title: Neurotrophic requirements of human motor neurons defined using amplified and purified stem cell-derived cultures.
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.
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America; Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Minho, Portugal; ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães, Minho, Portugal.
authors.forename: Nuno Jorge
authors.initials: NJ
authors.lastname: Lamas
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America.
authors.forename: Bethany
authors.initials: B
authors.lastname: Johnson-Kerner
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America.
authors.forename: Laurent
authors.initials: L
authors.lastname: Roybon
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America.
authors.forename: Yoon A
authors.initials: YA
authors.lastname: Kim
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Garcia-Diaz
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America.
authors.forename: Hynek
authors.initials: H
authors.lastname: Wichterle
authors.affiliation: Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, New York, New York, United States of America; Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, New York, United States of America; Department of Rehabilitation and Regenerative Medicine, Columbia University Medical Center, New York, New York, United States of America; Department of Pathology and Cell Biology, Columbia University Medical Center, New York, New York, United States of America; Department of Neurology, Columbia University Medical Center, New York, New York, United States of America; Department of Neuroscience, Columbia University Medical Center, New York, New York, United States of America; Columbia Stem Cell Initiative, Columbia University Medical Center, New York, New York, United States of America; Columbia Translational Neuroscience Initiative, Columbia University Medical Center, New York, New York, United States of America.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Henderson
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D020934:Ciliary Neurotrophic Factor / Q000494:pharmacology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000819:agonists / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D015870:Gene Expression; D051100:Glial Cell Line-Derived Neurotrophic Factor / Q000494:pharmacology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009046:Motor Neurons / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000577:Amides; D019208:Brain-Derived Neurotrophic Factor; D020934:Ciliary Neurotrophic Factor; D051100:Glial Cell Line-Derived Neurotrophic Factor; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D005576:Colforsin; C579455:BDNF protein, human; D000242:Cyclic AMP; D054460:rho-Associated Kinases; D015056:1-Methyl-3-isobutylxanthine