Next, serial transplantation experiments of CD9+
Amyloid beta-Protein (1-15) were performed for further confirmation of their in vivo stem cell properties ( Fig. 2B). Following the first transplantation, enlarged spleens (YAMN90 and Reh) or bone marrows (ARH77) were removed from the euthanized mice and minced to obtain tumor cells, which were then cultured for three additional days to reduce the contamination of the recipient mouse cells and sorted again for a second transplantation. FACS analysis of the leukemic cells revealed that CD9+ cells generated both CD9+ and CD9? cells in each cell line ( Fig. 2B, left panels). Following the same procedure, the leukemic
cells from the second recipient mice were cultured and analyzed (middle panels). Then the CD9+ cells were subsequently transplanted into the third recipient mice, resulting in the reconstitution of the same expression pattern of CD9 in each cell line (right panels). The results of our serial transplantation experiments are summarized in Table 1. Thus, the serial transplantation capacity and higher tumorigenic potential, essential characteristics for CSCs, were demonstrated in the CD9+ population of each cell line.
