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. 2021 Mar 5;371(6533):1019-1025.
doi: 10.1126/science.abe2485.

Combined liver-cytokine humanization comes to the rescue of circulating human red blood cells

Yuanbin Song #  1   2   3 , Liang Shan #  4   5 , Rana Gbyli #  1   2 , Wei Liu  1   2 , Till Strowig  6   7 , Amisha Patel  1   2 , Xiaoying Fu  1   2   8 , Xiaman Wang  1   2   9 , Mina L Xu  10 , Yimeng Gao  1   2 , Ashley Qin  1   2 , Emanuela M Bruscia  11 , Toma Tebaldi  1   2   12 , Giulia Biancon  1   2 , Padmavathi Mamillapalli  1   2 , David Urbonas  6 , Elizabeth Eynon  6 , David G Gonzalez  13 , Jie Chen  6 , Diane S Krause  2   10   14 , Jonathan Alderman  6 , Stephanie Halene #  15   2 , Richard A Flavell #  4   16
Affiliations

Combined liver-cytokine humanization comes to the rescue of circulating human red blood cells

Yuanbin Song et al. Science. .

Abstract

In vivo models that recapitulate human erythropoiesis with persistence of circulating red blood cells (RBCs) have remained elusive. We report an immunodeficient murine model in which combined human liver and cytokine humanization confer enhanced human erythropoiesis and RBC survival in the circulation. We deleted the fumarylacetoacetate hydrolase (Fah) gene in MISTRG mice expressing several human cytokines in place of their murine counterparts. Liver humanization by intrasplenic injection of human hepatocytes (huHep) eliminated murine complement C3 and reduced murine Kupffer cell density. Engraftment of human sickle cell disease (SCD)-derived hematopoietic stem cells in huHepMISTRGFah -/- mice resulted in vaso-occlusion that replicated acute SCD pathology. Combined liver-cytokine-humanized mice will facilitate the study of diseases afflicting RBCs, including bone marrow failure, hemoglobinopathies, and malaria, and also preclinical testing of therapies.

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Conflict of interest statement

Competing interests: R.A.F., S.H., L.S., and Y.S. are inventors on a patent related to this study (PCT/US20/42475). R.A.F. provides consultancy to Zai labs and Glaxo-Smith-Kline.

