Neurotrophic Requirements of Human Motor Neurons Defined Using Amplified and Purified Stem Cell-Derived Cultures
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{"title"=>"Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures", "type"=>"journal", "authors"=>[{"first_name"=>"Nuno Jorge", "last_name"=>"Lamas", "scopus_author_id"=>"23100224900"}, {"first_name"=>"Bethany Johnson", "last_name"=>"Kerner", "scopus_author_id"=>"56404362400"}, {"first_name"=>"Laurent", "last_name"=>"Roybon", "scopus_author_id"=>"8146519100"}, {"first_name"=>"Yoon A.", "last_name"=>"Kim", "scopus_author_id"=>"56403530300"}, {"first_name"=>"Alejandro Garcia", "last_name"=>"Diaz", "scopus_author_id"=>"56403973400"}, {"first_name"=>"Hynek", "last_name"=>"Wichterle", "scopus_author_id"=>"6603364179"}, {"first_name"=>"Christopher E.", "last_name"=>"Henderson", "scopus_author_id"=>"7202491087"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"84908587490", "doi"=>"10.1371/journal.pone.0110324", "pui"=>"600247560", "pmid"=>"25337699", "scopus"=>"2-s2.0-84908587490", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\r1932-6203 (Linking)"}, "id"=>"37388c78-1d02-38c8-8915-145940777461", "abstract"=>"Human motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.", "link"=>"http://www.mendeley.com/research/neurotrophic-requirements-human-motor-neurons-defined-using-amplified-purified-stem-cellderived-cult", "reader_count"=>35, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>3, "Student > Doctoral Student"=>3, "Researcher"=>6, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>1, "Other"=>2, "Student > Master"=>7, "Student > Bachelor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>3, "Student > Doctoral Student"=>3, "Researcher"=>6, "Student > Ph. D. Student"=>11, "Student > Postgraduate"=>1, "Other"=>2, "Student > Master"=>7, "Student > Bachelor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>3, "Biochemistry, Genetics and Molecular Biology"=>7, "Medicine and Dentistry"=>3, "Agricultural and Biological Sciences"=>15, "Neuroscience"=>4, "Pharmacology, Toxicology and Pharmaceutical Science"=>3}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>3}, "Neuroscience"=>{"Neuroscience"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>15}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>3}}, "group_count"=>2}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1728093"], "description"=>"<p>(A) The plating efficiency of FACS-purified human motor neurons after 24 hours is not increased in the presence of Y-27632. (B) Y-27632 enhances the survival of FACS-purified human motor neurons in a 7-day survival assay. Scale bar = 200 µM. (C) Dose-dependent effects of Y-27632 on human motor neuron survival, expressed relative to the basal condition (0 µM). Values shown as mean ± s.e.m., n≥5 (t-test, *p<0.05; **p<0.01).</p>", "links"=>[], "tags"=>["increase motor neuron", "rodent motor neurons", "bdnf", "ibmx", "Human Motor Neurons Defined", "motor neurons", "novel neurotrophic agents", "ec", "motor neuron survival", "motor neuron progenitor proliferation", "cntf", "gdnf"], "article_id"=>1212990, "categories"=>["Biological Sciences"], "users"=>["Nuno Jorge Lamas", "Bethany Johnson-Kerner", "Laurent Roybon", "Yoon A. Kim", "Alejandro Garcia-Diaz", "Hynek Wichterle", "Christopher E. Henderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0110324.g006", "stats"=>{"downloads"=>1, "page_views"=>32, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Y_27632_is_also_a_survival_factor_for_human_motor_neurons_/1212990", "title"=>"Y-27632 is also a survival factor for human motor neurons.