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Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro
Accepted Manuscript Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro Dr. Sofia B. Leite, Tiffany Roosens, Adil El Taghdouini, Inge Mannaerts, Ayla J. Smout, Mustapha Najimi, Etienne Sokal, Fozia Noor, Christophe Chesne, Leo A. van Grunsven, Prof. PII:
S0142-9612(15)00922-9
DOI:
10.1016/j.biomaterials.2015.11.026
Reference:
JBMT 17203
To appear in:
Biomaterials
Received Date: 24 September 2015 Revised Date:
30 October 2015
Accepted Date: 13 November 2015
Please cite this article as: Leite SB, Roosens T, El Taghdouini A, Mannaerts I, Smout AJ, Najimi M, Sokal E, Noor F, Chesne C, van Grunsven LA, Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.11.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Novel human hepatic organoid model enables testing of drug-induced liver
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fibrosis in vitro
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Sofia B. Leite , Tiffany Roosens , Adil El Taghdouini , Inge Mannaerts , Ayla J. Smout , Mustapha Najimi , 2 3 4 1* Etienne Sokal , Fozia Noor , Christophe Chesne , Leo A. van Grunsven
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*Correspondence to: Prof. Leo van Grunsven ([email protected]) or Dr. Sofia Batista Leite ([email protected]). Faculty of Medicine and Pharmacy, Laarbeeklaan 103, 1090 Brussel, Building D Ground Floor Room D022/025B. TEL (+32) 2 477 44 09;
Liver Cell Biology Laboratory, Vrije Universiteit Brussel (VUB), Belgium
Laboratory of Pediatric Hepatology and Cell Therapy, Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain (UCL), Belgium
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Biochemical Engineering Institute, Saarland University, Germany
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Biopredic International, France
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List of abbreviations (by order of appearance): HSC, Hepatic Stellate Cell; Hep, HepaRG; 3D, threedimensional; Hep/HSC, Hep and HSC co-culture, hepatic organoid; PDGFR-β, Platelet derived growth factor receptor β; CYP, cytochrome P450; RIF, Rifampicin; BNF, β-Naphtoflavone; PB, Phenobarbital; MRP2, Multidrug resistance-associated protein 2; CMFDA, 5-chloromethylfluorescein diacetate; 2D, twodimensional; mRNA, messenger Ribonucleic Acid; dCT, deltaCT, gene expression relative to housekeeping gene; TGFβ, Transforming Growth Factor β; LPS, Lipopolysaccharide; APAP, Acetaminophen; ATP, Adenosine Triphosphate; αSMA, alpha Smooth Muscle Actin; HDAC, Histone deacetylase; CK, Cytokine mixture; VPA, Valproic Acid; CCl4, Carbon Tetrachloride; EC50, Half Maximal Effective Concentration; OCR, Oxygen consumption rate; ECAR, Extracellular acidification; Oligo, Oligomycin; FCCP, Trifluoromethoxy carbonylcyanide phenylhydrazone; A+R, Antimycin A and Rotenone; MTX, Methotrexate; ALT, Alanine Aminotransferase; AOP, Adverse Outcome Pathway; DILI, Drug-induced liver injury.
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ABSTRACT
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Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic
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stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method
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allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG
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(Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting
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scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte
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and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21
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days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner.
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After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol
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and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen
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secretion and deposition. Acetaminophen was identified by these organoids as an inducer of
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hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic
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organoid culture model is the first that can detect hepatocyte-dependent and compound-induced
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HSC activation, thereby representing an important step forward towards in vitro compound
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testing for drug-induced liver fibrosis.
