Fig 2: Injection of ALL EVs in vivo affects maintenance of murine HSC.A NSG mice were injected with fluorescent EVs (ATTO488 EVs) by i.v. injections at 1010 particles/mouse. Mice were sacrificed 24 h after the injection, and florescence was determined by flow cytometry on Lin−, LSK (c-Kit+ Sca1+) and SLAM (LSK CD150+ CD48−) cells. B Procedure followed to treat NSG mice with ALL EVs or control EVs, through three consecutive i.v. injections, at 1010 particles/mouse/every 10 days. Ten days after the third injection, mice were sacrificed to perform experiments on Sca1+ cells. C The absolute number of Sca1+ cells detected in BM. Data are shown as means ± SD; n = 4 mice. D The number of hematopoietic progenitors (CD45+ Sca1+ c-Kit+ cells) and HSC (SLAM; LSK CD150+ CD48− cells) were determined by flow cytometry. Data are shown as means ± SD; n = 4 mice. E Sca1+ cells (105 cells) isolated from mice were analyzed on semi-solid methylcellulose media for growth of murine hematopoietic CFU. Data show the percentage of total CFU and CFU-GM per Sca1+ cells, as well as the distribution among colonies. Data are shown as means ± SD; n = 4 mice. F Sca1+ cells (3 × 105 cells) were injected in C57BL/6.SJL (Ly.1) mice and the hematopoietic reconstitution was examined by flow cytometry, 4 weeks after the transplantation. Absolute number of CD45.2 total and Lin− BM cells. Data are shown as means ± SD; n = 3 transplanted mice. G Hematopoietic reconstitution for progenitors (LSK cells) and HSC (SLAM cells). Absolute number of CD45.2 cells in BM. Data are shown as means ± SD; n = 3 transplanted mice. On this figure, P value is measured by one-way Anova with Tukey’s multiple comparison test; **P < 0.01; ***P < 0.001; ns, non-significant.

Fig 3: Exhaustion of HSPC but not MSC in ALL PDX mice.Mice were injected with 5 × 105 cells (T-ALL) or 105 cells (B-ALL) and murine Sca1+ cells were recovered when mice were developing ALL disease (day 35). A The number of Sca1+ cells recovered from the BM of control NSG mice and PDX mice developing T-ALL or B-ALL. Data are shown as means ± SD; n = 5 mice. B Analysis of Sca1+ cells by flow cytometry showing loss of hematopoietic progenitors (CD45+ c-Kit+) and HSC (SLAM; CD150+ CD48−) in T-ALL and B-ALL PDX mice. Data are shown as means ± SD; n = 5 mice. C 25% of the Sca1+ cells were cultured in methylcellulose media and hematopoietic CFU were observed at day 7; scale bar represents 5 mm. Data are shown as means ± SD; n = 3 mice. D 75% of the Sca1+ cells were cultured in media for growth of mesenchymal CFU for 10 days, scale bar represents 1 cm. Data are shown as means ± SD; n = 3 mice. On this figure, P value is measured by one-way Anova with Tukey’s multiple comparison test; ***P < 0.001; ****P < 0.0001; ns non-significant.