85%. Although post-mortem studies have revealed reduced brain volume, cell size, spine density and abnormal neural distribution in the prefrontal cortex and hippocampus of SCZD brain tissue and neuropharmacological studies have implicated dopaminergic, glutamatergic and GABAergic activity in SCZD, the cell types affected in SCZD and the molecular mechanisms underlying the disease state remain unclear. To elucidate the cellular and molecular defects of SCZD, we directly reprogrammed fibroblasts from SCZD patients into human induced pluripotent stem cells (hiPSCs) and subsequently differentiated these disorder-specific hiPSCs into neurons (Supplementary Fig. 1). SCZD hiPSC neurons showed diminished neuronal connectivity in conjunction with decreased neurite number, PSD95-protein levels and glutamate receptor expression. Gene expression profiles of SCZD hiPSC neurons identified altered expression of many components of the cyclic AMP and WNT signalling pathways. Key cellular and molecular elements of the SCZD phenotype were ameliorated following treatment of SCZD hiPSC neurons with the antipsychotic loxapine. To date, hiPSC neuronal pathology has only been demonstrated in diseases characterized by both the loss of function of a single gene product and rapid disease progression in early childhood. We now report hiPSC neuronal phenotypes and gene expression changes associated with SCZD, a complex genetic psychiatric disorder.
Nature 05/2011; 473(7346):221-5. · 36.28 Impact Factor
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ABSTRACT: Adult neurogenesis, a particular form of plasticity in the adult brain, is under dynamic control of neuronal activity mediated by various neurotransmitters. Despite accumulating evidence suggesting that the neurotransmitter dopamine (DA) regulates proliferation of neural precursor cells in the neurogenic zones, whether and how it acts on newly generated neurons that integrate into the established network remains unknown. Using patch-clamp recordings from retrovirus-labeled newborn hippocampal dentate granule cells (DGCs) in acute mouse brain slices, we found that DA not only caused a long-lasting attenuation of medial perforant path (MPP) inputs to the young DGCs, but also decreased their capacity to express long-term potentiation (LTP). In contrast, DA suppressed MPP transmission to mature DGCs to a similar extent but did not influence their LTP expression. This difference was linked to activation of distinct subtypes of DA receptors in DGCs at different developmental stages. Our observations suggest that DA is particularly effective in modulating the activities of hyperexcitable young neurons, which may have important implications for the dentate function as a filter for incoming information to the hippocampus.
Journal of Neuroscience 03/2011; 31(11):4113-23. · 7.11 Impact Factor
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Shauna H Yuan,
Jody Martin,
Jeanne Elia,
Jessica Flippin,
Rosanto I Paramban,
Mike P Hefferan,
Jason G Vidal, Yangling Mu,
Rhiannon L Killian,
Mason A Israel,
Nil Emre,
Silvia Marsala,
Martin Marsala,
Fred H Gage,
Lawrence S B Goldstein,
Christian T Carson
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ABSTRACT: Neural induction of human pluripotent stem cells often yields heterogeneous cell populations that can hamper quantitative and comparative analyses. There is a need for improved differentiation and enrichment procedures that generate highly pure populations of neural stem cells (NSC), glia and neurons. One way to address this problem is to identify cell-surface signatures that enable the isolation of these cell types from heterogeneous cell populations by fluorescence activated cell sorting (FACS).
We performed an unbiased FACS- and image-based immunophenotyping analysis using 190 antibodies to cell surface markers on naïve human embryonic stem cells (hESC) and cell derivatives from neural differentiation cultures. From this analysis we identified prospective cell surface signatures for the isolation of NSC, glia and neurons. We isolated a population of NSC that was CD184(+)/CD271(-)/CD44(-)/CD24(+) from neural induction cultures of hESC and human induced pluripotent stem cells (hiPSC). Sorted NSC could be propagated for many passages and could differentiate to mixed cultures of neurons and glia in vitro and in vivo. A population of neurons that was CD184(-)/CD44(-)/CD15(LOW)/CD24(+) and a population of glia that was CD184(+)/CD44(+) were subsequently purified from cultures of differentiating NSC. Purified neurons were viable, expressed mature and subtype-specific neuronal markers, and could fire action potentials. Purified glia were mitotic and could mature to GFAP-expressing astrocytes in vitro and in vivo.
These findings illustrate the utility of immunophenotyping screens for the identification of cell surface signatures of neural cells derived from human pluripotent stem cells. These signatures can be used for isolating highly pure populations of viable NSC, glia and neurons by FACS. The methods described here will enable downstream studies that require consistent and defined neural cell populations.
PLoS ONE 01/2011; 6(3):e17540. · 4.09 Impact Factor
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ABSTRACT: Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment.
Cell 11/2010; 143(4):527-39. · 32.40 Impact Factor
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ABSTRACT: Neural stem cells (NSCs) in the adult brain continuously supply new neurons to the hippocampal dentate gyrus (DG) and the olfactory bulb (OB). Recent studies indicate that the progression from neural precursor cells (NPCs) to mature neurons is tightly controlled by coordinate cell-intrinsic programs and external signals within the neurogenic niche. In this review, we summarize both classes of regulatory factors involved in distinct stages of adult neurogenesis, including proliferation and lineage differentiation of NSCs, migration of neuroblasts and integration of newborn neurons. A full understanding of the wide variety of signaling pathways will ultimately provide precise targets for therapeutic applications.
