Forced expression of the Ikaros 6 isoform in human placental blood CD34+ cells impairs their ability to differentiate toward the B-lymphoid lineage by Cécile Tonnelle, Florence Bardin, Christine Maroc, Anne-Marie Imbert, Fanny Campa, Ali Dalloul, Christian Schmitt, and Christian Chabannon Blood Volume 98(9):2673-2680 November 1, 2001 ©2001 by American Society of Hematology

Experimental design. Experimental design. Experimental design to assess the biological consequences of transducing human placental blood CD34+ cells, either with a test retroviral vector that encodes the Ik6 isoform, along with the EGFP marker gene, or with a control vector that encodes EGFP only. Cécile Tonnelle et al. Blood 2001;98:2673-2680 ©2001 by American Society of Hematology

Western blot analysis of wild-type and transduced cells. Western blot analysis of wild-type and transduced cells. Whole protein extracts obtained from CB CD34+ cells transduced with the LZRS-EGFP retroviral vector (lanes 1 and 2) or LZRS-Ik6-EGFP (lanes 3 and 4) and from untransduced CB CD34+ cells (lanes 5 and 6) are revealed with (A) an anti-Ikaros antiserum (see “Materials and methods”) or (B) an anti-Grb2 polyclonal antibody. M indicates protein markers. At least 3 isoforms of Ikaros (Ik1, Ik2, Ik6) are endogeneously produced; lanes 3 and 4 show that after retroviral transduction the relative abundance of the Ik6 isoform is greatly increased (note that for these 2 lanes the signal for Grb2 is slightly less than for other lanes). Cécile Tonnelle et al. Blood 2001;98:2673-2680 ©2001 by American Society of Hematology

Comparison of lymphoid or myeloid antigen expression in transduced cells after in vitro differentiation. Comparison of lymphoid or myeloid antigen expression in transduced cells after in vitro differentiation. Percentage of cells positive for CD10 or CD19 (B lineage) or CD14 or CD33 (myeloid lineage) in the “lymphocyte” gate of 6- to 8-week-old MS-5 cocultures, initiated with sorted EGFP+ placental CD34+ cells, transduced with LZRS-Ik6-EGFP or LZRS-EGFP (paired experiments). Five of 6 experiments showed a decrease of CD10+ or CD19+ cells and an increase of CD14+ and CD33+ cells in cultures initiated with CD34+ cells transduced with LZRS-Ik6-EGFP when compared with cultures initiated with LZRS-EGFP transduced CD34+ cells. Cécile Tonnelle et al. Blood 2001;98:2673-2680 ©2001 by American Society of Hematology

Flow cytometry analyses of human transduced hematopoietic cells after in vitro culture. Flow cytometry analyses of human transduced hematopoietic cells after in vitro culture. After 6 weeks, coculture with the MS-5 murine stromal cell line, initiated with sorted EGFP+ placental CD34+cells, transduced with LZRS-EGFP (top) or LZRS-Ik6-EGFP (bottom), were stained with antihuman mAbs against CD10, CD19, and CD33 conjugated with PE. In test cultures initiated with sorted CD34+ cells transduced with the LZRS-Ik6-EGFP retroviral vector, a small percentage of cells expressing CD10 and CD19 can be identified; residual B cells in these cultures expressed low levels of EGFP. Cécile Tonnelle et al. Blood 2001;98:2673-2680 ©2001 by American Society of Hematology

Percentages of CD19+ and CD33+cells 6 to 9 weeks after injection of CB CD34+ cells in 3 goups of NOD-SCID mice. Percentages of CD19+ and CD33+cells 6 to 9 weeks after injection of CB CD34+ cells in 3 goups of NOD-SCID mice. Percentage of cells expressing CD19 or CD33 recovered from the bone marrow of NOD-SCID mice that were killed 6 to 9 weeks after tail injection of fresh CD34+ CB cells (primary CD34+ cells that did not undergo amplification and transduction steps), or cultured CD34+ cells that underwent LZRF-EGFP transduction (control cells), or CD34+ cells tranduced with the LZRS-Ik6-EGFP retroviral vector (test cells). Detailed analysis of engraftment is provided in Tables 1 and2. Cécile Tonnelle et al. Blood 2001;98:2673-2680 ©2001 by American Society of Hematology

Flow cytometry analyses of human hematopoietic cells engrafted in NOD-SCID mice. Flow cytometry analyses of human hematopoietic cells engrafted in NOD-SCID mice. Six weeks after injection in a control animal injected with CD34+ cells transduced with the LZRS-EGFP retroviral vector (A), CD19+ cells are more abundant than CD33+cells and both populations express significant levels of the EGFP marker. The same pattern is observed in one of the test animals injected with the LZRS-Ik6-EGFP retroviral vector (B). In another test animal injected with CD34+ cells transduced with the LZRS-Ik6-EGFP retroviral vector (C), a different pattern is seen. A small percentage of cells expressing CD19 can be identified; only rare CD19+ cells express EGFP, in contrast with CD33+ cells recovered from the same animal. Similar numbers of events were acquired during flow cytometry analysis for all lines. For detailed analyses, see Table 2. Cécile Tonnelle et al. Blood 2001;98:2673-2680 ©2001 by American Society of Hematology