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-22 03:40:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/1728090"], "description"=>"<p>(A) Y-27632-supplemented cultures contain increased numbers of OLIG2-positive cells at day 31+9. Scale bar = 50 µM. (B) Time-dependent increase in numbers of OLIG2-expressing progenitors in the presence of Y-27632. Data normalized to control at day 31+1; mean ± s.e.m., <i>n</i>>5 (t-test, **p<0.01). (C) OLIG2 progenitors at day 31+9 stained for BrdU. Scale bar = 25 µM. (D) Percent of OLIG2 precursors that are BrdU-positive at day 31+9 (mean ± s.e.m., <i>n</i> = 4). (E) Hb9::GFP-expressing motor neurons at day 31+9 stained for BrdU. Scale bar = 25 µM. (F) Percent motor neurons that are BrdU-positive at day 31+9 (mean ± s.e.m., <i>n</i> = 4). (G) The total number of cells in culture is increased at day 31+9 following Y-27632 treatment of hESC RUES1 and hiPSC18c. Values are mean ± s.e.m., <i>n</i>≥3 (t-test, *p<0.05). (H) Numbers of OLIG2 precursors increase significantly at day 31+9 following Y-27632 treatment of hiPSC 18c. Values are mean ± s.e.m., <i>n</i>≥3 (t-test, *p<0.05). (I) Numbers of motor neurons identified by staining for endogenous HB9 increase significantly at day 31+9 following Y-27632 treatment of hESC RUES1 and hiPSC 18c. Values are mean ± s.e.m., <i>n</i>≥3 (t-test, *p<0.05). (J) Cultures from healthy control hESCs (RUES1) or hiPSCs (18c) immunostained for the motor neuron marker HB9 and the pan-neuronal marker β-III tubulin. Y-27632 increases the number of motor neurons in each case. Scale bar  =  25 µM.</p>", "links"=>[], "tags"=>["increase motor neuron", "rodent motor neurons", "bdnf", "ibmx", "Human Motor Neurons Defined", "motor neurons", "novel neurotrophic agents", "ec", "motor neuron survival", "motor neuron progenitor proliferation", "cntf", "gdnf"], "article_id"=>1212987, "categories"=>["Biological Sciences"], "users"=>["Nuno Jorge Lamas", "Bethany Johnson-Kerner", "Laurent Roybon", "Yoon A. Kim", "Alejandro Garcia-Diaz", "Hynek Wichterle", "Christopher E. Henderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0110324.g003", "stats"=>{"downloads"=>1, "page_views"=>93, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Y_27632_enhances_proliferation_of_motor_neuron_progenitors_in_hESC_and_hiPSC_derived_motor_neuron_cultures_/1212987", "title"=>"Y-27632 enhances proliferation of motor neuron progenitors in hESC- and hiPSC-derived motor neuron cultures.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-22 03:40:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/1728089"], "description"=>"<p>(A) Screening of 160 compounds for their potential to increase the number of human motor neurons in hESC cultures at day 31+13. Compounds were tested in quadruplicate at a single concentration (10 µM). Values are plotted as mean fold difference in motor neuron numbers relative to the negative control condition (No NTFs). The Rho-kinase (ROCK) inhibitor Y-27632 was the compound showing the highest capacity to increase the number of human motor neurons. (B) Y-27632 increases the number of fluorescent hESC-motor neurons in mixed cultures in a dose-dependent manner. Cells were cultured in the absence of neurotrophic factors and in the presence of increasing concentrations of Y-27632. Values shown as mean ± s.e.m., <i>n</i> = 4. (C) Representative images of hESC-motor neuron cultures at day 31+13 grown under neurotrophic factor deprivation (No NTFs), neurotrophic factor supplementation (NTFs + F + I) and Y-27632 (10 µM). Scale bar = 25 µM. (D) Time-dependent increase in the number of motor neurons in the presence (green) but not absence (blue) of Y-27632 (10 µM), with a peak effect at day 31+9. Values shown as mean ± s.e.m., <i>n</i>>5 (t-test, *p<0.05; **p<0.01). (E) Y-27632 also increases the total number of cells in culture. Mean ± s.e.m., <i>n</i> = 3. (F) Hb9::GFP-positive neurons continue to express motor neuron markers HB9 and ISL1 after treatment with Y-27632 for 9 days. Scale bar = 50 µM. (G) Supplementation of cultures with Y-27632 (red line) leads to increased numbers of human motor neurons expressing endogenous ISL1 at day 31+9. Mean ± s.e.m., <i>n</i> = 3 (**p<0.01).