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.
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.
Cooper
Oliver
O
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
Hargus
Gunnar
G
Deleidi
Michela
M
Blak
Alexandra
A
Osborn
Teresia
T
Marlow
Elizabeth
E
Lee
Kristen
K
Levy
Adam
A
Perez-Torres
Eduardo
E
Yow
Alyssa
A
Isacson
Ole
O
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
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