title: Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.
abstract: The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/β-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.
authors.affiliation: Neuroregeneration Laboratories, Harvard Medical School/McLean Hospital, NINDS Udall Parkinson's Disease Research Center of Excellence, 115 Mill Street, Belmont, MA 02478, USA. ocooper@mclean.harvard.edu
authors.forename: Oliver
authors.initials: O
authors.lastname: Cooper
authors.forename: Gunnar
authors.initials: G
authors.lastname: Hargus
authors.forename: Michela
authors.initials: M
authors.lastname: Deleidi
authors.forename: Alexandra
authors.initials: A
authors.lastname: Blak
authors.forename: Teresia
authors.initials: T
authors.lastname: Osborn
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Marlow
authors.forename: Kristen
authors.initials: K
authors.lastname: Lee
authors.forename: Adam
authors.initials: A
authors.lastname: Levy
authors.forename: Eduardo
authors.initials: E
authors.lastname: Perez-Torres
authors.forename: Alyssa
authors.initials: A
authors.lastname: Yow
authors.forename: Ole
authors.initials: O
authors.lastname: Isacson
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002478:Cells, Cultured; D004298:Dopamine / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D051524:Fibroblast Growth Factor 8 / Q000494:pharmacology*; D053823:Hedgehog Proteins / Q000235:genetics / Q000378:metabolism*; D051542:Hepatocyte Nuclear Factor 3-beta / Q000378:metabolism; D006801:Humans; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010300:Parkinson Disease; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D014212:Tretinoin / Q000494:pharmacology*; D051155:Wnt1 Protein / Q000494:pharmacology
chemical_list: C496456:FGF8 protein, human; C496502:FOXA2 protein, human; D053823:Hedgehog Proteins; C509239:SHH protein, human; C495247:WNT1 protein, human; D051155:Wnt1 Protein; D051542:Hepatocyte Nuclear Factor 3-beta; D051524:Fibroblast Growth Factor 8; D014212:Tretinoin; D004298:Dopamine