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. 2014 Sep 22;211(10):1925–1935. doi: 10.1084/jem.20140131

Figure 2.

Figure 2.

Cotransplantation of healthy HSPCs with AML MNCs can competitively reduce leukemic progression. (A) Experimental design to study competitive niche repopulation between HSPCs enriched from pairs of healthy human CB donors. (B) Gating strategy and representative flow cytometry plots showing relative donor cell proportions within human populations before and after co-injection into xenograft recipients. Top left plot shows that CD2+ T cells were absent from all injected Lin− populations, precluding any immune reactions within the recipient mice. (C) Scatter plot of flow cytometry data showing that in mouse BM grafts, the relative donor CD45+ cell frequencies closely recapitulated their original proportions within injected CD34+ populations, consistently across four independent experiments using different CB donor pairs. Dotted lines represent 95% confidence intervals. Each data point represents an individual transplanted mouse. ***, P < 0.0001, linear regression. (D) Experimental design to study the effects of BM niche competition between healthy and leukemic human repopulating cells. AML cell numbers were kept constant with increasing dose titrations of CB Lin− cells. (E) Example gating strategy to assess the relative reconstitution of primary AML samples in the presence of competing CB-HSPCs, expressed as a proportion of the total human graft. (F) Flow cytometry data showing that HSPC competition reduces the leukemic contribution to human grafts in a cell dose-dependent manner for all AML samples tested (three independent experiments). Cotransplantation of CB Pair #1 is also shown for comparison. Shaded areas represent 95% confidence intervals. Each data point represents an individual mouse. *, P < 0.05; **, P < 0.01; ***, P < 0.001, linear regression. (G) Example gating strategy to assess the absolute BM reconstitution levels of AML samples in the presence of CB-HSPC competition. (H) Flow cytometry data showing that absolute leukemic reconstitution is reduced by HSPC competition in a cell dose-dependent manner. Shaded areas represent 95% confidence intervals. Each data point represents an individual mouse. *, P < 0.05, linear regression. (I) Flow cytometry data showing that relative to control mice transplanted with AML cells alone, the total leukemic reconstitution of AML Patient #1 is reduced in the presence of competing HSPCs at all cell doses tested (single transplant n = 2, cotransplant n = 8). ***, P < 0.0005, unpaired Student’s t test. (J) Flow cytometry data showing that absolute CB reconstitution levels are also influenced dose-dependently by competing CB donor cells only if human chimerism levels are high. Each data point represents an individual mouse. *, P < 0.05, linear regression. (K) Experimental design to study the effects of BM niche competition on the self-renewal ability of AML L-ICs by serial transplantation. (L) Flow cytometry data showing that in the presence of CB competition, leukemic chimerism after serial transplantation is either lower than or equal to leukemic reconstitution in primary recipients. Leukemic engraftment in secondary recipients was normalized to that of primary recipients (dotted line). Relative engraftment of control AML cells transplanted in the absence of CB is shown in gray. Each data point represents an individual mouse. (M) Representative flow cytometry plots gated on human CD45 show robust normal human reconstitution subsequent to therapeutic irradiation-conditioned CB Lin− transplantation of AML-engrafted mice (top), followed by AML recurrence after serial transplantation (bottom). Plots are representative of 6 mice that received irradiation-conditioned HSPC transplantation 3 wk after disease initiation, all of which had persistent leukemia after treatment.