Peripheral blood CD34⁺ cells efficiently engraft human cytokine knock-in mice

Modeling the human hemato-lymphoid system in human hematopoietic stem and progenitor cell (HSPC) xenografted mice has advanced our understanding of hematopoiesis and leukemogenesis and allowed testing novel therapeutic approaches.^1-4  Although granulocyte colony-stimulating factor (G-CSF)–mobilized CD34⁺ peripheral blood (PB) cells are clinically widely used for autologous and allogenic hematopoietic stem cell transplantation,⁵  these cells, in contrast to fetal liver (FL) and cord blood (CB) CD34⁺ cells, have shown relatively poor engraftment at similar numbers in mice and are thus rarely used.^6,7  However, some cell-intrinsic pathways might differ in FL and CB compared with adult HSPCs, and fetal and neonatal cells are inappropriate to study individual patients’ acquired conditions.

We recently generated immunodeficient Rag2^−/−Il2rg^−/− mice with genes encoding human macrophage colony-stimulating factor (M-CSF),⁸  combined interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF),⁹  and thrombopoietin (TPO),¹⁰  knocked into the respective mouse loci, either individually or in combination (MISTRG).^3,11  Additionally, MISTRG mice carry a transgene encoding human signal regulatory protein α (SIRPα), known to bind human CD47 and thereby inhibiting mouse phagocytes of human cells.^12-14  We demonstrated that transplantation of human FLCD34⁺ cells into newborn MISTRG mice leads to high-level engraftment, multilineage differentiation, and functionality of innate immune cells.¹¹  Also, proof of principle engraftment of CB and PB CD34⁺ cells into newborn mice has been shown.¹¹ 

To test utility and value of these novel mouse strains for PB-derived human HSPC research, we compared engraftment, lineage differentiation, and tissue seeding of healthy adult mobilized PB human CD34⁺ cells and their progeny in various immune-deficient strains, including newborn and adult recipient MISTRG mice.

Humanized cytokine knock-in (KI) mice were generated as reported previously.¹¹  G-CSF–mobilized adult PB cells and CB cells were collected from healthy donors after obtaining informed consent. The study was approved by the ethics boards of the Canton of Zurich and Yale University Human Investigation Committee, respectively. Human (h) CD34⁺ cells were purified by density gradient centrifugation followed by positive immunomagnetic selection. Newborn and adult mice were sublethally irradiated. Newborn mice were intrahepatically injected with 300 000 to 500 000 (Zurich cohort) or 100 000 to 140 000 (Yale cohort) hCD34⁺ cells per mouse.^15,16  Adult mice were injected IV with 800 000 to 1 000 000 hCD34⁺ cells per mouse. Mice were bled at several time points and terminally analyzed 10 to 16 weeks posttransplantation. Experiments were approved by the Veterinäramt des Kantons Zurich and Yale University Institutional Animal Care and Use Committee. For further details, see supplemental Methods, available on the Blood Web site.

We first examined the engraftment of hCD34⁺ cells isolated from G-CSF mobilized PB upon intrahepatic injection into sublethally irradiated newborn NOD-scid (nonobese diabetic-severe combined immunodeficiency) Il2rg^−/− (NSG), hSIRPα-transgenic Rag2^−/−Il2rg^−/− (SRG),¹²  and mouse strains with human cytokine KI genes for hM-CSF combined with hTPO (MSTRG) or the addition of hIL-3/hGM-CSF (MISTRG).¹¹  PB analysis to determine hCD45⁺ cells at 4, 8, and 14 to 16 weeks revealed an increasing frequency of hCD45⁺ cells over time among total CD45⁺ cells in cytokine KI but not in control mice (supplemental Figure 1). When we compared percentage and absolute numbers of engrafted human cells in the bone marrow (BM) of recipient mice, the number was greatly increased in MSTRG and MISTRG in comparison with NSG and SRG mice (Figure 1A-B). Superior engraftment efficiency of cytokine KI mice was reproducible in an independent experimental cohort at Yale, where even lower numbers of PB CD34⁺ cells were transplanted (supplemental Figure 2A). Importantly, split donor sample transplantation into newborn cytokine KI vs NSG and SRG mice resulted in significantly higher engraftment in the KI group (Figure 1C). Although engraftment was highest in BM, %hCD45⁺ cells in PB and spleen of cytokine KI mice also exceeded engraftment of control groups (Figure 1D; supplemental Figure 3). Following transplantation of adult PB CD34⁺ cells, similar to previous studies using FL or CB CD34⁺ cells, we observed a lower frequency of hCD45⁺ cells in PB and spleen in comparison with BM in all recipient strains used. Interestingly, this difference was less prominent when transplanting CB CD34⁺ cells in newborn KI recipients (supplemental Figure 4). The reason for these differences needs to be further explored but might be in part attributable to extramedullary mouse hematopoiesis and/or differences in migration potential of human cells.¹¹  Irrespective of this observation and more importantly, adult recipient MISTRG mice transplanted with mobilized PB CD34⁺ cells also supported substantial overall hCD45⁺ and myeloid cell engraftment in BM, PB, spleen, and liver with a trend toward superiority compared with NSG mice (Figure 1E; supplemental Figure 5).