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Keywords: organoid; liver fibrosis; DILI; in vitro; hepatic stellate cell; Hepatocyte; HepaRG; APAP; Methotrexate; Allyl alcohol
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INTRODUCTION
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Hepatic stellate cells (HSCs) are the major collagen producing cells during conditions of
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sustained hepatic injury (either metabolic, cholestatic, viral or toxic), when hepatocyte damage
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triggers a cascade of events leading to activation of quiescent HSCs into a myofibroblastic
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(activated) HSC state1. HSC activation is mediated by a plethora of pathways that finally result in
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increased secretion of extracellular matrix proteins, such as collagens, that accumulate as scar
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tissue (fibrosis) within the liver parenchyma and to liver cirrhosis in a later stage2. To date, the
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best in vitro fibrosis models consist of mono-layer cultures of freshly isolated rodent HSCs in
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regular tissue culture dishes which leads to “spontaneous” HSC activation3. Obvious limitations
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of these cultures are the rodent background and the un-controlled and hepatocyte damage-
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independent activation of the HSCs, making these cultures less suitable for pro- and anti-fibrotic
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compound testing translatable to human.
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We developed a novel three-dimensional (3D) human co-culture model where both hepatocyte
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functionality and HSC quiescence can be maintained for at least 21 days. This novel system
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allows hepatotoxicity testing as well as drug-provoked and hepatocyte-dependent HSC
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activation and fibrosis.
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MATERIAL AND METHODS
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Cell culture
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HepaRG/HSC cell culture. Human liver non-parenchymal fractions isolation were obtained from
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the Agreed Hepatocytes & Hepatic Stem cell Bank (Saint-Luc Hospital and Université Catholique
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de Louvain). This raw material (obtained after written and signed informed consent) was
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processed for Hepatic Stellate Cell (HSC) isolation and prepared for culture as previously
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described4-6. Cells from two different donors were used, both male, healthy and under 13 years
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old. For a thorough characterization of the cells we refer to El Taghdouini et. al.7 and Coll & El
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Taghdouini et. al.4. For the 3D cultures cells were used between passages 5 and 10.
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Differentiated cryopreserved HepaRG® cells were obtained from Biopredic. On day 0 of culture,
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HSCs were trypsinized and HepaRG (Hep) thawed according to the instructions of the provider.
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For the generation of the cell spheroids, 96-well plates treated with cell-repellent (Greiner) were
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used. Mono- and co-culture suspensions were prepared and seeded in the cell densities shown
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below:
Hep seeding 5 (x10 cells/ml)
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After 15-30 min in the incubator, plates were placed on an orbital shaker, where stirring was kept
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at 80 rpm for the entire culture period. The outer wells were filled with 100 µl of PBS to minimize
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liquid evaporation. Culture medium was refreshed (90%) every second day. 3D Hep/HSC co-
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cultures and control mono-cultures were cultured for 21 days. Cross diameter of the spheroids
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never exceeded 200 µm.
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HepaRG/HSC culture medium. Day 0 cells were resuspended in HepaRG thawing medium and
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from day 1 to 21 cells were kept in the HepaRG culture medium 0% DMSO (Biopredic), unless
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otherwise stated. In the APAP proof-of-concept studies, medium was replaced by HepaRG
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Induction/Toxicity medium with 0.1% (v/v) DMSO (Sigma-Aldrich, St. Louis, MO, USA) during
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compound incubation. In the repeated versus single compound exposure assays, on day 8, the
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HepaRG culture medium 0% DMSO was replaced by the Serum-free HepaRG maintenance
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medium described in Klein et. al. 20138 and kept in this medium until the end of culture
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The remaining material and methods are available in supplementary information.
