*2.6. Ex Vivo Expanded CD34*<sup>+</sup> *Cells Maintain Their Colony-Forming Capacity*

As CFU indicates the presence of HSPCs, the colony forming potential (colony-forming unit/burst-forming unit-erythroid (CFU/BFU-E), CFU-granulocyte/macrophage (CFU-GM), and CFU-granulocyte/erythroid/macrophage/megakaryocytes (CFU-GEMM), and total CFU) of freshly isolated CD34<sup>+</sup> cells or CD34<sup>+</sup> cells, expanded in the absence or presence of Ech A (10 μM) or NAC (5 mM), was investigated (Figure 6). The frequency of CFU/BFU-E (Ech A, 173.3 ± 3.51; NAC, 181.7 ± 0.58) and total CFU (Ech A, 248.0 ± 5; NAC, 262.7 ± 1.53) in the 10 μM Ech A or 5 mM NAC group was significantly higher than that in the unexpanded (CFU/BFU-E, 91.3 ± 6.11; total CFU, 145.7 ± 6.03) or control expanded group (CFU/BFU-E, 99.3 ± 3.06; total CFU, 157.7 ± 3.06). The frequency of CFU-GM (Ech A, 73.33 ± 2.08; NAC, 80 ± 2) and CFU-GEMM (Ech A, 1.33 ± 0.58; NAC, 1 ± 0) was not vastly different from the unexpanded (CFU-GM, 52.67 ± 4.04; CFU-GEMM, 1.67 ± 0.58) or control group (CFU-GM, 57.3 ± 3.06; CFU-GEMM, 1 ± 0) (Figure 6A). Although the number of CFU/BFU-E was higher in the Ech A and NAC expanded groups, the size of CFU/BFU-E was larger in the unexpanded group (Figure 6B). Therefore, the addition of 10 μM Ech A certainly improved ex vivo expansion of HSPC numbers, but the potency may have slightly decreased.

**Figure 6.** Ex vivo expanded CD34+ cells maintain their colony-forming capabilities. Purified PB-CD34<sup>+</sup> non-expanded cells, or cells expanded ex vivo in the presence or absence of NAC or Ech A for 4 days were evaluated for pluripotency using hematopoietic colony-forming assays in semi-solid methylcellulose media. (**A**) Colony-forming unit/burst-forming unit-erythroid (CFU/BFU-E), CFU-granulocyte/ macrophage (CFU-GM), and CFU-granulocyte/erythroid/macrophage/megakaryocytes (CFU-GEMM) per 500 CD34<sup>+</sup> cells plated were enumerated after 14 days of culture. (**B**) Representative images of CFU/BFU-E, CFU-GM, and CFU-GEMM for each experimental condition. Magnification: ×200.
