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

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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.