</p>", "links"=>[], "tags"=>["increase motor neuron", "rodent motor neurons", "bdnf", "ibmx", "Human Motor Neurons Defined", "motor neurons", "novel neurotrophic agents", "ec", "motor neuron survival", "motor neuron progenitor proliferation", "cntf", "gdnf"], "article_id"=>1212986, "categories"=>["Biological Sciences"], "users"=>["Nuno Jorge Lamas", "Bethany Johnson-Kerner", "Laurent Roybon", "Yoon A. Kim", "Alejandro Garcia-Diaz", "Hynek Wichterle", "Christopher E. Henderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0110324.g002", "stats"=>{"downloads"=>2, "page_views"=>94, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_ROCK_inhibitor_Y_27632_increases_human_motor_neuron_numbers_in_hESC_derived_motor_neuron_cultures_/1212986", "title"=>"The ROCK inhibitor Y-27632 increases human motor neuron numbers in hESC-derived motor neuron cultures.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-22 03:40:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/1728092"], "description"=>"<p>(A) Whole-well imaging of live motor neurons labeled with calcein-AM captured using the Plate Runner (left two panels). Surviving human motor neurons were counted in whole culture wells in an automated manner using MetaMorph (red tracing, right two panels). Scale bar = 200 µm. (B) Y-27632-expanded motor neurons show enhanced survival in the presence of a cocktail of neurotrophic factors. Values shown as mean ± s.e.m., n>5 (t-test, ***p<0.001). (C) GDNF, (D) BDNF and (E) CNTF alone (blue lines) enhance the survival of expanded FACS-purified human motor neurons. The addition of F+I significantly potentiates the survival-inducing activity of GDNF at high concentrations. Values shown as mean ± s.e.m., n>4 (t-test, *p<0.05; **p<0.01; ***p<0.001). Asterisks on individual points represent significance of difference with No-NTF control (white rectangle in the curve); asterisks on bars represent significant differences between a given concentration of NTF and the corresponding value for NTF + F+ I. (F) The cocktail of neurotrophic factors (NTFs) enhances the survival of expanded FACS-purified human motor neurons in a dose-dependent manner in the presence of 10 µM forskolin plus 100 µM IBMX. Values shown as mean ± s.e.m., n≥5 (t-test, **p<0.01; ***p<0.001).</p>", "links"=>[], "tags"=>["increase motor neuron", "rodent motor neurons", "bdnf", "ibmx", "Human Motor Neurons Defined", "motor neurons", "novel neurotrophic agents", "ec", "motor neuron survival", "motor neuron progenitor proliferation", "cntf", "gdnf"], "article_id"=>1212989, "categories"=>["Biological Sciences"], "users"=>["Nuno Jorge Lamas", "Bethany Johnson-Kerner", "Laurent Roybon", "Yoon A. Kim", "Alejandro Garcia-Diaz", "Hynek Wichterle", "Christopher E. Henderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0110324.g005", "stats"=>{"downloads"=>2, "page_views"=>37, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Purified_human_motor_neurons_show_a_potent_response_to_known_neurotrophic_factors_/1212989", "title"=>"Purified human motor neurons show a potent response to known neurotrophic factors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-22 03:40:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/1728091"], "description"=>"<p>(A) Y-27632 supplementation for 3 days leads to a 1.8-fold increase in motor neuron yield judged by FACS analysis. Data normalized to controls without Y-27632. Values are mean ± s.e.m., n>5 (t-test, **p<0.01). (B) Nine-day treatment with Y-27632 gives a ∼5-fold increase in motor neuron yield as compared to controls without Y-27632, as quantified by flow cytometry. Values are mean ± s.e.m., n>5 (t-test, **p<0.01). (C) FACS purification of Hb9::GFP motor neurons expanded with Y-27632 for 3 days. Representative FACS gating used to retrieve an almost pure (>95%) population of human motor neurons. (D) FACS-purified motor neurons at day 31+3+1 stained for GFP (green), and a combination of HB9 and ISL1 (“pan-MN”; white nuclei).