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Lentiviral vector transduction of human hematopoietic progenitor cells has shown transgene expression for extended periods after transplantation into NOD/SCID mice, however, the transduction of a true hematopoietic stem cells with the capacity to differentiate into both myeloid and lymphoid cells has not yet been proven. In this study we transplanted lentivirally transduced human cord blood CD34+ cells in limiting dilution into NOD/SCID mice. Transplantation of transduced cells in limiting dilution generated GFP+ lymphoid and myeloid progeny cells that may have arisen from single SRC. Five groups of primary transplant recipients receiving 400,000; 100,000; 35,000; 12,500; and 5,000 cells showed a linear relationship between the level of engraftment, as measured by FACS for CD45, and cell dose. The mice receiving low cell doses of 35,000; 12,500; or 5,000 cells, showed 3 of 4; 2 of 4; and 0 of 4 mice positive for engraftment respectively. Transduction efficiency (T.E.) as measured by FACS of the bone marrow from low celldose positive engrafting mice was 53+25% (n=5), with all mice showing the presence of GFP. Similar T.E. were seen in CFU-GM colonies grown from the same bone marrow with a mean of 56+47%. Lineage analysis of mice bone marrow by FACS showed GFP expression in both the lymphoid (CD19) and myeloid (CD15, CD33) populations. A unique mouse showed GFP expression in 91% of human cells as measured by FACS, and 100% of CFU-GM colonies. The low engraftment level of this mouse (4.2%), and the complete transduction of all human cells within the mouse suggest that this mouse was reconstituted by a single lentivirally transduced SRC. A cell-dose limiting dilution into secondary transplant recipients showed lentiviral transduction of more primitive SRC. Each primary recipient mouse was transplanted into 3 secondary recipients. Two of 13 secondary recipients were positive for engraftment indicating that both positive mice were likely to have been repopulated by a single SRC. FACS analysis of bone marrow derived from these mice revealed that 5% and 64% of cells expressed GFP and 0% and 74% of the CFU-GM derived from these mice express the transgene. This study shows that the lentiviral vector is capable of high efficiency transduction of candidate human hematopoietic stem cells that have the capacity to differentiate into both myeloid and lymphoid cells.


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