Research ArticleAPPLIED SCIENCES AND ENGINEERING

Transforming the spleen into a liver-like organ in vivo

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Science Advances  10 Jun 2020:
Vol. 6, no. 24, eaaz9974
DOI: 10.1126/sciadv.aaz9974
  • Fig. 1 Spleen remodeling.

    (A) Illustration of spleen remodeling. (B) The spleen (white dotted) before and after translocation (micro-CT). (C) Gross view of a normal and translocated spleen. (D) Mean SW/BW of normal and translocated spleens 7 days after translocation. (E) Microarray analysis of gene expression between normal and translocated spleens. IRCs, immune-related cytokines. (F to O) The translocated spleens injected with phosphate-buffered saline (PBS) or STH three times over 7 days with their (F) gross view; (G) SW/BW; (H) gene expression compared by microarray; (I) expression of typical genes of ECM, chemokines, growth factors (GFs), and cytokines; (J) contents of hydroxyproline, COL1, and COL4, plus the hardness of the spleens (n = 8 for COL4); (K) hematoxylin and eosin (H&E) and (L) Masson’s trichrome staining; (M) expression of COL1, COL4, and α–smooth muscle actin (α-SMA) (inset scale bar, 200 μm); (N) levels of growth factors and cytokines [enzyme-linked immunosorbent assay (ELISA); values normalized to PBS group]; and (O) three-dimensional (3D) reconstructed micro-CT images showing vascularization, with the vessel area measured. (P) Average proportion of different cell populations in the spleens treated with PBS or STH. Images are representative of three independent experiments. Results are shown as means ± SEM (n = 5 unless otherwise noted). Statistics: (D, G, J, N, and O) Student’s t test. TNF-α, tumor necrosis factor–α; IFN-γ, interferon-γ; TGF-β1, transforming growth factor–β1; EGF, epidermal growth factor; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor. Photo credit: Lintao Wang, Nanjing University.

  • Fig. 2 Establishment of immunosuppression in the remodeled spleen.

    (A) Scheme of xenografting HepG2 cells into the spleen. (B) Genome-wide microarray profiling of the spleen 8 hours after HepG2 transplantation. Signaling pathways (KEGG, left) and gene expression (heat map, right) related to immune rejection in STH/PBS-treated spleens. NOD, nucleotide oligomerization domain; JAK-STAT, Janus kinase–signal transducers and activators of transcription. (C) Serum concentration of immunoglobulins and cytokines and (D) CD4+/CD8+ T cell proportion in peripheral blood (statistics on the right) in mice 1 week after HepG2 transplantation. (E) Scheme of assessing long-term rejection of xenografted HepG2, with (F) the determination of growth factors and cytokines in the spleen. (G) Scheme of analyzing the response in OT-1 mice, whose (H) expression level of IFN-γ by spleen cells 4 days after stimulation with OVA-expressing HepG2 (OVA-HepG2) cells and (I) CD8+ T cell proportion expressing IFN-γ in the spleen (statistics on the right). Furthermore, OT-1 mice with OVA-HepG2 cell transplantation and long-term STH treatment were analyzed against the group with STH injection ceased for 14 days: (J) IFN-γ expression by the spleen cells and (K) CD8+ T cell proportion expressing IFN-γ. Images are representative of three independent experiments. Results are shown as means ± SEM (n = 5). Statistics: (F) one-way and (C and D) two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons post hoc test and (H to K) Student’s t test.

  • Fig. 3 Hepatocyte transplantation in the STH-remodeled spleen.