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92 RESULTS
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Characterization of human liver organoid cultures
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We use cryopreserved differentiated HepaRGs (Heps) as functional hepatocyte-like cells and
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primary human HSCs for the development of the human 3D hepatic organoids. Differentiated
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HepaRG, widely characterized in the last decade, show a close correlation with human
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hepatocyte functions9 which are improved when cultured in 3D10, 11. HSCs are isolated from the
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non-parenchymal fraction of human livers, expanded in culture and frozen down as previously
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described4, 6. Liver organoids are generated by mixing the two single-cell suspensions in a ratio
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of 1Heps:2HSCs in non-attachment round-bottom 96 well-plate wells with orbital stirring. This
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allows for quick generation of spheroids with an optimal diameter of ≤200 µm, representing the
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maximum physiological distance between a cell and a blood vessel12 and precludes the
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formation of necrotic cores13 (Fig. 1A). The diameter variation at day 7 and 21 among the co-
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culture spheroids is 190µm ± 10 µm (n=100) while only 4% of cells incorporate EdU during a 48h
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exposure (Suppl. Fig. 1A), illustrating the non-proliferative nature of the cells. The 3D Hep/HSC
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spheroids demonstrate a segregated organization with a concentration of HSCs in the core of
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the spheroid (PDGFR-β positive cells, with a small elongated nucleus, Fig. 1B) while
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hepatocyte-like cells (CYP3A4 positive cells, with large round nuclei, Fig. 1B) accumulate
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preferentially in the periphery. Phase I hepatocyte metabolic capacity is demonstrated by
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exposing the cells to prototype cytochrome P450 inducers for CYP3A, CYP1A2 and CYP2C9.
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Even though there are less hepatocyte-like cells in the co-culture spheroids, the inducibility of
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the cells shows little variation between mono- and co-cultures of Heps (Fig. 1D). Likewise, CYP-
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induction values are in agreement with CYP-inducing capacities of Heps maintained in similar
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conditions (Suppl. Table 4). Additionally, CYP3A4 staining of 3D Hep/HSC spheroids indicates
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the presence of mature hepatocytes (± 20% of all spheroid cells is CYP3A4 positive, n=4). Note
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that differentiated HepaRGs are 50% hepatocyte-like while the other 50% are cholangiocyte-like
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cells14, thus only 1/6 of the spheroid can be hepatocyte-like cells (Fig. 1B). Excretion of the
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MRP2 substrate CMFDA15 and its accumulation in canulicular structures is only observed after
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fixation, underlining the good elimination capacity of the spheroids (Fig. 1C, arrows). Albumin
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secretion is similar in 3D Hep and 3D Hep/HSC cultures, but is manifold higher than in 2D Hep
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cultures (Fig. 1E). Finally, mRNA levels of hepatocyte markers such as ALB, CY3A4, GSTA1
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and SLCO1B1 is maintained in 3D Hep/HSC cultures at comparable levels to Hep mono-
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cultures and in some cases higher than 2D Hep (Fig. 1F) despite the lower amount of
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hepatocyte-like cells. In the presence of HepaRG culture medium, human 3D HSCs still activate
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from day 7 onwards (Fig. 1G) while in 3D Hep/HSC co-cultures they do not, as can be
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evidenced by the low mRNA levels of ACTA2, COL1A1 and LOX on day 21. Due to the
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mesenchymal origin HepaRG still express these markers, but in much lower extent when
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compared with the HSC-containing cultures (Fig. 1G). Nevertheless the HSCs in the co-culture
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spheroids are still capable of responding to pro-fibrogenic stimuli such as TGFβ and LPS
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(Suppl. Fig. 1B and C), known to have direct effects on HSCs.
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Taken together, these experiments demonstrate functionality of both cell types in 3D Hep/HSC
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cultures and we therefore refer to these cultures as human hepatic organoids. Despite the good
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cellular profile of these organoids after 7 days in culture, we mainly focus on 21 day culture
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characterization which perhaps allows long term repeated dose toxicity testing.
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Drug-induced hepatic injury by acetaminophen
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- Single exposure of human hepatic organoids to Acetaminophen
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To show the drug responsiveness of the organoids we used Acetaminophen (APAP) and
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compared it to the respective mono-cultures for specificity of the response. APAP was selected
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for its kinetic stability, known effective in vitro concentrations, specific toxicity in hepatocytes and
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high human relevance16. After a 24h APAP exposure at day 20, we observe only small
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differences in toxicity between the 3D organoids and the 3D Hep cultures (Fig. 2A,B),
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suggesting a good CYP2E1 metabolism in both cultures17. Nevertheless, there is a tendency for
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higher toxicity in the organoids, more pronounced on day 21 than on day 7 (Suppl. Table 5).
