Regulating dopaminergic development of mouse embryonic stem cells using small molecules

The degeneration of substantia nigral dopaminergic (mDA) neurons is responsible for the resting tremors and difficulties in fine motor control seen in Parkinson’s disease. Embryonic stem cell (ESC)-derived mDA neurons provide an ideal opportunity to model mDA development in vitro. However, current mouse differentiation protocols are inefficient possibly as a result of the influence of endogenous signalling pathways. In theory, the ability to identify cell types that have undergone specification of neural lineage should make it easier to identify upstream regulators of that lineage. In this study dopaminergic progenitor specific reporter cell lines expressing -lactamase or luciferase under the control of the Lmx1a promoter were used to investigate protein kinase signalling pathways involved in dopaminergic neural specification. Screening experiments using a small kinase inhibitor library revealed that inhibition of Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF) and DNA-dependent protein kinase (DNA-PK) signalling pathways promoted Lmx1a expression using Lmx1a reporter mESC lines. Moreover, additional experiments revealed that DNA-PKi-mediated increases in Lmx1a+ progenitors upregulated Notch signalling suggesting that DNA-PK signalling may alter neurogenic potential of developing progenitors. Co-culture differentiation revealed that DNA-PKi alone positively regulated Lmx1a expression. However, as the generation of Nurr1+ post-mitotic dopaminergic progenitors could not be enhanced using small molecules identified from early screens suggests that these compounds may exclusively regulate earlier specification pathways such as Lmx1a. Intriguingly, only progenitors with compromised EGF signalling were able promote generation of dopaminergic (TH+) neurons. Lastly, inhibition of endogenous Smad signalling pathways sped up neural induction and promoted lineage specification within mESCs, albeit within a narrow time frame in a monolayer. Specific inhibition of the Smad1/5/8 pathway induced a floor plate (FP) transcription factor profile with an upregulation of Corin, Shh and FoxA2 genes and increased the number of TH+ neurons. The FP molecular signature was further enhanced by the addition of small molecules CHIR and SAG that promote Wnt and Shh signalling pathways, respectively. Although these studies demonstrated that midbrain and FP specification could be regulated in mESCs using inhibitors for DNA-PK and Smad signalling pathways, co-incubation with DNA-PK and Smad inhibitors did not support ventral mDA specification. This may be due to the immature or neurogenic state generated by DNA-PK inhibition to support neural specification driven by inhibition of Smad pathways. These studies, demonstrate that DNA-PK signalling has a, previously unrecognised, role in regulating neural specification and that endogenous Smad signalling can rapidly specify neuroectoderm and regional subtype specification in a monolayer. These studies provide insight into the complex regulation of endogenous signalling networks during neural differentiation of mESCs and how they may be regulated using targeted small molecules.