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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. <t>Heatmap</t> showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.
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Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. Heatmap showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.

Journal: bioRxiv

Article Title: Precursor of chemically expanded hepatocytes (pre-cHep) with 1-million-fold expansion potential and liver repopulation capacity

doi: 10.64898/2026.05.15.725446

Figure Lengend Snippet: Characterization of cHep. A. Direct differentiation of cryopreserved pre-cHep using a protocol equivalent to 3D PHH spheroid formation. B. Representative images of 3D PHH and cHep. C. Principal component analysis (PCA) plot including cHep generated from day 30 (D30) J03, J06, and J13 L4 pre-cHep, 3D PHH, and published hepatocyte-like cells (HLC). D. Heatmap showing expression of 264 liver-enriched genes. E–H. Distance-based similarity scores based on gene expression associated with xenobiotic metabolism (E), amino acid biosynthesis (F), bile acid biosynthesis (G), and the urea cycle (H). I. Immunofluorescence staining for albumin, HNF4α, CYP2E1, and ASGR1. J. Cholyl-lysyl-fluorescein (CLF) staining.

Article Snippet: Hierarchical clustering and gene expression heatmap analysis of 264 liver-enriched genes from Human Protein Atlas (gene list in Supplementary Table S3) also showed that D30 cHep clustered closely with 3D PHH, although expression of some genes remained lower than in uncultured PHH and liver samples ( ).

Techniques: Generated, Expressing, Gene Expression, Immunofluorescence, Staining

Gene expression analysis of HLC. Distance-based similarity score and gene expression heatmap with KEGG gene sets: Fatty acid metabolism (A, D), Retinol metabolism (B, E) and Complement coagulation cascade (C, F).

Journal: bioRxiv

Article Title: Precursor of chemically expanded hepatocytes (pre-cHep) with 1-million-fold expansion potential and liver repopulation capacity

doi: 10.64898/2026.05.15.725446

Figure Lengend Snippet: Gene expression analysis of HLC. Distance-based similarity score and gene expression heatmap with KEGG gene sets: Fatty acid metabolism (A, D), Retinol metabolism (B, E) and Complement coagulation cascade (C, F).

Article Snippet: Hierarchical clustering and gene expression heatmap analysis of 264 liver-enriched genes from Human Protein Atlas (gene list in Supplementary Table S3) also showed that D30 cHep clustered closely with 3D PHH, although expression of some genes remained lower than in uncultured PHH and liver samples ( ).

Techniques: Gene Expression, Coagulation