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Upon APAP exposure, Heps disappear from the periphery of the organoids, probably by
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necrosis-mediated cell death, while the ones remaining in a ring surrounding the HSC core are
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caspase 3-positive apoptotic cells (Fig. 2E), representing the second path of APAP-induced
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hepatocyte injury18. Although not known as a pro-fibrotic agent, metabolization of APAP by
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functional hepatocytes in the organoid leads to toxicity mainly in hepatocytes and not HSCs, but
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might lead to hepatocyte-injury dependent HSC activation. Indeed, we detect a strong dose-
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dependent up-regulation of HSC activation-associated mRNAs COL1A1, COL3A1 and LOXL2
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only in the hepatic organoids (Fig. 2C), suggesting HSC activation in response to hepatocyte
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injury. In Hep mono-cultures, APAP exposure also leads to a 2-5 fold increase of collagen
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mRNA levels, however the basal levels of collagen expression are 10-100 fold lower in 3D Hep
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cultures when compared to the organoids (Suppl. Fig. 2A), highlighting the activation state of
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the HSCs, the main producers of collagen in the liver1. Measurements of C-terminal pro-collagen
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I peptide in the culture supernatant show an APAP-dependent increase of collagen protein only
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in the organoid cultures (Fig. 2D and Suppl. Fig. 2B). ACTA2 mRNA levels do not change upon
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APAP exposure in any of the cultures suggesting that the indirect APAP-induced HSC activation
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is independent of TGFβ, since ACTA2 transcription depends largely on TGFβ signaling19. Yet,
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we can detect a slight increase in αSMA protein levels in the center of the organoids after APAP
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exposure (Fig. 2E). Additionally, we confirm collagen production, by immuno-staining and by
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detection of cross-linked collagens (Sirius-red) in sections of hepatic organoids (Fig. 2E).
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- Hepatic organoids recapitulate several aspects of liver fibrosis
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Our results with APAP exposed organoids suggest that besides the known hepatotoxicity, APAP
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has the capacity to induce HSC activation through hepatocyte injury. This prompted us to
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investigate drug-dependent HSC activation further by addressing in these organoids three in
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vivo features of HSC activation: (i) enhanced HSC activation due to inflammation20
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inhibition of HSC activation by histone deacetylase (HDAC) inhibition; and (iii) recovery from
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HSC activation. We mimic the cytokine release from immune cells22 by performing the APAP
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exposure in the presence of a cytokine mixture (CK: TNFα, IL1β, IFNα and IFNγ). While CK
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alone (0 mM APAP+CK) has no effect on the culture read-outs, when combined with APAP, the
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toxic effect is potentiated and the up-regulation of COL1A1, COL3A1 and LOXL2 mRNA is
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increased (Fig. 3A, B). We then used valproic acid (VPA), an HDAC inhibitor that inhibits HSC
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activation induced by CCl4 in mice23. Similar to in vivo, VPA does not affect APAP toxicity in the
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organoids while up-regulation of the activation markers at the highest APAP concentrations was
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inhibited (Fig. 3C, D). Finally, organoids cultured for 3 additional days after APAP washout,
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show a decrease in HSC activation markers (Fig. 3E) indicative of HSC recovery24.
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APAP seems to affect HSC mono-cultures only at the highest concentrations (EC50 = 31.4
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±11mM). Instead of the CYP-mediated metabolization of APAP into NAPQI which is the main
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toxic pathway in hepatocytes, this effect could also be linked to mitochondrial oxidative stress25,
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assessed the mitochondrial functions and glycolytic activity by measuring the oxygen
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consumption rate (OCR) and the extracellular acidification rates (ECAR) respectively (Fig. 4A).