    (A) Implantation of liver cells to remodeled spleens. (B to E) Hepatocytes from green fluorescent protein (GFP) transgenic mice were transplanted to the remodeled spleens for 8 weeks, followed by (B) FACS counting of GFP+ cells, (C) SW/BW, (D) bioluminescence imaging and quantification, (E) H&E staining (asterisks, bile infarcts), (F) gross view, and (G) immunostaining for GFP (star, spleen-liver boundary). DAPI, 4′,6-diamidino-2-phenylindole. (H to J) Long-term growth of the allograft hepatocytes (24 weeks), with (H) GFP signal in the spleen sections, (I) H&E staining and immunostaining for cytokeratin 7 (for bile duct), and (J) liver zonation analysis using glutamine synthetase (GS) immunostaining. (K to Q) Human primary hepatocytes alloHEPs or hiPS-HEPs were transplanted to the remodeled spleens in mouse for 8 weeks, followed by (K) human-specific genomic DNA (gDNA) detection, (L) SW/BW, (M) determination of human serum albumin (HSA), (N) gross view, (O) H&E staining (stars, bile infarcts), (P) immunostaining of HSA and Ku80 in the spleen (stars, spleen-liver boundary), and (Q) fluorescent in situ hybridization (FISH) analysis of the spleen sections for human GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Images are representative of three independent experiments. Results are shown as means ± SEM (n = 5). Statistics: (M) one-way and (B, D, and K) two-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test and (C and L) Student’s t test. Photo credit: Lintao Wang, Nanjing University.

  • Fig. 4 The remodeled spleen in mice supported the growth of xenograft HepG2 cells.

    (A) Schematic diagram of intrasplenical injection and assay of the growth of transspecies HepG2 cells in the STH-remodeled or PBS-treated spleen. (B) Bioluminescence imaging and corresponding quantitative analysis based on photon counts gated over the spleen. (C to F, I, and K) The spleens treated as in (A) were analyzed at 8 weeks after HepG2 transplantation. (C) Representative gross view and (D) micro-CT scanning, with the section highlighting maximal splenic cross-sectional area; red dotted area indicated the liver-like region. (E) H&E staining of the spleens with EdU labeling identifying cells under proliferation. (F) Representative confocal microscopy images of the spleen stained for HSA or Ku80 and expressing GFP. The star indicated the boundary of spleen and liver tissue. (G) FACS-based quantitative analysis of GFP transgenic HepG2 cells in the spleen treated as in (A) at the indicated time. (H) Weight of the spleen treated as in (A) at the indicated time. (I) Representative immunostaining of HSA across the whole frozen spleen sections. (J) The levels of HSA in the spleens treated as in (A) at the indicated time. (K) Representative FISH of human GAPDH in the spleens. Images are representative of three independent experiments. Results are shown as means ± SEM (n = 5 per group). Statistics: (B, G, H, and J) two-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test. Photo credit: Lintao Wang, Nanjing University.

  • Fig. 5 Function of the hepatized spleen.

    (A to D) Transplantation of alloHEPs into transformed spleens, followed by (A) Oil red O and PAS staining of the spleen, (B) ICG clearance test, (C) frozen spleen sections, and (D) mice survival after 90% hepatectomy. (E to H) Xenografting of human hepatocytes (HepG2 or hiPS-HEP) into transformed spleens: (E) liver-specific metabolic profiling (quantitative PCR; n = 3 for liver samples), (F) serum HSA concentration after HepG2 transplantation, (G) serum HSA concentration 15 days after hiPS-HEP transplantation, and (H) human-specific debrisoquine (DB) metabolite formations (n ≥ 5). AUC, area under the curve; 4OHDB, 4-hydroxydebrisoquine. (I) Oil red O and PAS staining of the spleen. (J) Frozen spleen sections after ICG intravenous injections. (K to P) Examination of McA-RH7777 cells in transformed spleens. (K) Coimmunostaining for CYP26A1 with prothrombin or GS in the spleens 30 days after transplantation. (L) H&E staining of the hepatized spleen with or without d-galatosamine hydrochloride (d-Gal) exposure. (M) The prothrombin time (PT) and (N) the level of blood ammonia 6 hours after exposure to d-Gal. (O) Mice survival after d-Gal treatment or (P) 90% hepatectomy (n = 10). Images are representative of three independent experiments. Results are shown as means ± SEM (n = 5 unless otherwise noted). Statistics: (B, G, H, M, and N) one-way and (F) two-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test and (D, O, and P) log-rank test. HMG-CoA, hydroxymethylglutaryl-coenzyme A reductase; ACO, acyl-coenzyme A oxidase; PFK1, phosphofructokinase 1; GGT, gamma-glutamyltransferase.

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