Isolation and functional characterization of Rh^lo and Rh^hi cell populations.

(A) A cartoon showing 2 important populations of cells: LT-HSCs and multipotent progenitors (MPPs). The LT-HSCs can support long-term (up to 6 months) hematopoiesis in competitive repopulation assay. MPP cells can only briefly support (< 4 weeks) hematopoiesis in the same assay. Rhodamine-123 (Rh) was used to separate these 2 populations of cells. (B) Separation of Rh^loLin^−/loSca-1⁺c-kit⁺and Rh^hiLin^−/loSca-1⁺c-kit⁺cells using flow cytometry. R3: Lin^−/loSca-1⁺c-kit⁺ cells; R2: Rh^lo (15% of cells with the lowest staining); R4: Rh^hi (15% of cells with the highest staining). (C) Competitive repopulation assay. Either Rh^loLin^-/loSca-1⁺c-kit⁺or Rh^hiLin^-/loSca-1⁺c-kit⁺cells (250 cells) derived from the donor Ly5.2 mice were mixed with 4 × 10⁴ Lin^−/lo cells from congenic host strain Ly5.1,17 and injected to lethally irradiated (11 Gy [1100 rads]) Ly5.1 mice. Eight mice were used per group. Peripheral blood cells were harvested at different time points after transplantation as indicated. Flow cytometric analyses were performed using different specific cell surface markers (CD3, B220, Mac-1, and Gr-1) together with anti-Ly5.2 antibody. ○ indicates Rh^lo lymphoid lineage; ▵, Rh^lo myeloid lineage; ●, Rh^hi lymphoid lineage; ▴, Rh^hi myeloid lineage.