Midbrain dopaminergic neuron fate specification of pluripotent stem cells

Abstract

Disorders of dopaminergic neurons in the midbrain are associated with various brain diseases such as Parkinson’s disease (PD), for which there are no effective treatments. Pluripotent stem cells (PSCs) offer a remarkable potential for finding new therapeutic strategies because they are self-renewable and capable to differentiate into all cell types. However, biomedical applications of PSCs require knowledge of the molecular mechanisms directing PSCs into midbrain dopaminergic (mDA) cell fate. During development the generation of mDA neurons is induced by the extrinsic molecules SHH, FGF8 and WNT1 in the caudal region of the Otx2 positive domain, which marks the presumptive fore- and midbrain, and rostrally to the future hindbrain expressing Gbx2. In this study I have further investigated the role of FGF signalling in the generation of mDA neurons. Here, I have used mouse epiblast stem cells (EpiSCs). Based on their developmentally primed pluripotent state, we assumed that they represent a more suitable system when compared with mouse embryonic stem cells. I validated neural differentiation of EpiSCs as an alternative model to study neural development in vitro. I found that inhibition of the FGF/ERK activity at the onset of EpiSC differentiation initiated expression of Wnt and Shh and further induced ventral midbrain progenitor markers, accompanied by suppressing caudalisation as well as forebrain induction. To maintain ventral midbrain progenitor fate, cells required a period of endogeneous FGF/ERK signalling. Subsequent treatment of FGF8 and SHH, which restricts progenitors from adopting alternative fates, led to highly efficient production of authentic mDA neurons. These neurons exhibited functional, neuron-like properties and when implanted into the striatum of mouse PD model strongly restored parkinsonian features without any signs of overgrowth. In conclusion, a temporally controlled modulation of FGF/ERK activity during neural differentiation from PSCs is crucial for reliable and highly efficient generation of functional authentic mDA neurons.