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Comparison of the basal respiration, ATP production and spare respiratory capacity in the
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untreated control samples (Fig. 4B), suggests that the hepatic organoid cultures are more stable
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than the equivalent mono-cultures. Furthermore, when comparing Hep-containing cultures with
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the HSC mono-cultures, all parameters are roughly ten times higher. Especially in the case of
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ATP, this seems to indicate that Heps are the major production source in the organoids and
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suggests that the observed ATP-drop after APAP incubation in the hepatic organoids (Fig. 2B)
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is mainly a consequence of Hep toxicity. Additionally, at 24h, a shift towards a more glycolytic
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metabolism (higher ECAR and lower OCR) is only seen in 3D Hep mono-cultures, which in the
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HSC monocultures was already high, but not in the organoids. In the hepatic organoids, a
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general OCR decrease is observed, only after 24 hours of APAP exposure (Fig. 4C), while in the
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respective mono-cultures this is observed already after 12 hours. Together, this shows that there
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is little influence of APAP on mitochondrial functions in the hepatic organoids in contrast to the
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respective mono-cultures. These findings are in line with the mitochondrial resistance observed
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in hepatocytes during early stages of fibrosis in vivo27.
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Single and repeated exposure of hepatic organoids to Methotrexate and Allyl alcohol.
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To further explore the hepatic organoids as a read-out for drug-induced liver fibrosis we exposed
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the cultures to the reference pro-fibrotic compounds Allyl alcohol, known to induce fibrosis in
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rat28, and Methotrexate (MTX) which is classified to induce liver fibrosis in human subjects after
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prolonged exposure29. Since the fibrotic effect of these compounds in vivo is only observed upon
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long-term exposure, both compounds were tested in single and repeated doses (and compared
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to APAP for HSC activation (Suppl. Fig. 4A-C)). Serum-free culture conditions were established
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to avoid interference of the serum during repeated compound exposure30,8 (Suppl. Fig. 5A). The
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two exposure profiles are compared by assessing the cells on day 21, 24h after the last
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compound dose (Fig. 5A). Single MTX exposure does not affect cell viability at any of the tested
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concentrations (Fig. 5B). However, upon repeated exposure, the percentage of ATP production,
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even at the lowest concentration, decreases to less than 20%. Although repeated exposure to
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1.9 µM MTX leads to smaller spheroids, the amount of disaggregating cells around the spheroid
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is less than in organoids repeatedly exposed to 5 mM APAP (Fig. 5I and Suppl. Fig. 4D),
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suggesting that the lower ATP levels not necessarily represent cell death. While single exposure
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to MTX does not lead to an upregulation of activation markers at the mRNA level in the
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organoids, repeated exposure to MTX clearly induces HSC activation (Fig. 5C and Suppl. Fig.
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6B). Positive Collagen1 and αSMA staining in cross sections of the hepatic organoid however
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demonstrates that HSCs in the core of the spheroids are activated after both single and
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repeated exposures (Fig. 5J). Sirius-red stainings are more in line with the mRNA levels of
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activation showing a much stronger deposition of cross-linked collagen in the repeated dose
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MTX setting than after a single exposure.
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As for the case of Allyl alcohol, the clear dose-response observed at single exposure almost
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disappears upon repeated exposure, letting us to presume induced toxicity resistance of the
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cells (Fig. 5E). This can be explained by the inhibition of its metabolism to acrolein31 observed
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during rat and mice exposures32. In single exposure, despite the drop in ATP at high
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concentrations it does not affect the spheroid integrity (Fig. 5I,J). Both operational exposures
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show an increased tendency of mRNA levels of HSC activation (Fig. 5F and Suppl. Fig. 6C).