Current opinion in neurobiology 08/2010; 20(4):416-23. · 7.21 Impact Factor
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ABSTRACT: Long interspersed element 1 (LINE-1 or L1) retrotransposons have markedly affected the human genome. L1s must retrotranspose in the germ line or during early development to ensure their evolutionary success, yet the extent to which this process affects somatic cells is poorly understood. We previously demonstrated that engineered human L1s can retrotranspose in adult rat hippocampus progenitor cells in vitro and in the mouse brain in vivo. Here we demonstrate that neural progenitor cells isolated from human fetal brain and derived from human embryonic stem cells support the retrotransposition of engineered human L1s in vitro. Furthermore, we developed a quantitative multiplex polymerase chain reaction that detected an increase in the copy number of endogenous L1s in the hippocampus, and in several regions of adult human brains, when compared to the copy number of endogenous L1s in heart or liver genomic DNAs from the same donor. These data suggest that de novo L1 retrotransposition events may occur in the human brain and, in principle, have the potential to contribute to individual somatic mosaicism.
Nature 09/2009; 460(7259):1127-31. · 36.28 Impact Factor
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ABSTRACT: The hippocampal formation is widely studied in part because of its distinct and highly laminar organization as well as its demonstrated fundamental role in learning and memory. The dentate gyrus of the hippocampal formation is one of two recognized brain regions that continually generate new neurons in adulthood. In this chapter, we review the basic structure of hippocampal cellular components and circuitry, the properties of stem cells and their progeny in the dentate gyrus, and the known mechanisms and timing of their maturation and integration into the adult circuitry. We also address the functional implication of neurogenesis in the adult hippocampus.
Current Topics in Developmental Biology 02/2009; 87:149-74. · 6.00 Impact Factor
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ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. ALS can be induced by mutations in the superoxide dismutase 1 gene (SOD1). Evidence for the non-cell-autonomous nature of ALS emerged from the observation that wild-type glial cells extended the survival of SOD1 mutant motor neurons in chimeric mice. To uncover the contribution of astrocytes to human motor neuron degeneration, we cocultured hESC-derived motor neurons with human primary astrocytes expressing mutated SOD1. We detected a selective motor neuron toxicity that was correlated with increased inflammatory response in SOD1-mutated astrocytes. Furthermore, we present evidence that astrocytes can activate NOX2 to produce superoxide and that effect can be reversed by antioxidants. We show that NOX2 inhibitor, apocynin, can prevent the loss of motor neurons caused by SOD1-mutated astrocytes. These results provide an assay for drug screening using a human ALS in vitro astrocyte-based cell model.
Cell stem cell 01/2009; 3(6):649-57. · 23.56 Impact Factor
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ABSTRACT: Sensory experience plays an instructive role in the development of the nervous system. Here we showed that visual experience can induce persistent modification of developing retinotectal circuits via spike timing-dependent plasticity (STDP). Pairing light stimuli with spiking of the tectal cell induced persistent enhancement or reduction of light-evoked responses, with a dependence on the relative timing between light stimulus and postsynaptic spiking similar to that for STDP. Using precisely timed sequential three-bar stimulation to mimic a moving bar, we showed that spike timing-dependent LTP/LTD can account for the asymmetric modification of the tectal cell receptive field induced by moving bar. Furthermore, selective inhibition of signaling mediated by brain-derived neurotrophic factor and nitric oxide, which are respectively required for light-induced LTP and LTD, interfered with moving bar-induced temporally specific changes in the tectal cell responses. Together, these findings suggest that STDP can mediate sensory experience-dependent circuit refinement in the developing nervous system.
Neuron 05/2006; 50(1):115-25. · 14.74 Impact Factor
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ABSTRACT: Local GABA (gamma-aminobutyric acid) circuits contribute to sensory experience-dependent refinement of neuronal connections in the developing nervous system, but whether GABAergic synapses themselves can be rapidly modified by sensory stimuli is largely unknown. Here we report that repetitive light stimuli or theta burst stimulation (TBS) of the optic nerve in the developing Xenopus retinotectal system induces long-term potentiation (LTP) of glutamatergic inputs but long-term depression (LTD) of GABAergic inputs to the same tectal neuron. The LTD is due to a reduction in presynaptic GABA release and requires activation of presynaptic NMDA (N-methyl-D-aspartate) receptors (NMDARs) and coincident high-level GABAergic activity. Thus, the presynaptic NMDAR may function as a coincidence detector for adjacent glutamatergic and GABAergic activities, leading to coordinated synaptic modification by sensory experience.
Nature Neuroscience 04/2006; 9(3):372-80. · 15.53 Impact Factor
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ABSTRACT: SUMMARY Amyotrophic lateral sclerosis (ALS) is a neurodegen- erativediseasecharacterizedbymotorneurondeath. ALS can be induced by mutations in the superoxide dismutase 1 gene (SOD1). Evidence for the non- cell-autonomous nature of ALS emerged from the observation that wild-type glial cells extended the survival of SOD1 mutant motor neurons in chimeric mice. To uncover the contribution of astrocytes to human motor neuron degeneration, we cocultured hESC-derived motor neurons with human primary astrocytes expressing mutated SOD1. We detected a selective motor neuron toxicity that was correlated with increased inflammatory response in SOD1-mu- tated astrocytes. Furthermore, we present evidence that astrocytes can activate NOX2 to produce super- oxide and that effect can be reversed by antioxi- dants. We show that NOX2 inhibitor, apocynin, can prevent the loss of motor neurons caused by SOD1- mutated astrocytes. These results provide an assay for drug screening using a human ALS in vitro astro- cyte-based cell model.
