Journal: Stem Cell Reports

Article Title: Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis

doi: 10.1016/j.stemcr.2024.04.006

Figure Lengend Snippet: Interspecies chimera-derived hepatocytes exhibited mature hepatic function in vitro (A) Representative fluorescence images showing that GFP mouse ESCs derived from 129 mouse strain contribute to the liver in the 8-week-old mouse-rat chimera. Arrowheads indicate the GFP signals. Top scale bar, 5 mm; bottom scale bar, 1 mm. (B) Representative immunofluorescence images showing the expression of hepatocyte markers, including ALB, FAH, and ASGR1/2, in the liver tissue of mouse-rat chimeras. Red, ALB, FAH, or ASGR1/2; blue, Hoechst. Scale bar, 50 μm. (C) Live GFP-positive hepatocytes obtained by flow sorting with DAPI at a rate of up to 20.6%. FITC, fluorescein isothiocyanate. (D) Lipid accumulation in chimeric hepatocytes (Chimera Hep) analyzed by Nile red staining. Scale bar, 50 μm. (E) Glycogen storage in chimeric hepatocytes (Chimera Hep) analyzed by periodic acid-Schiff staining. Scale bar, 50 μm. (F) The capacity for urea synthesis in chimeric hepatocytes (Chimera Hep) is normal. WT hepatocytes (WT Hep) are directly used as a positive control. ( n = 3 wells independent replicates per group). Results were the mean ± SEM. n.s., no significance on unpaired 2-tailed t test. (G) Representative immunofluorescence images showing the expression of ALB in chimeric hepatocytes. Red, ALB; blue, Hoechst. Scale bar, 50 μm.

Article Snippet: Hepatocytes were plated at a density of 3 × 10 4 cells/well in 96-well plates coated with collagen I (354236; Corning, NY, USA) and cultured overnight in complete culture medium of mouse hepatocytes (CM-M033; Procell, Wuhan, China).

Techniques: Derivative Assay, In Vitro, Fluorescence, Immunofluorescence, Expressing, Staining, Positive Control

Journal: Stem Cell Reports

Article Title: Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis

doi: 10.1016/j.stemcr.2024.04.006

Figure Lengend Snippet: Gene expression profiles indicated that interspecies chimera-derived hepatocytes were more similar to endogenous hepatocytes (A) Flow chart for interspecies chimera-derived hepatocytes obtained by in situ collagenase perfusion and flow sorting and gene expression profiling of interspecies chimera-derived hepatocytes by RNA-seq. (B) Heatmap of expression levels of hepatocyte marker genes in chimeric hepatocytes (Chimera), mouse WT hepatocytes (WT) as positive controls, and mouse embryonic fibroblast (MEF) as negative controls. For the full list, see Figure S3 A. (C) Heatmap of expression levels of hepatocyte marker genes among mouse WT hepatocytes (WT), chimeric hepatocytes (Chimera), and SD rat WT hepatocytes (SD) for comparison, which proves chimeric hepatocytes belong fully to murine origin. For the full list, see Figure S3 B. (D) Hierarchical clustering analysis of the gene expression of mouse WT hepatocytes (WT), chimeric hepatocytes (Chimera), iHep cells (iHep), in vitro differentiated hepatocytes (IV diff), and WT hepatocytes reported (WT Ref.). (E) Heatmap of expression levels of hepatocyte marker genes among chimeric hepatocytes, iHep cells, and in vitro differentiated hepatocytes reported for comparison. WT hepatocytes are used as positive controls. For the full list, see Figure S3 C. (F) Enriched GO terms of upregulated biological processes involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes. (G) Enriched GO terms of downregulated biological processes involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes. (H) Enriched KEGG pathways of upregulated signaling pathways involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes. (I) Enriched KEGG pathways of downregulated signaling pathways involved in chimeric hepatocytes and WT hepatocytes compared with iHep cells and in vitro differentiated hepatocytes.

Article Snippet: Hepatocytes were plated at a density of 3 × 10 4 cells/well in 96-well plates coated with collagen I (354236; Corning, NY, USA) and cultured overnight in complete culture medium of mouse hepatocytes (CM-M033; Procell, Wuhan, China).

Techniques: Gene Expression, Derivative Assay, In Situ, RNA Sequencing, Expressing, Marker, Comparison, In Vitro, Protein-Protein interactions

Journal: Stem Cell Reports

Article Title: Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis

doi: 10.1016/j.stemcr.2024.04.006

Figure Lengend Snippet: Mouse chronic liver fibrosis was alleviated after interspecies chimera-derived hepatocyte transplantation (A) Preparation of mice with chronic fibrotic liver injury and chimeric hepatocyte transplantation. CCl 4 is used to model chronic fibrosis injury in NOD SCID mice with a corn oil injection group as concurrent controls. At the end of 6 weeks of CCl 4 injection, interspecies chimera-derived hepatocytes are injected into the spleens of chronic liver fibrosis models, with WT hepatocytes injected as a positive control and PBS injected as a negative control. (B) ALT and AST activities in CCl 4 -treated mice measured and normalized to the values from NOD SCID mice injected with corn oil before and after fibrosis modeling. ( n = 3 mice in each group at each time point). Results were the mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and n.s., no significance on unpaired 2-tailed t test. (C) Typical liver images with (CCl 4 ) or without (corn oil) CCl 4 treatment. (D) Typical liver images after PBS (w/PBS) or WT hepatocytes (w/WT Hep) or chimera hepatocytes (w/Chimera Hep) injection on day 7 in CCl 4 -treated mice compared with NOD SCID mice injected with corn oil (corn oil). Arrowheads indicate GFP signals. Scale bar, left 1 cm, right 0.5 mm. (E) ALT and AST activities in PBS-transplanted (w/PBS), WT Hep-transplanted (w/WT Hep), and Chimera Hep-transplanted (w/Chimera Hep) mice measured and normalized to the values of NOD SCID mice injected with corn oil (corn oil) on day 7 after injection. ( n = 3 mice in each group). Results were the mean ± SEM. ∗ p < 0.05 on unpaired 2-tailed t test. (F) Pathological section of H&E staining on day 7 after injection. Arrowheads indicate ballooning degeneration and the presence of apoptotic cells. Scale bar, 100 μm. (G) Pathological section of Masson staining on day 7 after injection. Arrowheads indicate remarkably increased collagen fibers. Scale bar, 100 μm. (H) GFP hepatocyte localization of representative immunofluorescence images on day 7 after injection. Red, ALB; blue, Hoechst. Scale bar, left 100 μm, right 50 μm.

Article Snippet: Hepatocytes were plated at a density of 3 × 10 4 cells/well in 96-well plates coated with collagen I (354236; Corning, NY, USA) and cultured overnight in complete culture medium of mouse hepatocytes (CM-M033; Procell, Wuhan, China).

Techniques: Derivative Assay, Transplantation Assay, Injection, Positive Control, Negative Control, Staining, Immunofluorescence