Longterm Multilineage Human Hematopoietic Repopulation of Untreated KIT-Mutant Immunodeficient Mice

Background: Assessment of the growth and differentiation of human hematopoietic cells in immunodeficient mice has become vital to much basic and translational research. Historically, the primary focus has been to quantify different types of repopulating cells, based on the diversity and longevity of their clonal outputs in transplanted mice, assuming the results would be relevant to clinical transplants in humans. With the development of mice that lack B, T and NK cells on a variety of backgrounds and that have normal life expectancies but differences in cellular DNA repair ability, an increasing application is to use mice repopulated with human cells to interrogate and perturb mechanisms controlling normal, genetically modified and malignant cell behavior. We have previously shown that C57Bl6 mice homozygous for the W⁴¹ mutation of c-kit are fertile with a normal lifespan but have a functionally compromised hematopoietic stem cell (HSC) population. This enables single transplanted syngeneic HSCs to be detected at high frequency in these mice when they have been given a sublethal irradiation dose. Importantly, the HSC-derived clones produced in these mice display the same growth, self-renewal and differentiation abilities as in myeloablated recipients that require a co-transplant of normal mouse bone marrow (BM) cells to support their survival. We now report the development and improved repopulation by human cord blood (CB) CD34⁺ cells of mice that have the same genetically determined B, T, NK immunodeficiency as NOD/Rag1^-/--IL2Rγc^-/- (NRG) into which a homozygous W⁴¹gene has been introduced.

This was achieved by crossing and backcrossing the progeny of NRG x C57Bl6-W⁴¹/W⁴¹ matings and selecting mice that were homozygous for the Sirpα allele of the NOD mouse, the null Rag1 and null IL2Rgamma chain, genes of the NRG mouse and the W⁴¹ gene to obtain all of these on an otherwise mixed NODxC57Bl/6 background (NRG-W41 mice). The NRG mouse was chosen because the Rag1 KO has no effect on the radiosensitivity of other tissues as is the case with the scid (S) gene in the NSG mouse. As a result, use of the NRG mouse allows exploitation of the radioprotective effect of a reduced irradiation dose rate and hence delivery of a selectively higher dose to the HSCs of the host.

Results: Initial studies showed that parental NRG mice given 900 cGy split or spread continuously over 3 hrs show similar repopulation by human CD34⁺ CB cells as NSG mice given 315 cGy, but are more robust with consistent longterm survival. We then performed a pilot experiment using the same transplant design (2x10⁴ CD34⁺ CB cells/mouse) to compare chimerism obtained in NRG-W41 mice given an estimated “equivalent” radiosensitizing regimen of 150 cGy. The levels of multiple lineages of human cells measured in the BM and spleen 20 weeks post-transplant revealed these were greatly increased in the NRG-W41 mice (>95% human CD45⁺ cells in the BM vs 40% in NRG mice). Kinetic analysis of human cells in the blood also showed an enhanced output of human myeloid and B-lymphoid cells over time (5-fold higher in the NRG-W41 mice after >3 weeks). Particularly notable was the selectively increased (20-fold) and sustained output of human glycophorin A⁺ (GPA⁺) erythroid cells in the NRG-W41 mice (5% human GPA⁺ cells in the BM of 20-week NRG-W41 mice given 150 cGy and 5x10⁴ CD34⁺ CB cells/mouse vs 0.25% in the BM of the matched NRG mice given 900 cGy). A similar marked increase (20-fold) was seen on the level of circulating human platelets (SSC^lowCD41⁺ CD61⁺ cells) in comparable groups of transplanted NRG-W41 and NRG mice. We then investigated the extent of repopulation achievable in untreated NRG-W41 recipients. We therefore transplanted mice of both strains with 5x10⁴ CD34⁺CB cells each and have now followed the levels of human cells in their circulation and BM for up to 20 weeks. Human cells were barely detectable at 3 weeks post-transplant in either strain, but then in the unirradiated NRG-W41 mice only, their levels (all lineages) increased to close to those attained in NRG mice given 900 cGy.

Conclusion: NRG-W41 mice support robustly enhanced and long term generation in vivo of a wide range of human hematopoietic cell types including erythrocytes and platelets, with high levels of chimerism achieved even in unirradiated primary recipients transplanted with relatively low numbers of human CD34⁺ CB cells.