Fig. 1: Mitochondrial augmentation occurs in human HSPCs and is a dose-dependent process.

a Representative confocal microscopy images of human CD34⁺ stained with MitoTracker Orange and subsequently augmented with GFP-labeled mitochondria isolated from HeLa cells. Images were taken at 30 min, 8 h, and 24 h post augmentation; scale bar, 5 µm. Mitochondrial dose used was 4.4 mU CS activity per million cells. b–d Healthy donor CD34⁺ cells were augmented for 21 h with increasing doses of placenta-derived mitochondria. b Illustration of method of assessing percent of exogenous mtDNA content using NGS. Single nucleotide variants which differentiate between donor and recipient cell mtDNA are identified, and percent representation of donor mtDNA variants ((placenta-derived mitochondria batch 1 (PLC1) or 2 (PLC2)) in augmented cells are calculated. The average percent defined by all variants identified is reported as the percent exogenous mtDNA. c Exogenous mtDNA content quantified by NGS (average values ± SEM following augmentation with 0.88 mU CS (n = 4), 4.4 mU CS (*p = 0.0402 vs. NT cells, n = 4) or 8.8 mU CS (**p = 0.0020 vs. NT cells, n = 3) are presented (p values calculated by Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison test, p = 0.0001 for the difference between three experimental groups (K–W stat = 13)). d COX-1 levels were determined using ELISA (0.88 mU CS, n = 3; 4.4 mU CS, n = 3, *p = 0.0199 vs. NT cells, calculated by Kruskal–Wallis ANOVA followed by Dunn’s multiple comparison test. p = 0.0071 for the difference between three experimental groups (K–W stat = 6.73). e The ability of blood (BLD) or placenta (PLC) derived mitochondria to utilize tryptamine was assessed using home-made tryptamine coated plates (n = 3 per BLD or PLC-derived mitochondria). f The ability of augmented or non-augmented healthy donor HPSCs to utilize tryptamine as a mitochondrial substrate was assessed using MitoPlate assay in HSPCs which were either non-augmented (n = 3), augmented with blood-derived mitochondria (HSPCs + BLD mito at 0.88 or 4.4 mU CS, n = 3), or augmented with placenta-derived mitochondria (HSPCs + PLC mito at 4.4 mU CS, n = 3). g Complex IV-dependent respiration in HSPCs augmented with placenta-derived mitochondria, 21 h post augmentation. Data presented as mean ± SEM. n = 3, p = 0.052 for 4.4 mU CS. h Healthy donor’s CD34⁺ cells were augmented with placenta-derived mitochondria. Ability to form colonies was tested 14 days (±2) post augmentation. n = 3 placental mitochondria batches. BLD blood, CS citrate synthase, HSPCs hematopoietic stem and progenitor cells, NGS next-generation sequencing, NT non-treated, OCR oxygen consumption rate, PLC placenta, PLC1 placenta-derived mitochondria batch 1, PLC2 placenta-derived mitochondria batch 2.