>95% of the FACS-purified cells in culture are Hb9::GFP positive. Scale bar  =  25 µM. (E) Even following FACS sorting, some contaminant cells were able to proliferate and form colonies that interfered with survival assays (left panel). Uridine/Fluorodeoxyuridine (U/FdU) (each at 1 µM) successfully prevented the proliferation (right panel).</p>", "links"=>[], "tags"=>["increase motor neuron", "rodent motor neurons", "bdnf", "ibmx", "Human Motor Neurons Defined", "motor neurons", "novel neurotrophic agents", "ec", "motor neuron survival", "motor neuron progenitor proliferation", "cntf", "gdnf"], "article_id"=>1212988, "categories"=>["Biological Sciences"], "users"=>["Nuno Jorge Lamas", "Bethany Johnson-Kerner", "Laurent Roybon", "Yoon A. Kim", "Alejandro Garcia-Diaz", "Hynek Wichterle", "Christopher E. Henderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0110324.g004", "stats"=>{"downloads"=>5, "page_views"=>56, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_FACS_sorting_of_amplified_cultures_yields_a_pure_preparation_of_viable_human_motor_neurons_/1212988", "title"=>"FACS-sorting of amplified cultures yields a pure preparation of viable human motor neurons.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-22 03:40:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/1728088"], "description"=>"<p>(A) Live fluorescent human motor neurons derived from the Hb9::GFP reporter line at day 31+13 after growth with a cocktail of neurotrophic factors (NTFs). (B) Automated quantification of fluorescent cells with significant neurite outgrowth (SNO) using the Neurite Outgrowth module of MetaMorph software; cells counted are identified with a red overlay. Motor neurons were considered to have significant neurite outgrowth when their overall neurite length exceeded 75 µm (scale bar). (C) Representative image of immunostained Hb9::GFP hESC-motor neuron cultures at day 31+13 after growth with a cocktail of neurotrophic factors (NTFs). Scale bar = 50 µM. (D) Number of cells with significant neurite outgrowth (SNO) when grown with (red bars) or without (blue bars) neurotrophic factors, expressed as a percentage of numbers at day 31+1. The increase in motor neuron numbers after day 31+7 in NTF-supplemented cultures suggests ongoing neurogenesis. Surviving fluorescent GFP-positive motor neurons with SNO shown as mean ± s.e.m., <i>n</i>>5 (t-test, ***p<0.001, *p<0.05). (E) BrdU-positive Hb9::GFP-positive motor neurons (arrows) at day 31+15 confirming the presence of newborn human motor neurons in culture. Scale bar = 50 µM. (F) The percentage of Hb9::GFP-positive motor neurons that were BrdU-positive at day 31+15 is not changed by NTFs but (G) total numbers of BrdU-positive motor neurons are increased with NTFs. Bars indicate mean ± s.e.m., <i>n</i> = 3 (t-test, *p<0.05; n.s.  =  not significant).</p>", "links"=>[], "tags"=>["increase motor neuron", "rodent motor neurons", "bdnf", "ibmx", "Human Motor Neurons Defined", "motor neurons", "novel neurotrophic agents", "ec", "motor neuron survival", "motor neuron progenitor proliferation", "cntf", "gdnf"], "article_id"=>1212985, "categories"=>["Biological Sciences"], "users"=>["Nuno Jorge Lamas", "Bethany Johnson-Kerner", "Laurent Roybon", "Yoon A. Kim", "Alejandro Garcia-Diaz", "Hynek Wichterle", "Christopher E. Henderson"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0110324.g001", "stats"=>{"downloads"=>5, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Ongoing_birth_of_motor_neurons_in_hESC_derived_cultures_is_stimulated_by_neurotrophic_factors_/1212985", "title"=>"Ongoing birth of motor neurons in hESC-derived cultures is stimulated by neurotrophic factors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-10-22 03:40:07"}

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Relative Metric

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