Efficient Conversion of Astrocytes to Functional Midbrain Dopaminergic Neurons Using a Single Polycistronic Vector
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{"title"=>"Efficient conversion of astrocytes to functional midbrain dopaminergic neurons using a single polycistronic vector", "type"=>"journal", "authors"=>[{"first_name"=>"Russell C.", "last_name"=>"Addis", "scopus_author_id"=>"23567670500"}, {"first_name"=>"Fu Chun", "last_name"=>"Hsu", "scopus_author_id"=>"7202881062"}, {"first_name"=>"Rebecca L.", "last_name"=>"Wright", "scopus_author_id"=>"7403911658"}, {"first_name"=>"Marc A.", "last_name"=>"Dichter", "scopus_author_id"=>"7006051123"}, {"first_name"=>"Douglas A.", "last_name"=>"Coulter", "scopus_author_id"=>"7102017232"}, {"first_name"=>"John D.", "last_name"=>"Gearhart", "scopus_author_id"=>"36960096900"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-83055173820", "pui"=>"363060339", "doi"=>"10.1371/journal.pone.0028719", "isbn"=>"1932-6203; 1932-6203", "sgr"=>"83055173820", "pmid"=>"22174877"}, "id"=>"4f3feeff-6c79-31d1-a169-a2e428827bb4", "abstract"=>"Direct cellular reprogramming is a powerful new tool for regenerative medicine. In efforts to understand and treat Parkinson's Disease (PD), which is marked by the degeneration of dopaminergic neurons in the midbrain, direct reprogramming provides a valuable new source of these cells. Astrocytes, the most plentiful cells in the central nervous system, are an ideal starting population for the direct generation of dopaminergic neurons. In addition to their potential utility in cell replacement therapies for PD or in modeling the disease in vitro, astrocyte-derived dopaminergic neurons offer the prospect of direct in vivo reprogramming within the brain. As a first step toward this goal, we report the reprogramming of astrocytes to dopaminergic neurons using three transcription factors - ASCL1, LMX1B, and NURR1 - delivered in a single polycistronic lentiviral vector. The process is efficient, with 18.2±1.5% of cells expressing markers of dopaminergic neurons after two weeks. The neurons exhibit expression profiles and electrophysiological characteristics consistent with midbrain dopaminergic neurons, notably including spontaneous pacemaking activity, stimulated release of dopamine, and calcium oscillations. The present study is the first demonstration that a single vector can mediate reprogramming to dopaminergic neurons, and indicates that astrocytes are an ideal starting population for the direct generation of dopaminergic neurons.", "link"=>"http://www.mendeley.com/research/efficient-conversion-astrocytes-functional-midbrain-dopaminergic-neurons-using-single-polycistronic", "reader_count"=>112, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Researcher"=>27, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>36, "Student > Postgraduate"=>3, "Student > Master"=>20, "Other"=>3, "Student > Bachelor"=>9, "Lecturer > Senior Lecturer"=>1, "Professor"=>3}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Researcher"=>27, "Student > Doctoral Student"=>8, "Student > Ph. D. Student"=>36, "Student > Postgraduate"=>3, "Student > Master"=>20, "Other"=>3, "Student > Bachelor"=>9, "Lecturer > Senior Lecturer"=>1, "Professor"=>3}, "reader_count_by_subject_area"=>{"Engineering"=>2, "Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>9, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>71, "Medicine and Dentistry"=>7, "Neuroscience"=>15, "Physics and Astronomy"=>1, "Psychology"=>2, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>7}, "Neuroscience"=>{"Neuroscience"=>15}, "Physics and Astronomy"=>{"Physics and Astronomy"=>1}, "Psychology"=>{"Psychology"=>2}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>71}, "Computer Science"=>{"Computer Science"=>1}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>9}, "Unspecified"=>{"Unspecified"=>2}}, "reader_count_by_country"=>{"South Korea"=>1, "United States"=>1, "China"=>1, "Japan"=>1, "Italy"=>1, "Australia"=>1}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/704809"], "description"=>"<p>RMP: resting membrane potential; AP freq: frequency of spontaneous action potentials; I<sub>Na</sub> and I<sub>K</sub>: Maximum sodium and potassium currents; IR: input resistance.</p>", "links"=>[], "tags"=>["properties", "astrocyte-derived", "dopaminergic"], "article_id"=>375168, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.t001", "stats"=>{"downloads"=>1, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Electrophysiological_properties_of_astrocyte_derived_dopaminergic_neurons_/375168", "title"=>"Electrophysiological properties of astrocyte-derived dopaminergic neurons.