title: Differentiation of spinal motor neurons from pluripotent human stem cells.
abstract: We have devised a reproducible protocol by which human embryonic stem cells (hESCs) or inducible pluripotent stem cells (iPSCs) are efficiently differentiated to functional spinal motor neurons. This protocol comprises four major steps. Pluripotent stem cells are induced to form neuroepithelial (NE) cells that form neural tube-like rosettes in the absence of morphogens in the first 2 weeks. The NE cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) or purmorphamine in the next 2 weeks. These progenitor cells further generate post-mitotic, HB9-expressing motoneurons at the 5th week and mature to functional motor neurons thereafter. It typically takes 5 weeks to generate the post-mitotic motoneurons and 8-10 weeks for the production of functional mature motoneurons. In comparison with other methods, our protocol does not use feeder cells, has a minimum dependence on proteins (purmorphamine replacing SHH), has controllable adherent selection and is adaptable for scalable suspension culture.
authors.affiliation: Department of Anatomy and Department of Neurology, School of Medicine and Public Health, Waisman Center, The WiCell Institute, University of Wisconsin Madison, Madison, WI, USA.
authors.forename: Bao-Yang
authors.initials: BY
authors.lastname: Hu
authors.forename: Su-Chun
authors.initials: SC
authors.lastname: Zhang
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D006801:Humans; D051379:Mice; D009046:Motor Neurons / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D013127:Spinal Nerves / Q000166:cytology*; D013997:Time Factors
references.citation: Cell. 2002 Aug 9;110(3):385-97
references.pmid: 12176325
references.citation: Neuron. 2001 Sep 13;31(5):757-71
references.pmid: 11567615
references.citation: Nat Biotechnol. 2005 Feb;23(2):215-21
references.pmid: 15685164
references.citation: Genes Dev. 2005 Jan 15;19(2):282-94
references.pmid: 15655114
references.citation: Dev Biol. 2004 Nov 1;275(1):124-42
references.pmid: 15464577
references.citation: Nat Biotechnol. 2001 Dec;19(12):1129-33
references.pmid: 11731781
references.citation: Cell. 1997 Jul 11;90(1):169-80
references.pmid: 9230312
references.citation: Development. 2009 May;136(9):1443-52
references.pmid: 19363151
references.citation: J Neurosci. 2007 Mar 21;27(12):3069-77
references.pmid: 17376968
references.citation: Stem Cells. 2009 Aug;27(8):1741-9
references.pmid: 19544434
references.citation: Stem Cells. 2009 Apr;27(4):806-11
references.pmid: 19350680
references.citation: Stem Cells. 2005 Jun-Jul;23(6):781-90
references.pmid: 15917474
references.citation: Exp Neurol. 2005 Dec;196(2):224-34
references.pmid: 16198339
references.citation: Nat Med. 2006 Nov;12(11):1259-68
references.pmid: 17057709
references.citation: Stem Cells. 2008 Apr;26(4):886-93
references.pmid: 18238853
references.citation: Glia. 2005 Feb;49(3):385-96
references.pmid: 15538751
references.citation: Methods Mol Biol. 2009;549:51-8
references.pmid: 19378195
references.citation: Nat Neurosci. 2005 Mar;8(3):288-96
references.pmid: 15696161
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12543-8
references.pmid: 15310843
references.citation: Neuron. 2000 Feb;25(2):317-29
references.pmid: 10719888
references.citation: J Comp Neurol. 1987 Dec 15;266(3):332-59
references.pmid: 2447134
references.citation: Neuron. 2000 Feb;25(2):331-43
references.pmid: 10719889
references.citation: Stem Cells. 2007 Jun;25(6):1511-20
references.pmid: 17332508
references.citation: J Neurosci. 2005 Nov 2;25(44):10064-73
references.pmid: 16267213
references.citation: Brain Pathol. 2006 Apr;16(2):132-42
references.pmid: 16768754
references.citation: Proc Natl Acad Sci U S A. 2003 May 13;100(10):5828-33
references.pmid: 12724518