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While at the mRNA level the difference between single and repeated exposure to Allyl alcohol is
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not as outspoken as for MTX, at the protein level, there is a strong fibrotic response in the
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repeated exposures leading to a ∼100 fold increase of secreted collagen at day 14 (Fig. 5G) and
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a strong accumulation and cross-linked collagen at the end of the experiment, shown by Sirius-
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red and collagen stainings (Fig. 5J).
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For both Allyl alcohol and MTX, repeated exposure shows high collagen secretion at day 14, 48h
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after the 3rd exposure (Fig. 5D,G). At the end of the culture, the 24h pro-collagen secretion is
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comparable to the single exposure in the case of Allyl alcohol, but lower in the case of MTX (Fig.
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5D). For both compounds, nearly no collagen secretion is detected in the 3D mono-cultures (Fig.
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5H).
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Finally, APAP caused toxicity and HSC activation both in single and repeated-exposures, with a
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shift to lower APAP concentrations for the repeated dose (EC50 from 10.4 ±2.3 mM to 2.25 ±0.35
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mM APAP; Suppl. Fig. 4A). HSC activation followed with a peak at around 5-10 mM for the
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repeated exposure instead of 20-40 mM (Suppl. Fig. 4B and Suppl. Fig. 6A).
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Once again, the capacity of the cultures to recover from the insult was analyzed 3 days after
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compound washout, however recovery was only observed in APAP single exposures and 63 µM
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MTX repeated exposures (Suppl. Fig. 7) which most likely reflects the mechanism by which
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these compounds induce HSC activation.
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Pro-fibrotic effect of APAP in vivo
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Acute liver failure as a consequence of exposure to high doses of APAP has been widely
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reported in animals and humans33, but so far there are no reports describing a fibrotic response
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of APAP. Since our results with hepatic organoids indicated that APAP can indirectly induce
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HSC activation, we next investigated the in vivo fibrotic potential of APAP in mice. Blood
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analysis 24h post-injection demonstrates increased alanine transaminase (ALT) levels indicating
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hepatocyte toxicity upon exposure of regular chow-fed mice to 300-500 mg/kg APAP (Fig. 6A).
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300mg/kg APAP is already sufficient for HSC activation within 24 hours, demonstrated by
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increased mRNA levels of Acta2, Col1a1 and Lox in freshly isolated HSCs (Fig. 6B). Bi-weekly
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injections of APAP for 4 and 8 weeks (300 mg/Kg), results in the formation of scar tissue and
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increased αSMA expression in BALB/c and C57BL/6 mice, but has the tendency to decline over
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time (Fig. 6C and Suppl. Fig. 8). Indeed, when compared to CCl4-induced liver injury, APAP
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treatment appears to be less fibrogenic (Fig. 6C, D).
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DISCUSSION
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The microenvironmental factors that can affect a cell’s performance in culture are countless,
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varying from chemical medium composition, mechanical factors or even diffusion rates. In the
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case of HSCs, rigidity of the matrix to which they are attached at least partly determines their
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activation state3. We report, for the first time, metabolically active human hepatic organoids in
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which HSCs maintain a quiescent-like state for 21 days while retaining their capacity to respond
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to pro-fibrotic compounds directly and in a hepatocyte-dependent manner.
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In the last years, simplified models for the use in chemical risk assessment have been
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developed, the so-called Adverse Outcome Pathways - AOP (https://aopkb.org) which should
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finally lead to an integration of all AOPs into one large network34. A good predictive in vitro
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method should at least recapitulate some of the key events of such AOP (Suppl. Fig. 9). The
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developed human hepatic organoid consisting of differentiated HepaRGs and primary HSCs is to
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our knowledge the first in vitro method that mimics drug-induced liver fibrosis and could be used
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to further optimize the AOP of liver fibrosis.
