Differential Secondary Reconstitution of In Vivo-Selected Human SCID-Repopulating Cells in NOD/SCID versus NOD/SCID/γ chain Mice

Abstract

Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chain(null) mice to support long-term engraftment of MGMT(P140K)-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMT(P140K)-transduced CD34(+) cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMT(P140K)-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMT(P140K)-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chain(null) mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chain(null) mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.

Figure 1

Comparison of human hematopoietic cell engraftment in NSG versus NOD/SCID mice. Nonmanipulated umbilical cord blood CD34⁺ cells (1 × 10⁴–5 × 10⁴ per mouse) were transplanted into sublethally irradiated (300 cGy) NSG or NOD/SCID mice and human cell engraftment in the bone marrow (% human CD45⁺ cells) analyzed 8 weeks later by flow cytometry. Multi-lineage engraftment was similar in both strains (data not shown). n = 4 mice per cohort; *NS2 versus NOD/SCID, P < .001.

Figure 2

Overview of primary and secondary reconstitution experiments in NSG versus NOD/SCID mice. Human umbilical cord blood CD34⁺ cells were transduced with a GALV-pseudotyped oncoretroviral vector, SF1-MGMT^P140K-IRES-EGFP (MO1 = 5) and transduced cells transplanted into sublethally irradiated (300 cGy) NSG or NOD/SCID mice. One month after transplantation, cells were selected in vivo with 2 cycles of 20 mg/kg 6BG followed one hour later with 5 mg/kg BCNU. At 4 months after transplantation, the bone marrow was harvested and analyzed for the presence of human cells by flow cytometry. Bone marrow cultures were prestimulated in IL-6 and SCF for 48 hours and then equal numbers of human bone-marrow cells were transplanted into secondary recipient NSG and NOD/SCID mice. At 2 months after transplant, the secondary mice were analyzed for the presence of human cells via flow cytometry.

Figure 3

Comparison of in vivo selected MGMT^P140K-transduced human cells in NSG and NOD/SCID mice. The starting cell number was 4 × 10⁵ cells per transplanted mouse. MGMT^P140K-transduced CD34⁺ cells were transplanted into NS2 and NOD/SCID mice and selected in vivo as outlined in Figure 2. The BM was harvested and analyzed for the level of total human cell engraftment (%huCD45⁺ cells) and the level of transduced human cells (%huCD45⁺EGFP⁺) by flow cytometry. The BM cellularity was similar between the NSG and NOD/SCID mice and the differences in total human engraftment in NSG versus NOD/SCID mice were not significant (P > .05). The data presented here are from 1 transplant experiment (n = 3-4 mice in vehicle-treated cohorts and n = 8 mice in 6BG/BCNU-treated cohorts). Similar results were obtained in a second independent transplant experiment.

Figure 4

Analysis of human CD34⁺ cells in the bone marrow of primary recipient vehicle- and 6BG/BCNU-treated mice. (a) The percentage of total human CD34⁺ cells and (b) CD34⁺ human cells that are EGFP⁺ (huCD34⁺EGFP⁺) was determined by flow cytometry at 8 weeks after drug treatment. *P < .001, vehicle versus 6BG/BCNU treated.

Figure 5

Representative example of human multilineage engraftment in 6BG/BCNU-treated primary recipient mice. The bone marrow was harvested at 8-weeks after drug treatment, and the level of total and transduced human progenitors (CD34⁺), B-lymphoid (CD19⁺), and myeloid (CD33⁺) cells were determined via flow cytometry. The level of transduced cells was similar between NOD/SCID and NSG mice. The percentage in the upper right-hand quadrant represents the percentage of the human progenitor, B-lymphoid, or myeloid cells that are EGFP⁺.

Figure 6

Analysis of human cell engraftment in secondary recipient NOD/SCID and NSG mice. (a) The percentage of total human cells (%huCD45⁺) in the bone marrow and (b) the percentage of the huCD45⁺ cells that are EGFP⁺ (%huCD45⁺EGFP⁺) were analyzed at 8 weeks after transplant into secondary recipient NOD/SCID and NSG mice as outlined in Figure 2. *P < .001, NSG versus NOD/SCID. The date presented are from 1 transplant experiment (n = 3 secondary recipient mice transplanted with BM from vehicle-treated cohorts and n = 4–8 secondary mice transplanted with BM from 6BG/BCNU-treated cohorts. Similar results were obtained in a second independent transplant experiment.

Figure 7

Analysis of human CD34⁺ cells in secondary recipient NSG mice transplanted with BM from vehicle- or 6BG/BCNU-treated mice. (a) The percentage of total human CD34 cells (%huCD34⁺) (b) the percentage of the human CD34⁺ cells that are EGFP⁺ (%huCD34⁺EGFP⁺) in the bone marrow were analyzed at 8 weeks after transplant into secondary recipient NSG mice by flow cytometry. The source of the bone-marrow cells that were transplanted into the secondary recipient mice were derived from vehicle- or 6BG/BCNU-treated mice primary NSG mice. *P < .001, total CD34⁺ cells-vehicle versus 6BG/BCNU treated; *P < .05, transduced CD34⁺ cells-vehicle versus 6BG/BCNU treated.

Figure 8

Representative example of multilineage engraftment in secondary recipient NSG mice transplanted with BM from vehicle- or 6BG/BCNU-treated mice. The percentage of total human (CD45⁺) and transduced human progenitors (CD34⁺), B-lymphoid (CD19⁺), and myeloid (CD33⁺) cells in the bone marrow were determined via flow cytometry at 8 weeks after transplant in secondary recipient NSG mice. The percentage in the upper right-hand quadrant represents the percentage of the human cells that are EGFP⁺ in the progenitor, B-lymphoid, or myeloid cell subsets. The source of the bone-marrow cells that were transplanted into the secondary recipient mice were derived from vehicle- or 6BG/BCNU-treated mice primary NSG mice.