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-12-09 01:26:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/704834"], "description"=>"<p>TaqMan assays for RT-PCR.</p>", "links"=>[], "tags"=>["assays"], "article_id"=>375198, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.t003", "stats"=>{"downloads"=>11, "page_views"=>3, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_TaqMan_assays_for_RT_PCR_/375198", "title"=>"TaqMan assays for RT-PCR.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-12-09 01:26:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/704695"], "description"=>"<p>(A) Action potential firing characteristics of induced dopaminergic neurons. Five overlapping traces are depicted derived from whole-cell current clamp recording of a representative induced dopaminergic neuron, elicited in response to hyperpolarizing and depolarizing current injection, increased from −10 to +40 pA in 10 pA increments. (B) Voltage-dependent sodium currents in induced dopaminergic neurons. Membrane potential was initially held at −90 mV and incrementally increased from −60 to +20 mV in 5 mV depolarizing steps. (C) Spontaneous action potential firing, consistent with a dopaminergic neuron pacemaker phenotype. The recording was conducted at resting membrane potential (−51 mV). (D) Dopamine release quantified by HPLC. Membrane depolarization was induced with 56 mM KCl at 17 days post-infection. 0F: uninfected astrocytes; ALN: induced dopaminergic neurons. (E) <i>MAP2</i>-GCaMP3 reveals rhythmic oscillations of intracellular calcium. Left panel displays a single frame of <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028719#pone.0028719.s002\" target=\"_blank\">Movie S1</a>, a recording of oscillating levels of intracellular calcium, which is presented as a histogram of GCaMP3 fluorescence intensity in the adjacent panel.</p>", "links"=>[], "tags"=>["characterization", "astrocyte-derived", "dopaminergic"], "article_id"=>375049, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.g004", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Functional_characterization_of_astrocyte_derived_dopaminergic_neurons_/375049", "title"=>"Functional characterization of astrocyte-derived dopaminergic neurons.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-09 01:24:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/704429"], "description"=>"<p>Astrocyte-derived dopaminergic neurons, 17 (A) to 19 (B,C) days post-induction. (A) Efficient conversion of astrocytes to dopaminergic neurons demonstrated by immunocytochemistry for the neuronal marker TUJ1 (type III beta tubulin) and the dopaminergic marker tyrosine hydroxylase (Th); the pan-nuclear marker DAPI is also shown. (B) Th positive neurons express the potassium channel Girk2, a marker of A9 (substantia nigra) dopaminergic neurons. (C) Punctate synaptophysin (Syp) expression in tyrosine hydroxylase (Th) positive neurons, indicating the potential to form synaptic connections. Scale bars 50 µm.</p>", "links"=>[], "tags"=>["characterization", "astrocyte-derived", "dopaminergic"], "article_id"=>374787, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.g002", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Immunocytochemical_characterization_of_astrocyte_derived_dopaminergic_neurons_/374787", "title"=>"Immunocytochemical characterization of astrocyte-derived dopaminergic neurons.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-09 01:19:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/704263"], "description"=>"<p>(A–B) Evaluation of transcription factor combinations to induce reprogramming. RT-PCR results for a subset of the 74 transcription factor combinations tested for their ability to induce expression of DOPA decarboxylase (Ddc) and tyrosine hydroxylase (Th) in mouse embryonic fibroblasts after 7 days of factor expression. 0F: uninfected control; A: ASCL1; B: BRN2; L: LMX1B; N: NURR1; P: PITX3. The combination of ASCL1, LMX1B, and NURR1 (ALN) was the only combination to give robust expression of both Ddc and Th. (C) Polycistronic vector containing open reading frames of human ASCL1, LMX1B, and NURR1 linked by viral 2A sequences. (D) Complete cleavage at 2A peptide sites confirmed by <i>in vitro</i> transcription and translation (TnT) of lentiviral plasmids in the presence of biotinylated lysine. Streptavidin-HRP Western blot shows all newly synthesized protein. Lane 1: No DNA TnT control. Lanes 2–4: TnT performed on original single-factor plasmids. Lane 5: TnT for polycistronic ALN plasmid. Band intensity is proportional to the number of lysine residues present in each protein sequence.