by Suzanne M. Vercauteren, and Heather J. Sutherland Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo by Suzanne M. Vercauteren, and Heather J. Sutherland Blood Volume 104(8):2315-2322 October 15, 2004 ©2004 by American Society of Hematology

Relative Notch1, Notch2, Notch3, and Notch4 expression in human hematopoietic marrow progenitor subsets as compared with unfractionated marrow (uF) as measured by real-time PCR. Normal marrow cells were suspended in Hanks solution with 5% human serum at 5 t... Relative Notch1, Notch2, Notch3, and Notch4 expression in human hematopoietic marrow progenitor subsets as compared with unfractionated marrow (uF) as measured by real-time PCR. Normal marrow cells were suspended in Hanks solution with 5% human serum at 5 to 10 × 106/mL and incubated for 30 minutes with CD34–fluorescein isothiocyanate (CD34-FITC) (gift from Dr P. Lansdorp) used at 4 μg/mL and CD38-PE (Becton Dickinson) used according to the manufacturer's instructions. Separate aliquots were stained with an irrelevant mouse immunoglobulin G1–FITC (IgG1-FITC) and IgG1-PE antibody (both from Becton Dickinson) as an isotype control. Viable CD34+CD38–, CD34+CD38+, and CD34– cells were sorted and used for RNA extraction. The cDNA of unsorted marrow and of marrow sorted for CD34 and CD38 was used for real-time PCR with Notch primers and SybrGreen. Notch expression in the sorted subpopulations was compared with its unfractionated population (uF) to allow comparisons and relative expression of Notch1 through Notch4 in the progenitors expressed. Error bars indicate the standard error of the mean. The expression of the sorted population was compared with the unsorted population in a paired t test. *P ≤ .05. Notch1 is indicated by ▪; Notch2, by □; Notch3, by ▧; and Notch4, by ▦. Suzanne M. Vercauteren, and Heather J. Sutherland Blood 2004;104:2315-2322 ©2004 by American Society of Hematology

Decreased colony-forming ability of Notch1-IC– and Notch4-IC–transduced normal marrow (BM) and cord cells compared with MIG-transduced cord blood cells. Decreased colony-forming ability of Notch1-IC– and Notch4-IC–transduced normal marrow (BM) and cord cells compared with MIG-transduced cord blood cells. Normal marrow cells (panel A) (Notch1-IC, n = 3; Notch4-IC, n = 4) and cord blood (panel B) (Notch1-IC, n = 4; Notch4-IC, n = 5) were retrovirally transduced with MIG, Notch1-IC, and Notch4-IC and sorted for GFP. Cultures were initiated with 1000 GFP+ normal marrow cells (panel A) or 500 cord blood cells (panel B) placed into methylcellulose in the presence of IL-3, IL-6, G-CSF, GM-CSF, and SF, and colonies were counted 10 days after initiation. A paired t test was performed to determine statistical differences between colony numbers in Notch1-IC or Notch4-IC and MIG. Error bars indicate the standard error of the mean. *P ≤ .05. Erythroid colonies are indicated by ▪; granulocyte and/or macrophage colonies, by ▦; and total colonies, by ▧. Suzanne M. Vercauteren, and Heather J. Sutherland Blood 2004;104:2315-2322 ©2004 by American Society of Hematology

Increased engraftment of Notch4-IC–transduced cord cells compared with MIG-transduced cord cells in β2-microglobulin–/– NOD/SCID mice. Increased engraftment of Notch4-IC–transduced cord cells compared with MIG-transduced cord cells in β2-microglobulin–/– NOD/SCID mice. Lineage-depleted cord cells were retrovirally transduced with MIG and Notch4-IC (n = 4) and injected into sublethally irradiated β2-microglobulin–/– NOD/SCID mice. Every 3 weeks, bone marrow aspirations were performed, and cells were stained with CD45 and GFP and analyzed by FACS to determine human engraftment. A paired t test was used to determine differences in engraftment. Error bars indicate the standard error of the mean. *P ≤ .05. CD45+GFP+ cells are indicated by ▪; CD45+GFP– cells, by ▦; total CD45+ cells, by ▧. Suzanne M. Vercauteren, and Heather J. Sutherland Blood 2004;104:2315-2322 ©2004 by American Society of Hematology

Representative FACS profile of Notch4-IC–transduced cord cells. Representative FACS profile of Notch4-IC–transduced cord cells. FACS profile demonstrates that Notch4-IC–transduced cord cells give rise to a CD4+CD8+ population in the bone marrow of β2-microglobulin NOD/SCID mice. Marrow from week-9 and week-12 β2-microglobulin NOD/SCID mice receiving transplants of MIG- and Notch4-IC (n = 4)–transduced cord blood cells was analyzed for phenotype by FACS. Murine marrow cells were stained with CD4-PE and CD8–allophycocyanin (CD8-APC) along with propidium iodide (PI). (A) Marrow from MIG mouse within PI gates. (B) Marrow from Notch4-IC mouse within PI gates. Suzanne M. Vercauteren, and Heather J. Sutherland Blood 2004;104:2315-2322 ©2004 by American Society of Hematology