Patterning effects of FGF17 and cAMP on generation of dopaminergic progenitors for cell replacement therapy in Parkinson’s disease. [scRNA-Seq]

Cell replacement therapies using human pluripotent stem cell-derived dopaminergic (DA) progenitors are currently in clinical trials for treatment of Parkinson’s disease (PD). Recapitulating developmental patterning cues, such as fibroblast growth factor 8 (FGF8), secreted at the midbrain-hindbrain boundary (MHB), is critical for the in-vitro production of authentic midbrain DA progenitors. Here, we explore the application of alternative MHB-secreted FGF-family members, FGF17 and FGF18, for DA progenitor patterning. We show that while FGF17 and FGF18 both recapitulate DA progenitor patterning events, FGF17 induced expression of key DA progenitor markers at higher levels than FGF8 and transplanted FGF17-patterned progenitors fully reversed motor deficits in a rat PD model. Early activation of the cAMP pathway mimicked FGF17-induced patterning, although strong cAMP activation also came at the expense of EN1 expression. In summary, we identified FGF17 as a promising candidate for more precise and robust DA progenitor patterning, with potential to improve cell products for treatment of PD. To compare the ability of different MHB-associated FGFs from the FGF8 subfamily to fine-tune hPSC-derived ventral midbrain (VM) DA progenitors, we added either FGF8 or FGF17 from day 9 to 16 for the production of DA progenitors. On day 9 the cells were treated either with FGF8 or FGF17 for 15 min, 1 h, 4 h, and 24 h (n=3). We performed bulk RNA sequencing for these treatments to investigate immediate differences in FGF response pathways. On differentiation day 16 we performed scRNA-seq on hashtag-labeled (hto) DA progenitors patterned with either FGF8 or FGF17 (n=4).