Fig. 1: Generation of iPSC-derived MMUT-deficient neurons from individuals with methylmalonic aciduria.
From: Mitochondrial dysfunction drives a neuronal exhaustion phenotype in methylmalonic aciduria
a Schematic of the affected metabolic pathway in MMUT deficiency. Created in BioRender. b Brightfield and epifluorescent acquisitions of pluripotency (NANOG, SSEA4, SOX2) and proliferation (Ki67) markers in representative iPSC cultures from control and patient lines. Scale, 50 μm. c Ectoderm lineage indicated by beta-tubulin III (TUJ1) and nestin (NES) positive cells after 12 days in vitro (DIV). Endoderm lineage indicated by anti-α-Fetoprotein (AFP) positive cells after 4 days in vitro (DIV). Mesoderm lineage indicated by alpha-smooth muscle actin (SMA) and brachyury positive cells after 4 DIV. Scale, 10 μm. d Comparison of the anti-MMUT (green) staining pattern from representative patient fibroblast-derived iPSCs compared to controls. Representative images come from cell line Ct1.2 and Pt1.1 For reference, anti-TOMM20, a mitochondrial protein, is also shown (red). Scale, 50 μm. e Western blotting analysis of derived iPSCs using anti-MMUT. MMUT is anticipated at 83 kDa. The loading control ACTB is anticipated at 42 kDa. Uncropped membranes are available in Supplementary Fig. 12. f Propionate incorporation of two Ct1.1 and Ct1.2 wildtype iPSC sub-clones and Pt1 and Pt2 patient cell lines. Data points represent independent measurements taken from 3 separate cultures per cell line. Mean control iPSC incorporation values were 22.71 and 24.36 pmol/mg.protein/16 h. Patient iPSC incorporation values were 0.77 pmol/mg.protein/16 h in Pt1 and 0.44 pmol/mg.protein/16 h in Pt2. P-value was tested using Mann–Whitney U-test (**, < 0.01). Error is presented as standard deviation.