We next compared immature human HSPC engraftment and maintenance in different newborn recipients. Cytokine KI mice showed significantly increased relative and absolute numbers of human hematopoietic progenitor cells (CD34⁺CD38⁺) and hematopoietic stem cells containing CD34⁺CD38⁻ cells in BM (Figure 1F-G). Thus, we conclude that, in line with prior in vitro and in vivo observations,^17-19  human cytokines such as M-CSF, IL-3/GM-CSF, and TPO support maintenance and differentiation of human HSPCs.

The percentage of human CD33⁺ myeloid cells in the BM was not significantly different between newborn recipients, whereas absolute numbers were higher in KI than in conventional mice (Figure 2A-B; supplemental Figure 2B), regardless of a comparable BM cellularity (supplemental Figure 6). Importantly, hCD33⁺ cells were significantly increased in PB as well as nonlymphoid tissues such as liver and lung in the KI group (Figure 2B,D-E; supplemental Figure 7), and human CD68⁺ myeloid cells were detectable in nonlymphoid tissues such as lung, liver, colon, and skin of KI but not of control mice (Figure 2F). In adult recipients, we observed a trend toward superior myeloid development in different organs (supplemental Figure 5).

CD14⁺CD16⁻ cells are classical monocytes, whereas CD14⁺CD16⁺ and CD14^dimCD16⁺ cells are categorized as nonclassical “patrolling” monocytes.²⁰  The number of both CD14⁺CD16⁺ and CD14^dimCD16⁺ nonclassical monocytes in BM of the KI group was higher than in the control group (Figure 2C). In addition, CD141⁺ (BDCA-3⁺) dendritic cells with cross-presentation activity were significantly increased in the BM of KI mice (supplemental Figure 8).

As previously reported, we observed splenomegaly because of extramedullary mouse hematopoiesis in highly engrafted animals.¹¹  Immunohistochemical staining of BM of an engrafted MISTRG mouse (32% hCD45⁺ cells) confirmed the presence of human macrophages and showed evidence of hemophagocytosis, underlining human myeloid cell functionality (supplemental Figure 9). Interestingly, and in contrast to MISTRG mice engrafted with 1 × 10⁵ hCD34⁺ FL cells,¹¹  mice engrafted with up to 5 × 10⁵ mobilized PB CD34⁺ cells survived clinically healthy until terminal analysis up to 16 weeks. This might be a result of lower human BM engraftment levels, leading to relatively less reduction of mouse hematopoiesis and less phagocytosis of mouse cells by human phagocytes.¹¹ 

Analysis of human lymphocyte subsets revealed that the frequency of natural killer cells in BM and PB was significantly higher in KI mice (supplemental Figure 10A). Also, the CD4/8 ratio of T cells in the BM of MSTRG and MISTRG mice was higher, whereas naïve and memory CD4⁺ T-cell subsets and B-cell subsets were similar between KI and control groups (supplemental Figure 10B-C). Of interest, thymus hCD45⁺ cells and thymocyte development in KI mice were significantly enhanced as compared with control mice (Figure 2G).

Taken together, these data indicate that human cytokine KI mice support the development of mature lineage cells, both myeloid and lymphoid, from adult mobilized PB CD34⁺ cells in hematopoietic, lymphoid, and nonlymphoid tissues. Even though this study focused on newborn recipients, we are also showing confirmatory proof-of-principle data from adult recipient cytokine KI mice, a setting that might, because of easier experimental handling, facilitate clinical translational research. Indeed, it will now become feasible to use these and other humanized mice for individualized patient sample–derived in vivo research that allows preclinical, likely predictive testing of pathophysiology and novel therapeutic strategies in a personalized manner in infectious and neoplastic diseases^11,21 

This work was supported by a Hanne-Liebermann research fellowship (J.M.E.); the University of Zurich clinical research priority program Human Hemato-Lymphatic Diseases (M.G.M.); the Bill and Melinda Gates Foundation; the National Cancer Institute, National Institutes of Health (grant CA156689) (R.A.F. and M.G.M.); the US Department of Defense (grant CA120128) (S.H.); and the State of Connecticut Department of Public Health (grant RFP 2014-0135) (S.H.).

Contribution: Y. Saito, J.M.E., Y. Song, and S.H. designed research, performed experiments, analyzed data, and wrote the manuscript; A. Rafiei, A. Rongvaux, D.K., and M.H. performed experiments; and R.A.F. and M.G.M. directed the study and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Markus G. Manz, Hematology, University Hospital and University of Zurich, Raemistr 100, CH-8091 Zurich, Switzerland; e-mail: markus.manz@usz.ch.