Figure 3. Stem and progenitor cell memory of leukemic exposure.

(A) CD45.1⁺ KSLs from either control or leukemic mice were sorted and placed in OP9 co-culture assay. Flow cytometry analysis of CD19 and Mac1 these cultures after 14 days is shown, n=4.

(B) Quantification of B cells (CD19⁺) and myeloid lineage cells (Mac1⁺) from OP9 co-cultures were plotted from CD45.1⁺ KSLs from either control or leukemic-exposed and CD45.2⁺ leukemic KSL 14 days after initiation, n=4. Error bars represent ± SEM.

(C) Cartoon showing control or leukemic-exposed CD45.1⁺ KSLs or HSCs (Lin⁻ ckit^Hi Sca1⁺ CD150⁺ CD48⁻) were sorted from the primary chimeras and transplanted into sublethally irradiated C57BL/6 (CD45.2) hosts. KSLs transplanted mice were analyzed at 10 weeks post transplant, while HSC transplanted mice were analyzed after 16⁺ weeks post transplant.

(D) CD45.2⁺ leukemic HSCs and CD45.1⁺ HSCs were sorted from control and/or leukemic mice and then labeled with CFSE (5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester). These cells were then transplanted into lethally irradiated mice for 12 hours at which time the femurs were analyzed for the presence of CFSE and CD45.1⁺ cells. Plots shows homing efficiency relative to the control HSCs, n=5. Error bars represent ± SD.

(E) Limiting dilution CRU (competitive repopulation unit) assay is shown. The indicated numbers of control or leukemic-exposed HSCs were transplanted along with 2×10⁵ BM of a competitor (CD45.2⁺) into lethally irradiated hosts (CD45.2⁺). Reconstitution was evaluated in the blood at 16 weeks post-transplantation (p value = 0.0001). Mice with CD45.1⁺ chimerism < 0.3% were considered as non-responders.

(F) For serial transplantation, 2×10⁶ sorted CD45.1⁺ bone marrow cells from either control and appropriately adjusted per HSC numbers of CD45.1⁺ cells from leukemic mice were transplanted into lethally irradiated CD45.2 hosts. Rounds of transplantations with performed at 16-week intervals. The proportion of peripheral blood generated from the CD45.1⁺ cells in recipient mice was determined for each round of transplantation. Bar graphs indicate chimerism after each round of transplantation, n = 4. Error bars represent ± SD.

(G) Peripheral blood samples from either 10 weeks post-transplant for CD45.1⁺ KSLs or 16 weeks post-transplant for CD45.1⁺ HSCs transplants, respectively, were stained with CD45.1, CD45.2, and DAPI for viability. The bars show percentages of the donor type cells (CD45.1) that expressed myeloid (Gr-1 and CD11b, filled), B (B220, striped), or T (CD4/8, open) lineage markers in the blood of individual recipients.