Additional file 1 of Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture
Creators: name: Akiyama, Saeko
name: Saku, Noriaki
name: Miyata, Shoko
name: Ite, Kenta
name: Toyoda, Masashi
name: Kimura, Tohru
name: Kuroda, Masahiko
name: Nakazawa, Atsuko
name: Kasahara, Mureo
name: Nonaka, Hidenori
name: Kamiya, Akihide
name: Kiyono, Tohru
name: Kobayshi, Tohru
name: Murakami, Yasufumi
name: Umezawa, Akihiro
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Developmental Biology
subject: Cancer
subject: 60506 Virology

description: Additional file 1. Figure S1 Details of the morphology and gene expression of proliferating hepatocytes. a Phase-contrast photomicrographs of primary human hepatocytes on irrMEF for 7 days. Two colonies are shown at low and high magnifications. b Relative gene expression by qRT-PCR in primary human hepatocytes (PHH2064) at passage 1 and 2 (Hep2064 P1 and P2). The data were normalized with the housekeeping gene UBC. Each relative value was calculated with respect to HepG2. Error bars indicate the standard deviation (n=3). "HepG2 (human hepatoma cell)," “Ad_Liver (human adult normal liver pools of five donors purchased from BioChain, R1234149-P)" and “Fresh_MH” (hepatocytes isolated from adult human liver in our laboratory) are used for comparison. c Gene expression by qRT-PCR in ProliHH from passage 1 to 21. The data were normalized with the housekeeping UBC gene. The expression level of each gene in PHH was set to 1.0. Error bars indicate the standard deviation (n=3). Figure S2. Evaluation of the effect of puromycin on mouse fetal fibroblasts (MEF) and ProliHH derived from other DILI patients. a Phase-contrast photomicrographs of MEF with exposure of puromycin (Puro: 0, 1, 2, 10, 50 and 100 µg/mL) for 3 days. Puromycin was added at 100% confluence. b Phase-contrast photomicrographs of puromycin-treated ProliHH at passage 5. c–e Phase-contrast photomicrographs of PHH (#2062) 3 days after exposure to 2 μg/mL puromycin. Puromycin was added when the cells reached confluence (Day 0) and removed 3 days after the addition (Day 3). The Day 4 image shows the cells 24 hours after puromycin removal (D). f Gene expression by qRT-PCR in puromycin-treated ProliHH (#2061 and #2062). The data were normalized by the housekeeping UBC gene. From left to right: non-treated ProliHH (Hep2061), puromycin-treated ProliHH (Hep2061+puro), non-treated ProliHH (Hep2062) and puromycin-treated ProliHH (Hep2062+puro). Cells were treated with 2 μg/mL puromycin for 3 days. Each relative value was calculated with respect to non-treated cells. Error bars indicate the standard deviation (n=3). The student's T test was performed for statistical analysis of two groups. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S3. Details of gene expression analysis and karyotyping of puromycin-treated proliferating hepatocytes, and hepatocyte selection with a low concentration (1 µg/mL) of puromycin. a Gene expression by qRT-PCR in puromycin-treated ProliHH from passage 1 to 25. The data were normalized with the housekeeping gene UBC. The expression level of each gene in PHH was set to 1.0. Error bars indicate the standard deviation (n=3). b Karyotypes of puromycin-treated ProliHH at passage 24. About 80% of the ProliHH were 46XX and no major abnormalities were found. c Growth curves of ProliHH treated with 1µg/mL puromycin (n=2, shown as green and yellow dots). Proliferative capacity was analyzed at each passage. Cells were passaged in the ratio of 1:4 for each passage. The numbers of cells were calculated as an average of 50 counts. "Population doubling" indicates the cumulative number of divisions of the cell population. d Phase-contrast photomicrographs of puromycin-treated ProliHH from passages 5, 7, 13, 21 and 25. e Microscopic view of ProliHH at passages 5, 13 and 25. HE stain. f Immunocytochemical analysis of puromycin-treated ProliHH with an antibody to Ki67 (cell proliferation marker). g A senescence-associated beta-galactosidase stain of puromycin-treated ProliHH at the indicated passages. The number of β-galactosidase-positive senescent cells increased at passage 25. h Karyotypes of puromycin-treated ProliHH at passage 24. Figure S4. Long-term analysis of ProliHH selected with a low concentration (1 µg/mL) of puromycin. a Gene expression by qRT-PCR in ProliHH at passage 4, 11 and 17. Puromycin-treated (+) and non-treated (-) ProliHH at passages 4, 11 and 17 were compared. The data were normalized with the housekeeping gene UBC. The expression level of each gene in PHH was set to 1.0. Error bars indicate the standard deviation (n=3). Student's T test was performed for statistical analysis of two groups. