Activated Notch1 expands primitive RAG-1^−/−hematopoietic cells in vivo and enhances the self-renewal of RAG-1^−/− hematopoietic stem cells in serial bone marrow transplantation.

Competitive repopulation of C57BL/6 Ly5.1 mice (n = 5 for each group), lethally irradiated and transplanted with 2000 RAG-1^−/−Sca1⁺lin⁻Ly5.2⁺bone marrow cells expressing control vector (MSCV-GFP) or activated Notch1 (MSCV-ICN/GFP) along with 1.5 × 10⁵Ly5.1⁺ competing bone marrow cells. Bone marrow cells were stained 5 months after transplantation. (A) Bone marrow cells were stained for Sca1 and lineage markers (CD3, CD4, CD8, B220, Mac1, Gr-1, and Ter119) or Sca1 and c-kit or c-kit and Thy1. Plots represent fluorescence intensity for the indicated markers on the x-axis and Sca1 or c-kit on the y-axis in the GFP⁺Ly5.2⁺ cell population of the bone marrow of 3 representative animals. Numbers in corners represent percent of events within that quadrant or gate. (B) Charts show the average percentage ± SEM of Sca1⁺lin⁻, Sca1⁺c-kit⁺, or c-kit⁺Thy1^low cells in the GFP⁺Ly5.2⁺ bone marrow population. The Student t test was used to analyze the data (Sca1⁺lin⁻, P = .002; Sca1⁺c-kit⁺, P = .01; c-kit⁺Thy1^low, P = .03). (C) Charts show the average of total Sca1⁺lin⁻ or Sca1⁺c-kit⁺ or c-kit⁺Thy1^low cells ± SEM in the GFP⁺Ly5.2⁺ bone marrow of animals receiving transplants. The Student t test was used to analyze the data. (Sca1⁺lin⁻, P = .03; Sca1⁺c-kit⁺, P = .06; c-kit⁺Thy1^low, P = .05). (D) After 1.5, 5, and 8 months the bone marrow of competitive repopulated animals was analyzed by flow cytometry for Sca1⁺lin⁻cells in GFP⁺Ly5.2⁺ bone marrow. Chart represents total number of Sca1⁺lin⁻ cells at different time points. (E) Bone marrow cells of competitive repopulated animals were pooled for each group 6 weeks after transplantation (first transplant; n = 5) containing equal numbers of Sca1⁺lin⁻GFP⁺Ly5.2⁺cells and again transplanted into lethally irradiated C57BL/6 Ly5.1 mice. The bone marrow was analyzed after a further 5 months by flow cytometry (second transplant; n = 4). Bone marrow cells were stained for Ly5.2, Sca1, and lineage markers (CD3, CD4, CD8, B220, Mac1, Gr-1, and Ter119). Total Sca1⁺lin⁻ cells, which were also GFP⁺Ly5.2⁺, were measured by flow cytometric analysis and total cell count of the bone marrow of competitive repopulated animals of the first and second transplant. Chart shows the average ± SEM of total Sca1⁺lin⁻GFP⁺Ly5.2⁺cells in the bone marrow.