Fig. 1: Characterisation of an enriched population of mDA NPCs and neurons.

a Differentiation protocol to generate mDA neurons. b Quantitative PCR at day 14–20 of differentiation showing mRNA for LMX1A, FOXA2, and EN1 relative to hiPSCs (ns P > 0.05, **P < 0.005). c Representative ICC images showing expression of FOXA2, LMX1A, and OTX2 (scale bar = 50 μm). d Quantification showing >80% of cells co-express FOXA2, LMX1A and OTX2 (ns P > 0.05, ordinary one-way ANOVA). e Quantification of ICC images showing the increase in expression of the mDA marker, TH over differentiation (weeks) (ns P > 0.05, ***P < 0.0005, ****P < 0.0001, ordinary one-way ANOVA). f Representative ICC images showing TH and TUJ1 expression after 41 days of differentiation (scale bar = 50 μm). g ICC Quantification showing approximately 80% cells express TH (ns P > 0.05, ordinary one-way ANOVA). h Representative dot plots of single-cell suspensions showing % TH and β-III Tubulin +ve cells (day 41). A negative control (DAPI only) was used to determine quantification thresholds (n = 10,000 events recorded per measurement). i Quantification of flow cytometry showing >80% of DAPI-positive cells co-express TH and β-III Tubulin. j A UMAP plot showing the 14 clusters identified from single-cell RNA-seq after day 48 of differentiation. Neuronal mDA (mDA1–8) clusters are in blue, and NPC clusters (NPCs1–5) are in red, orange and yellow. An unidentified cluster (N/A) is coloured in grey. k Heatmap showing expression of genes in clusters identified as mDA neurons (mDA1–8). Each line represents a cell from that cluster. All data plotted as ±s.e.m. All N numbers for each experiment can be found in Supplementary Table 5.