Protocol for HSC mobilization in mice

The mouse model of HSC mobilization was designed based on protocol used in human patients (Fig. 4a–b). A previously reported model of cyclophosphamide chemo-mobilization was used in this study [19]. Due to the apparent lack of a related animal model, we developed a new model of etoposide chemo-mobilization. Mice were injected intraperitoneally with 4 mg cyclophosphamide (≈ 200 mg/kg) on day 1 (D1) or with 0.8 mg etoposide (≈ 40 mg/kg) on days 1 and 2 (D1, D2). Subsequently, 5 μg human G-CSF (250 μg/kg per day; Leucostim prefilled syringe INJ, Dong-A Inc.) was administered daily as a single subcutaneous injection, on each successive day from day 3, for a total of 4 days. All mice were euthanized on D7 by cardiac puncture and cervical dislocation under anesthesia. On day 7 (D7), we isolated hematopoietic progenitor cells (HPCs) using an EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit and performed colony-forming unit (CFU) assays using MethoCult™ GF M3434 medium (both from Stem Cell Technologies, Vancouver, BC, Canada), according to the manufacturer’s instructions.

Mouse model of peripheral blood hematopoietic stem cell mobilization. a, b, c The mouse model of hematopoietic stem cell (HSC) mobilization was designed based on a protocol used in human patients (G-CSF only, n = 8; cyclophosphamide + G-CSF, n = 8; etoposide + G-CSF, n = 8). d On day 7 (D7) of the protocol, HPCs were isolated from the peripheral blood and CFUs (CFU-granulocytes, erythrocytes, monocytes, and megakaryocytes; CFU-granulocytes, macrophages; and burst-forming unit-erythroid) were counted. The presented pictures were obtained in the control group (G-CSF only). e The cyclophosphamide-treated (total 200 mg/kg) and etoposide-treated (total 80 mg/kg) groups showed a higher number of CFUs than the G-CSF only group (p = 0.021 and 0.003 after Bonferroni correction, respectively). No significant difference was observed in the total number of CFUs between the cyclophosphamide-treated (200 mg/kg) and etoposide-treated (80 mg/kg) groups (G-CSF only, n = 5; cyclophosphamide + G-CSF, n = 5; etoposide + G-CSF, n = 5). Note: ** p < 0.01 after Bonferroni correction; * p < 0.05 after Bonferroni correction. Note: Values are reported as the mean ± SEM. Abbreviations: S.C., subcutaneous injection; I.P., intraperitoneal injection; NS, normal saline; G-CSF, granulocyte colony-stimulating factor; CY, cyclophosphamide; ETO, etoposide; CFU, colony-forming unit; GEMM, granulocytes, erythrocytes, monocytes, and megakaryocytes; GM, granulocytes, macrophages; BFU-E, burst forming unit-erythroid; n.s., not significant