Loss of a striatal targets negatively impacts the size of hiPSC-derived VM progenitor grafts and the survival of DA neurons. Schematic sagittal view of the developing embryo illustrating the restrictive expression of PITX3, a key VM DA transcription factor (A). Monoalleleic targeting of enhanced GFP (eGFP) expression, driven under the PITX3 promotor region, used for the generation of a new PITX3-GFP iPSC cell line that enabled identification of VM DA neurons in vitro and in vivo (B). Specifically designed primers to amplify targeted PITX3 locus (C). Quantitative assessment of the proportion of cells expressing early (FOXA2, OTX2), intermediate (NURR1), and mature protein (TH, PITX3-GFP), reflective of VM progenitors and VM DA precursors and neurons (D). Quantitative PCR showing early (1), intermediate (2), and late (3) VM DA gene expression, confirming efficiency of the PITX3-GFP IPSC line to differentiate (E). Photomicrographs illustrating high expression of OTX2, FOXA2, and Nestin (F) as well as low proportions of off-target genes PITX2 and BARHL1 (G) confirm correct VM regional specification at day 13 of differentiation. By day 25, cultures show extensive GFP, TH, and FOXA2 co-expression (H) as well as expression of the DA precursor gene, NURR1 (I). Representative images show the placement of hiPSC-derived DA grafts (GFP⁺) within the 6OHDA (J) or 6OHDA+QA (K) lesioned striatum. After transplantation, the co-expression of TH with GFP confirms the robustness of the GFP⁺ reporter as a measure of VM DA neurons (L). GFP⁺ immunostaining reveals the impact of QA lesioning (N) on intrastriatal grafts of hiPSC-derived VM progenitors, compared with 6OHDA controls (M). High-magnification images from M, N illustrating A9-like (red arrow) and A10-like (blue arrow) DA neuron morphology within hiPSC-derived grafts (M', N'). Quantitative analysis reveals that ablation of striatal MSN results in significantly smaller grafts (O, p = 0.022), containing fewer GFP⁺ cells (P, p = 0.042) and consequently unchanged GFP⁺ cell density (Q, p = 0.494) in QA+6OHDA-lesioned compared with 6OHDA-lesioned animals. Data are mean ± SEM; *p < 0.05. n = 3 independent differentiations/time point (D, Ei–Eiii), n = 7–8/group (O–Q). Scale bars: 100 µm (F–I), 1 mm (J, K, M, N), and 50 µm (L, M', N').