Figures

Fig. 1.
Fig. 1.. Liver humanization prolongs the survival of infused huRBCs in circulation through elimination of muC3 and reduction of murine macrophages.
Equal numbers of fluorescently labeled huRBCs (labeled with carboxyfluorescein diacetate succinimidyl ester) and muRBCs (violet) were premixed and injected retro-orbitally into mice. (A and B) Ratios of infused huRBCs to muRBCs in PB (n = 15) (A) and in liver, spleen, lung, and BM (B) at designated time points (n = 8). (C) muC3 staining of infused huRBCs (red) and muRBCs (blue) 5 min after infusion into MISTRG mice (n = 11). APC, cells were stained with allophycocyanin. (D) Representative flow cytometry plots and histograms of murine F4/80 staining of infused huRBCs (red) and muRBCs (blue) collected from MISTRG livers at 60 min after infusion (n = 8). (E) Comparison of huRBC survival in PB of MISTRGFah (n = 6), muHepMISTRGFah (n = 8), and huHepMISTRGFah (n = 8) mice. (F) Representative flow plots and histograms of muC3 staining of infused huRBCs and muRBCs in PB at 5 min and 60 min after infusion. (G and H) Representative histologic images showing F4/80+ murine macrophages (G) and quantification of F4/80+ area (H) in the livers of muHepMISTRGFah (n = 6) and huHepMISTRGFah (n = 6) mice at 12 weeks after hepatocyte engraftment. Scale bars, 100 μm; original magnification, 10×. Histological images were quantified with ImageJ, and individual mice are represented by symbols. Data (means ± (SEM) are representative of three independent experiments with three different donors. P values were determined by Mann-Whitney U test: **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig. 2.
Fig. 2.. Enucleated, mature huRBCs circulate in huHepMISTRGFah mice.
(A and B) Representative flow cytometry plots (A) and quantitation (B) of human (huCD235+) and murine (muTer119+) erythroid cells in PB of muHepMISTRGFah (n = 8) versus huHepMISTRGFah (n = 13) mice. PE-Cy7, phycoerythin-Cy7 fluorophore. (C to F) Representative flow cytometry analysis and quantitation of human erythropoietic differentiation on the basis of huCD71 and huCD235a expression (C and D) and huCD49d and Band3 expression (E and F) in PB of muHepMISTRGFah (n = 8) versus huHepMISTRGFah (n = 13) mice. PacBlue, Pacific blue label; PerCP Cy5.5, peridinin chlorophyll protein Cy5.5. (G and H) Representative flow cytometry plots (G) and quantitation (H) of enucleated human RBCs, determined by Hoechst staining of the PB of muHepMISTRGFah (n = 8) versus huHepMISTRGFah (n = 13) mice. FSC-W, forward scatter width. Individual mice are represented by symbols; data are presented as means ± SEM. P values were determined by Mann-Whitney U test: n.s., not significant; *P < 0.05; **P < 0.01; ****P < 0.0001. Data are representative of three independent experiments with three different donors of hepatocytes and FL cells.
Fig. 3.
Fig. 3.. Liver humanization enhances human erythropoiesis.
(A and B) Representative flow cytometry plots (A) and quantitation (B) of human erythroid progenitors in BM of muHepMISTRGFah (n = 8) and huHepMISTRGFah (n = 13) mice. (C) Representative BM histology images of huCD235 staining from muHepMISTRGFah (n = 8) and huHepMISTRGFah (n = 13) mice. Scale bars, 100 μm; original magnification, 10×. (D and E) Representative flow cytometry plots (D) and quantitation (E) of erythroid lineage differentiation determined on the basis of huCD71 and huCD235 expression in BM of muHepMISTRGFah (n = 8) versus huHepMISTRGFah (n = 13) mice. GPA, glycophorin A. (F) Gating strategy in multispectral imaging flow cytometry of human EBIs in the huHepMISTRGFah mouse. BF, bright field. (G) Representative images of human EBIs. Enucleated reticulocytes in contact with the central macrophage are marked by white arrows. Scale bars, 10 μm; original magnification, 40× Individual mice are represented by symbols, and data are means ± SEM. P values were determined by Mann-Whitney U test: *P < 0.05; **P < 0.01; ****P < 0.0001. Data are representative of three independent experiments with three different donors of hepatocytes and FL cells.
Fig. 4.
Fig. 4.. Modeling sickle cell disease in huHepMISTRGFah mice.
(A and B) Representative multispectral imaging flow cytometry images of mature enucleated huRBCs (huCD235a+, Hoechst−) and muRBCs (muTer119+, Hoechst−) in the PB of SCD (A) and normal (B) BM CD34+ cell–engrafted huHepMISTRGFah mice (n = 3). (C and D) Representative images of human EBIs in the BM of SCD (C) and normal (D) BM CD34+ cell–engrafted huHepMISTRGFah mice (n = 6 for each BM). (E to H) Representative histologic images of tissues stained with human-specific anti–hemoglobin alpha (HBA) antibody from SCD (n = 6) and normal (n = 6) BM CD34+ cell–engrafted huHepMISTRGFah mice. Scale bars, 100 μm; original magnification, 20× Arrowheads in (E) mark lung alveolar hemorrhages and thrombosis, in (F) they mark erythroid precursor expansion, sickled erythrocytes in sinusoids, vascular occlusion, and thrombosis in the spleen, in (G) they mark sickled RBCs in sinusoids and microvascular thrombosis in liver, and in (H) they mark congestion of capillary loops and peritubular capillaries and engorged glomeruli in kidneys of SCD-engrafted huHepMISTRGFah mice. Data are representative of two independent experiments with two different donors of normal and SCD CD34+ cells.
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