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. b Gene expression by qRT-PCR in puromycin-treated ProliHH from passage 1 to 25. Data were normalized with the housekeeping gene UBC. The expression level of each gene in PHH was set to 1.0. Error bars indicate the standard deviation (n=3). C-E.Immunocytochemical analysis of puromycin-treated ProliHH at a low concentration ( (1µg/mL) with antibodies to ALB (C), HNF4A (D) and bile duct marker CK7 (E) at early and late passages (passage 5 and 25, respectively). Yellow circles indicate binuclear cells (E). F, G. Glycogen storage in puromycin-treated ProliHH (passage 5, 13 and 25) by PAS stain with (F) and without (G) diastase digestion. H-J. Expression of the genes for CYP1A2 (H), CYP2B6 (I) and CYP3A4 (J) in puromycin-treated ProliHH after exposure to omeprazole (H), phenobarbital (I) or rifampicin (J). The expression level of each gene without any treatment (DMSO) was set to 1.0. Expression levels were calculated from the results of independent (biological) triplicate experiments. Error bars indicate the standard error (n=3). Student's T test was performed for statistical analysis of two groups. ***p &lt; 0.001. K CYP3A4 activity of ProliHH (passage 5, 11, and 25). The CYP3A4 activity of PHH was set to 1.0. Error bars represent the standard errors (n=3). Each expression level was calculated from the results of independent (biological) triplicate experiments. Error bars indicate the standard error (n=3). *p &lt; 0.05, NS: p &gt; 0.05. Figure S5. Detailed expression analysis and comprehensive analysis of hepatic progenitor cell markers in proliferating hepatocytes. A, B.Expression of the genes for hepatic progenitor cell markers by qRT-PCR in non-treated (A) and puromycin-treated (B) ProliHH. Data were normalized with the housekeeping gene UBC. The expression level of each gene in HepG2 cells was set to 1.0, as a positive control. Error bars indicate the standard deviation (n=3). C Immunocytochemical analysis of non-treated (Puro(-)) and puromycin-treated (Puro(+)) ProliHH with antibodies to AFP and CK19 at the early passage (passage 5). D Heat map showing the expression levels of top fetal hepatocytes upregulated genes (gene list: https://www.nature.com/articles/s41467-019-11266-x) in PHH (PHH2055 and PHH2064), iPSC-derived hepatocyte-like cells (HLC.KI and HLC.O), non-treated ProliHH (Puro(-)) and puromycin-treated ProliHH (Puro(+)_x1 and Puro(+)_x2). The colored bars show the signal strength scaled by the z score. E, F.Gene sets enriched in either (FDR &lt; 0.05, P &lt; 0.05) compared to non-treated and puromycin-treated ProliHH. Gene sets for the TCA cycle and pentose phosphate circuit (E), and epithelial–mesenchymal transition (EMT) (F) were identified. "non": non-treated ProliHH, "+puro": puromycin-treated ProliHH. Figure S6. ProliHH treated with low concentrations (1 µg/mL) of puromycin restore the expression level of liver-related genes after long-term culture to the level of the initial stage of hepatocyte culture. A. Hepatic maturation protocol. Puromycin-treated ProliHH (Puro(+)) at early passage (Passage 5) and late passage (Passage 25) at 70% confluence were exposed to 1 or 2 µg/mL puromycin for 3 days. ProliHH were then seeded onto low adhesion plates for spheroid formation one day after removal of puromycin. The cells were cultured to form spheroids for 10 days and subjected to further analysis. B-G.Histology of spheroids generated from ProliHH at each passage (B, D, E: Early passage (Passage 5); C, F, G: late passage (Passage 25)). "Puro(+)": Spheroids generated from ProliHH treated with 1 μg/ml puromycin. B, C: HE stain; D, F: PAS stain; E, G: PAS stain with diastase digestion. H, I. Immunohistochemistry of spheroids generated from puromycin-treated ProliHH at each passage (H: Early passage (passage 5); I: late passage (passage 25)) with antibodies to albumin (ALB), cytochrome p450 3A4 (CYP3A4) and multidrug resistance-associated protein 2 (MRP2). "Puro(+)": Spheroids generated from ProliHH treated with 1 μg/ml puromycin. Figure S7. ProliHH treated with low concentrations (1 µg/mL) of puromycin restore the expression level of liver-related genes after long-term culture to the level of the initial stage of hepatocyte culture. A.Gene expression levels were analyzed by qRT-PCR. Puromycin-treated ProliHH were subjected to spheroid culture conditions for 10 days (Figure S6A). RNAs were isolated from spheroids generated from puromycin-treated ProliHH at passage 5 and passage 25. The data were normalized by the housekeeping gene UBC. The expression level of each gene in ProliHH without any treatment in PHH was set to 1.0. "U.D.": undetectable. Each expression level was calculated from the results of independent (biological) triplicate experiments. Error bars indicate the standard error. The student's T test was performed for statistical analysis of two groups. *p &lt; 0.05, ***p &lt; 0.001. B.Urea synthesis of puromycin-treated ProliHH was analyzed at passage 5 and 25. Each expression level was calculated from the results of independent (biological) triplicate experiments. Error bars indicate the standard error. **p &lt; 0.01, ***p &lt; 0.001. C.ALB secretion of puromycin-treated ProliHH was analyzed at passage 5 and 25. Each expression level was calculated from the results of independent (biological) triplicate experiments. Error bars indicate the standard error. **p &lt; 0.01, ***p &lt; 0.001.