The human-sheep chimeras as a model for human stem cell mobilization and evaluation of hematopoietic grafts' potential

Abstract

Objective: To investigate whether the sheep xenograft model of human hematopoiesis can be used to mimic mobilization of human hematopoietic stem cells in vivo.

Material and methods: Sheep transplanted with 3.6 x 10(6) CD34+ from human adult bone marrow were mobilized 1.5 years posttransplantation with human granulocyte colony-stimulating factor for 5 days. At day 3 and 4 of mobilization, human cells were harvested from peripheral blood (PB) and bone marrow (BM) and were injected into secondary sheep recipients (n = 6) and these animals were analyzed for the presence of human cells in their BM and PB, starting at 3.5 months posttransplantation.

Results: Maximum mobilization of human cells in PB occurred at day 3, with a 21-fold increase in total numbers of human cells, and a recovery of 5.5 x 10(4)/mL CD34+. In the BM, maximal numbers of human cells were achieved at day 4, with a 6.3-fold increase and a recovery of 1.5 x 10(4)/mL CD34+ cells. PB and BM mobilized human cells were then transplanted into new sheep recipients, and analysis at 3.5 months posttransplantation demonstrated that levels of human cell engraftment in BM of the group transplanted with mobilized PB were significantly lower than those transplanted with BM cells (0.6% +/- 0.1% vs 8.0% +/- 1.8%). Furthermore, in sheep transplanted with mobilized PB, the levels of human cells in circulation remained 2.5-fold higher than the levels of human cells found in their BM.

Conclusion: Mobilization of human cells in the sheep model parallels human PB and BM hematopoietic stem cells (HSC) mobilization in healthy human donors in their ability to engraft, differentiate, and repopulate secondary hosts. Thus, this model can become a useful tool to study mobilization regimens, mechanisms, and quality of products obtained.

Figure 1. Human r-G-CSF is able to mobilize sheep white blood cells in control non-transplanted sheep

The results show that an efficient increase in total numbers of white blood cells in PB in all of the mobilized sheep (◆) (n=4) occurred when compared to day 0 of mobilization and a control un-mobilized sheep (■) (n=3) (p<0.01).

Figure 2. Selection of human chimeric animals for mobilization

3 animals that had been made chimeric by in utero HSC transplantation and displayed stable chimerism were selected to participate in this study. Flow cytometric analyses of the peripheral blood and bone marrow at the different time points post-transplant show consistent levels of human cell engraftment

Figure 3. Mobilization of human and sheep white blood cells in chimeric sheep

(A) Both human and sheep WBC were mobilized to the PB in the chimeric animals. Solid lines depict total number of WBC in the 2 animals that responded to G-CSF. One animal is represented by ■ while the other is represented by ▲. Doted lines represent the total number of human cells mobilized to the PB at each time point. In order to calculate the specific numbers of human cells mobilized, the total number of WBC was multiplied by the percentage of human CD45+ cells. Maximal mobilization of human cells to the PB occurred at days 2 and 3 of mobilization. The average of human cells mobilized at day 3 was significantly increased as compared with levels of human cells at day 0 (p<0.01). (B) Percentage of human CD45⁺ cells mobilized to the PB at each time point.

Figure 3. Mobilization of human and sheep white blood cells in chimeric sheep

(A) Both human and sheep WBC were mobilized to the PB in the chimeric animals. Solid lines depict total number of WBC in the 2 animals that responded to G-CSF. One animal is represented by ■ while the other is represented by ▲. Doted lines represent the total number of human cells mobilized to the PB at each time point. In order to calculate the specific numbers of human cells mobilized, the total number of WBC was multiplied by the percentage of human CD45+ cells. Maximal mobilization of human cells to the PB occurred at days 2 and 3 of mobilization. The average of human cells mobilized at day 3 was significantly increased as compared with levels of human cells at day 0 (p<0.01). (B) Percentage of human CD45⁺ cells mobilized to the PB at each time point.

Figure 4. Percentage of human CD45+ cells in BM of mobilized sheep

Percentage of human CD45⁺ increased in BM of mobilized sheep to reach a maximum at day 4 of mobilization.

Figure 5. Percentage of human cells at 3.5 months post-transplant in secondary sheep recipients transplanted with either mobilized BM or PB

The levels of human cells present in BM and in PB were significantly higher in sheep transplanted with CD45⁺ cells from BM than the ones transplanted with CD45⁺ cells from mobilized peripheral blood (p<0.01).

Figure 6. Levels of human cells in BM and PB of sheep transplanted with mobilized PB at 6 months post-transplant

In these animals the total levels of human cells persisted in higher levels in the PB in the BM. Also CD34⁺ cells in the animals transplanted with PBSC remained higher in circulation.