*2.1. Ech A Suppresses ROS Production and Promotes Expansion of PBMC-Derived CD34*<sup>+</sup> *Cells*

HSPCs reside in a hypoxic niche in the bone marrow, suggesting that HSPCs need to adopt unique metabolic properties, including intracellular ROS levels. We found that PB-derived CD34<sup>+</sup> cells (PB-CD34<sup>+</sup> cells) exhibit higher ROS levels than BM-derived CD34<sup>+</sup> cells (Figure 1A). We then examined whether Ech A can modulate ROS levels to cause an ex vivo expansion of PB-CD34<sup>+</sup> cells. To determine the optimal concentration of Ech A, G-CSF-mobilized PB mononuclear cells (PBMCs) were treated with different concentrations of Ech A (0, 1, 10, 20, 50, and 100 μM) for 24 h, and the CD34<sup>+</sup> cell number was analyzed by flow cytometry. Cells treated with 10 μM Ech A for 24 h showed approximately two-fold higher CD34<sup>+</sup> cell number than those in the control group (Figure S1A); therefore, we chose that condition for subsequent experiments. The effect of Ech A on the ex vivo expansion of PBMCs containing CD34<sup>+</sup> or purified PB-CD34<sup>+</sup> was investigated after 1 day or 4 days of culture, respectively (Figure 1B). The toxic reagent N-acetyl cysteine (NAC)—a well-known potent antioxidant—was used as the positive control. Immunophenotypic analysis showed a significantly higher percentage of CD34<sup>+</sup> cells and the CD34+ cell number in Ech A-treated group (PBMCs, 12.91% ± 3.22%, 2.05 ± 0.66-fold; PB-CD34<sup>+</sup> cells, 73.37% <sup>±</sup> 1.11%, 4.95 <sup>±</sup> 0.28-fold) than that in the control group (PBMCs, 7.07 <sup>±</sup> 0.66%, 0.87 <sup>±</sup> 0.08-fold; PB-CD34<sup>+</sup> cells, 67.27 <sup>±</sup> 1.79%, 3.71 <sup>±</sup> 0.18-fold). As shown in Figure 1C, Ech A dramatically suppressed intracellular ROS production in PBMCs and PB-CD34<sup>+</sup> cells. Additionally, H2O2 treatment showed dramatic suppression of PB-CD34<sup>+</sup> cell expansion that was recovered by Ech A treatment (Figure S2). These results suggest that Ech A promotes the ex vivo expansion of PBMC-derived CD34<sup>+</sup> cells by suppressing ROS levels.