</p>", "links"=>[], "tags"=>["polycistronic", "vector"], "article_id"=>374617, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.g001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transcription_factor_screen_and_polycistronic_vector_construction_/374617", "title"=>"Transcription factor screen and polycistronic vector construction.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-09 01:16:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/704581"], "description"=>"<p>Heat map of quantitative RT-PCR results comparing astrocyte-derived neurons magnetically sorted for a <i>MAP2</i>-CD4 reporter. Ast, uninfected astrocytes. ESN, mouse embryonic stem cell-derived neurons, generated via co-culture with PA6 stromal cells. Induced dopaminergic neurons express markers consistent with midbrain dopaminergic neurons. Color scale indicates change in Ct (threshold cycle) relative to the normalizing actin control. Higher delta Ct values correspond to lower relative gene expression, with every Ct decrease of 3.3 representing a ten-fold increase in relative expression.</p>", "links"=>[], "tags"=>["astrocyte-derived", "dopaminergic"], "article_id"=>374936, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.g003", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transcriptional_profile_of_astrocyte_derived_dopaminergic_neurons_/374936", "title"=>"Transcriptional profile of astrocyte-derived dopaminergic neurons.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-12-09 01:22:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/704877"], "description"=>"<p>Open Reading Frames and cDNAs cloned into FU-tetO-Gateway.</p>", "links"=>[], "tags"=>["frames", "cdnas", "cloned"], "article_id"=>375236, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0028719.t002", "stats"=>{"downloads"=>4, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Open_Reading_Frames_and_cDNAs_cloned_into_FU_tetO_Gateway_/375236", "title"=>"Open Reading Frames and cDNAs cloned into FU-tetO-Gateway.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2011-12-09 01:27:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/358366", "https://ndownloader.figshare.com/files/358394"], "description"=>"<div><p>Direct cellular reprogramming is a powerful new tool for regenerative medicine. In efforts to understand and treat Parkinson's Disease (PD), which is marked by the degeneration of dopaminergic neurons in the midbrain, direct reprogramming provides a valuable new source of these cells. Astrocytes, the most plentiful cells in the central nervous system, are an ideal starting population for the direct generation of dopaminergic neurons. In addition to their potential utility in cell replacement therapies for PD or in modeling the disease <em>in vitro</em>, astrocyte-derived dopaminergic neurons offer the prospect of direct <em>in vivo</em> reprogramming within the brain. As a first step toward this goal, we report the reprogramming of astrocytes to dopaminergic neurons using three transcription factors – ASCL1, LMX1B, and NURR1 – delivered in a single polycistronic lentiviral vector. The process is efficient, with 18.2±1.5% of cells expressing markers of dopaminergic neurons after two weeks. The neurons exhibit expression profiles and electrophysiological characteristics consistent with midbrain dopaminergic neurons, notably including spontaneous pacemaking activity, stimulated release of dopamine, and calcium oscillations. The present study is the first demonstration that a single vector can mediate reprogramming to dopaminergic neurons, and indicates that astrocytes are an ideal starting population for the direct generation of dopaminergic neurons.</p> </div>", "links"=>[], "tags"=>["astrocytes", "midbrain", "dopaminergic", "neurons", "polycistronic", "vector"], "article_id"=>130834, "categories"=>["Biochemistry", "Neuroscience", "Cell Biology", "Developmental Biology"], "users"=>["Russell C. Addis", "Fu-Chun Hsu", "Rebecca L. Wright", "Marc A. Dichter", "Douglas A. Coulter", "John D. Gearhart"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0028719.s001", "https://dx.doi.org/10.1371/journal.pone.0028719.s002"], "stats"=>{"downloads"=>0, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Efficient_Conversion_of_Astrocytes_to_Functional_Midbrain_Dopaminergic_Neurons_Using_a_Single_Polycistronic_Vector/130834", "title"=>"Efficient Conversion of Astrocytes to Functional Midbrain Dopaminergic Neurons Using a Single Polycistronic Vector", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-12-09 00:13:54"}

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Relative Metric

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