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. 2016 Oct 6;128(14):1829-1833.
doi: 10.1182/blood-2015-10-676452. Epub 2016 Aug 19.

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

Yasuyuki Saito  1   2 , Jana M Ellegast  1 , Anahita Rafiei  1 , Yuanbin Song  3 , Daniel Kull  4 , Mathias Heikenwalder  4   5 , Anthony Rongvaux  3   6 , Stephanie Halene  7   8 , Richard A Flavell  3   9 , Markus G Manz  1
Affiliations

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

Yasuyuki Saito et al. Blood. .

Abstract

Human CD34+ hematopoietic stem and progenitor cells (HSPCs) can reconstitute a human hemato-lymphoid system when transplanted into immunocompromised mice. Although fetal liver-derived and cord blood-derived CD34+ cells lead to high engraftment levels, engraftment of mobilized, adult donor-derived CD34+ cells has remained poor. We generated so-called MSTRG and MISTRG humanized mice on a Rag2-/-Il2rg-/- background carrying a transgene for human signal regulatory protein α (SIRPα) and human homologs of the cytokine macrophage colony-stimulating factor, thrombopoietin, with or without interleukin-3 and granulocyte-macrophage colony-stimulating factor under murine promoters. Here we transplanted mobilized peripheral blood (PB) CD34+ cells in sublethally irradiated newborn and adult recipients. Human hematopoietic engraftment levels were significantly higher in bone marrow (BM), spleen, and PB in newborn transplanted MSTRG/MISTRG as compared with nonobese diabetic/severe combined immunodeficient Il2rg-/- or human SIRPα-transgenic Rag2-/-Il2rg-/- recipients. Furthermore, newborn transplanted MSTRG/MISTRG mice supported higher engraftment levels of human phenotypically defined HSPCs in BM, T cells in the thymus, and myeloid cells in nonhematopoietic organs such as liver, lung, colon, and skin, approximating the levels in the human system. Similar results were obtained in adult recipient mice. Thus, human cytokine knock-in mice might open new avenues for personalized studies of human pathophysiology of the hematopoietic and immune system in vivo.

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Figures

Figure 1
Figure 1
Human cytokine KI mice support engraftment of human mobilized PB CD34+ cells. (A) Representative fluorescence-activated cell sorter (FACS) analysis of the frequency of mouse and human CD45+ cells in BM in newborn mice 14 weeks posttransplantation. Numbers besides gated areas indicate percentages of cells. (B) Engraftment of hCD45+ cells in BM of newborn mice 10 to 16 weeks posttransplantation. Left panel indicates percentage of hCD45+ cells among total CD45+ cells. Right panel indicates absolute number of hCD45+ cells per hind legs (2 femurs and tibias). Each symbol represents an individual mouse; bars indicate mean values; n = 11 (NSG), n = 3 (SRG), n = 22 (MSTRG), n = 4 (MISTRG). Conv, conventional; KI, human cytokine KI. *P < .05; ***P < .001 (1-way analysis of variance [ANOVA] Kruskal-Wallis test with Dunn’s multiple comparison test). (C) hCD45+ cell engraftment in BM of newborn mice from split, matched individual donors. Each symbol represents mean frequency of hCD45% cells among total CD45+ cells. Mean frequency of human CD45+ cells in Conv vs KI mice was significantly different. *P < .05 (Conv vs KI; paired t test). (D) Engraftment of hCD45+ cells in blood (left) and spleen (right) 10 to 16 weeks posttransplantation. Each symbol represents an individual mouse; bars indicate mean values; n = 11 (NSG), n = 3 (SRG), n = 22 (MSTRG), n = 4 (MISTRG). *P < .05; ***P < .001 (1-way ANOVA Kruskal-Wallis test with Dunn’s multiple comparison test). (E) Engraftment of hCD45+ cells in BM, blood, and spleen in adult mice 12 to 16 weeks after transplantation. Panel shows percentage of hCD45+ cells among total CD45+ cells in each organ. Each symbol represents an individual recipient engrafted with hCD45+ cells; bars indicate mean values; n = 2 (NSG), n = 4 (MISTRG). (F) Representative FACS plots for hCD34+CD38+ and hCD34+CD38− cells in newborn recipients gated on mCD45− Lineage (Lin)− hCD45+ cells. Numbers beside gate indicate percentages of cells. (G) Frequency (left 2 graphs) as well as absolute number (right 2 graphs) of hCD34+CD38+, hCD34+CD38− cells per hind legs. Each symbol represents an individual newborn recipient engrafted with hCD45+ cells; bars indicate mean values; n = 5 (Conv; 2 NSG and 3 SRG), n = 19 (KI; 15 MSTRG and 4 MISTRG). *P < .05 (Mann-Whitney U test).
Figure 2
Figure 2
Human cell engraftment in lymphoid and nonlymphoid tissues in newborn recipients. (A) Representative FACS plots for hCD19+ and hCD33+ cells gated on hCD45+ cells in BM (top panels) and PB (bottom panels). (B) Frequency and absolute number of hCD33+ cells in BM (left) and frequency of hCD33+ cells in PB (right). Each plot shows data from an individual recipient engrafted with hCD45+ cells. Numbers beside gates indicate percentages of cells; n = 5 (Conv; 2 NSG and 3 SRG), n = 20 (KI; 16 MSTRG and 4 MISTRG). *P < .05 (Mann-Whitney U test). (C) Frequency of classical (CD14+CD16−) and nonclassical monocytes (CD14+CD16+ or CD14dimCD16+) among lineage (Lin; CD3, CD19, CD20)− hCD45+ cells in BM of engrafted recipient mice; data show mean + standard error of the mean; n = 5 (Conv; 2 NSG and 3 SRG), n = 22 (KI; 18 MSTRG and 4 MISTRG). *P < .05; ***P<.001 (Mann-Whitney U test). (D) Representative FACS plots of recipient mice (in liver 14 weeks posttransplantation) for hCD45+ cells (top panels), CD33+ or CD19+ cells gated on hCD45+ cells (bottom panels). Numbers beside gates indicate percentages of cells shown. (E) Percentage of hCD45+ cells among total CD45+ cells in liver (left graph); each symbol represents an individual recipient engrafted with hCD45+ cells, and bars indicate mean values. CD33+, CD19+, CD3+, and NKp46+ population among hCD45+ cells in the liver of engrafted mice (right); data show mean + standard error of the mean; n = 5 (Conv; 2 NSG and 3 SRG), n = 25 (KI; 21 MSTRG and 4 MISTRG). *P < .05 (Mann-Whitney U test; left) and *P < .05 (for CD33+ cells, 1-way ANOVA Kruskal-Wallis test with Dunn’s multiple comparison test; right). (F) Immunohistological staining of human myeloid cells (hCD68+) in nonlymphoid tissues (liver, lung, colon, and skin) of recipient mice. Bars represent 20 μm. Images are representative of at least 3 mice analyzed per group. (G) Representative FACS plots for hCD45+ cells (top panels) and double-negative, double-positive, CD4+ single-positive, and CD8+ single-positive thymocytes among hCD45+ cells (bottom panels) in the thymus of indicated recipient mice. Number of hCD45+ cells in the thymus is shown (right). Each symbol represents an individual recipient engrafted with hCD45+ cells; bars indicate mean values; n = 4 (Conv; 2 NSG and 2 SRG), n = 9 (KI; 9 MSTRG). *P < .05; ***P < .001 (Mann-Whitney U test).
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