Fig. 5: LSCs in the spleen are dependent on macrophages and located in the red pulp.

A 3D confocal laser scanning microscopy of the spleen 10 days after CML induction. Representative images from 3 independent experiments with n = 6 mice are shown. B Experimental model. BL/6 CML mice were treated with clodronate liposomes (1 mg) or vehicle (PBS) every 5th day by intraperitoneal injection starting 3 days prior to CML induction. Spleen and BM were analyzed 18 days after CML induction. C Frequency of RPMs after clodronate treatment. D–H Spleen weight, numbers and frequencies of LSCs, LT-, ST-LSCs and L-lin⁻ cells. Pooled data from 2 to 3 independent experiments with n = 5–13 mice are shown. I Incorporation of BrdU in LSCs of the BM and spleen of clodronate or PBS treated CML mice (n = 6–8 mice). J In total, 1.5 × 10³ FACS-purified LSCs were co-incubated with RPMs for 48 h and subsequently plated into methylcellulose. Numbers of colonies were counted after 7 days. Pooled data of two independent experiments with n = 5–6 samples per group are shown. K For 1st platings, 1.5 × 10³ FACS-purified LSCs were co-incubated overnight with 1 × 10⁴ FACS-purified RPMs in the presence or absence of imatinib and plated into methylcellulose. Colonies were counted after 7 days. For 2nd platings, colonies were washed and 1 × 10⁴ cells were plated into methylcellulose. Colonies were counted after 7 days. One representative experiment with n = 3 biological replicates is shown. Data are displayed as mean ± SEM. Statistics: Unpaired student’s t test.