A novel competitive repopulation strategy to quantitate engraftment of ex vivo manipulated murine marrow cells in submyeloablated hosts

Abstract

Objective: Standard competitive repopulation assays have proven valuable in evaluating engraftment potential in ablated hosts, permitting comparisons between various test cell populations. However, no similar method exists to compare engraftment of test cells in submyeloablated hosts, which would be helpful given the applications of reduced-intensity conditioning for hematopoietic gene-replacement therapy and other cellular therapies. Here, we developed a novel assay to quantitate engraftment of hematopoietic stem cells in submyeloablated hosts.

Materials and methods: Engraftment of murine marrow cells transduced with retroviral vectors using two separate protocols was compared to engraftment of fresh untreated competitor cells within low-dose radiation-conditioned hosts using a "three-way" marking system, so that test, competitor, and host cell chimerism could be reliably determined posttransplantation.

Results: We demonstrate that the repopulating ability of marrow cells transduced using two distinct protocols was reduced approximately 10-fold compared to fresh competitor cells in submyeloablated hosts utilizing the novel "three-way" transplant assay.

Conclusions: Murine marrow cells transduced using a clinically applicable protocol acquire an engraftment defect in submyeloablated hosts, similar to cells transduced using a research protocol. We conclude that the submyeloablative competitive repopulation assay described here will be of benefit to comparatively assess the engraftment ability of manipulated hematopoietic stem cells using various culture protocols, such as to test the impact of modifications in transduction protocols needed to attain therapeutic levels of gene-corrected blood cells, or the effect of ex vivo expansion protocols on engraftment potential.

Figure 1

Engraftment of transduced lin⁻ cells in 300 and 550 cGy-conditioned hosts is diminished compared to fresh lin⁻ cells. (A) 10⁶ lin⁻ cells from untreated Bl/6 (CD45.2⁺) donors were either transplanted directly (fresh) or transduced (Td) with MFG-GFP or SFalltCD34 as described in the Materials and Methods prior to transplantation into 300 cGy-conditioned Boy J (CD45.1⁺) hosts. Total donor chimerism and the fraction of transgene-marked (Tg⁺) donor cells were determined by flow cytometry 4–6 months post-transplant. N = 10 hosts from 2 independent experiments for fresh cells; N = 13 hosts from 3 independent experiments for transduced cells. *, P = 0.0012 for transduced lin⁻ cell chimerism compared to that of fresh lin⁻ cells. (B) 10⁶ fresh or SFFV-91-LNGFR-transduced Bl/6 lin⁻ cells were transplanted into 550 cGy-conditioned Boy J hosts. N = 5 hosts for fresh cells; N = 4 hosts for transduced cells. †, P < 0.05 for chimerism of transduced lin⁻ cells in 550 cGy- vs. 300 cGy-conditioned hosts (panel A).

Figure 2

Transduced lin⁻ marrow cells display significantly impaired competitive repopulating ability in 1100 cGy-conditioned hosts. Transduced lin⁻ test cells from Bl/6 donors were mixed with freshly-isolated lin⁻ competitor cells from Boy J donors, and the mixtures were transplanted into 1100 cGy-conditioned Boy J hosts. In the experiment shown in panel (A), 3 × 10⁵ test cells were mixed with 3 × 10⁵ competitor cells prior to transplantation; in the experiment shown in panel (B), 6 × 10⁵ test cells were mixed with 2 × 10⁵ competitor cells. Test cell chimerism is shown 4–5 months post-transplant from two independent experiments. N = 4 hosts for each parameter. *, P < 0.012 comparing fresh test cell chimerism to that of transduced test cells.

Figure 3

Schematic of submyeloablative CRA. In this assay, test cells (in this illustration, transduced Bl/6 marrow cells) were mixed with fresh Boy J competitor cells and transplanted into 550 cGy-conditioned Bl/6 × Boy J F1 hosts. Note that because the three mouse strains have similar HSC function (see Results for details), any of the strains can be used as the source of test cells, competitor cells or hosts, as long as the strains remain consistent within each independent experiment. Thus, if submyeloablative CRAs are performed in parallel using untreated and transduced test cells, the engraftment of transduced test cells (relative to untreated control cells) can be deduced.

Figure 4

Marrow cells transduced using a 5-FU-containing protocol display markedly impaired competitive repopulating ability in 550 cGy-conditioned hosts. (A) Representative scatter plots showing peripheral blood leukocyte analysis from 550 cGy-conditioned Boy J (CD45.1⁺) hosts which received a mixture of 2 × 10⁶ fresh (left panel) or transduced (right panel) Bl/6 (CD45.2⁺) test cells and 2 × 10⁶ Bl/6 × Boy J F1 (CD45.1⁺ and CD45.2⁺) competitor cells 5 months post-transplant. (B) Composite test cell chimerism 5 months post-transplant from 550 cGy-conditioned hosts transplanted with fresh or transduced test cells. On the left side of the graph, data were normalized per 10⁶ transplanted cells; on the right side of the graph, chimerism was normalized per femur equivalent (FE) of cells transplanted, accounting for the enrichment of primitive-phenotype cells with this transduction protocol. N = 5 hosts for each group.

Figure 5

Transduced lin⁻ marrow cells display markedly impaired competitive repopulating ability in 550 cGy-conditioned hosts. (A) Representative scatter plots showing peripheral blood leukocyte analysis from 550 cGy-conditioned Bl/6 × Boy J F1 (CD45.1⁺ and 45.2⁺) hosts which received a mixture of fresh (left panel) or transduced (right panel) Boy J (CD45.1⁺) test cells and Bl/6 (CD45.2⁺) competitor cells 5 months post-transplant. (B) Test cell chimerism 5 months post-transplant from 550 cGy-conditioned hosts transplanted with fresh or transduced test cells is shown. N = 5 hosts for each group. *, P < 0.0001 comparing fresh test cell chimerism to that of transduced test cells.