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The HepaRG to HSC cell-ratio of 1:2 and the hepatocyte-specific medium guarantees HSC
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quiescense and keeps the hepatocytes functional. Although the used ratio is far from the
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physiological ratio, we believe that in these organoids the HSCs work as feeder cells to support
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hepatocyte functions35. The central accumulation of HSCs in the organoids is most likely due to
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their stronger capacity to aggregate at the time of spheroid formation; HSCs are cultured in 2D
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beforehand while the HepaRG cells are freshly thawn and immediately put in 3D co-culture. We
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did question the use of primary HSCs and differentiated HepaRG cells by culturing organoids
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consisting of the widely used HepG2 and LX-2 cells, hepatocyte and HSC lines respectively.
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Besides the fact that cells in these organoids keep proliferating, the LX-2 cells activate over time
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without injury and the organoids are much less sensitive to APAP, probably reflecting the low
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hepatocyte functionality of the HepG2 cells (Suppl. Fig. 10).
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The use of Allyl alcohol and MTX confirms the applicability of the method by showing HSC
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activation only in the organoids and not in 3D mono-cultures. The upregulation of some of the
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activated HSC-associated mRNAs in 3D Hep mono-cultures (albeit remaining at relatively low
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levels) might be due to an EMT-like process due to the treatment with MTX or Allyl alcohol.
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While EMT-like differentiation of hepatocytes is readily observed in vitro36, this does not
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contribute to liver fibrosis formation in vivo1.
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In contrast, repeated-dose exposure of the 3 tested compounds resulted in a significantly
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higher, and sometimes unique up-regulation of COL1A1, COL1A3 and LOXL2 in the organoids
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(Suppl. Fig. 11). These results demonstrate that only the hepatic organoids, and not their
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respective 3D mono-cultures, have the capacity to respond positively to the reference fibrotic
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compounds Methotrexate and Allyl alcohol.
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Even the hepatotoxic-independent HSC activation observed after repeated exposure to Allyl
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alcohol is hepatocyte-dependent since the effect is not observed in the HSC mono-cultures
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(Suppl. Fig. 11C). This highlights the potential of the hepatic organoids to not only “just” identify
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hepatotoxic compounds that might cause indirect HSC activation (i.e. APAP), but also by
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compounds that are not hepatotoxic but need the presence of functional hepatocytes to
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indirectly activate HSCs (i.e. Allyl alcohol at the tested concentrations). The in vivo confirmation
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of the unexpected fibrotic potential of APAP identified in the organoid cultures, highlights the
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potential to identify thus far unknown fibrotic compounds in vitro that can actually cause fibrosis
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in vivo. The lack of literature describing this fibrotic potential of APAP could be due to the much
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lower profibrotic capacity than for instance CCl4 and the decline of this effect over time. The
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report from Watelet and co-workers on preliminary APAP fibrosis evidence in humans37
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suggests that this assessment should perhaps be pursued.
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In conclusion, we have developed functional human hepatic organoids that can identify
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compounds that induce fibrosis, a drug-induced liver injury (DILI) rarely addressed in vitro unlike
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steatosis, choleastatis and phospholipidosis. This is a big step forward from the regular 2D HSC
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cultures that are generally used. The hepatic organoids are suitable for repeated dosage and are
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sensitive to the nature of the compounds by displaying differential toxicity and HSC activation
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profiles. These hepatic organoids represent a substantial improvement when screening for drug-
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induced liver fibrosis in terms of cost, animal use and prediction of liver fibrosis in human.
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Furthermore, this technology could stimulate the development of culture models representative
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of fibrosis in other organs such as lung and kidney, since these share common mechanisms38, 39.
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ACKNOWLEDGMENTS
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First we would like to acknowledge the finantial support from different entities: T. Roosens and A. El
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Taghdouini are funded by the Institute for the Promotion of Innovation through Science and Technology in
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Flanders (IWT-Vlaanderen; SB/13170 and SB/111008). I. Mannaerts is supported by a Fund of Scientific
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Research Flanders FWO-V post-doctoral fellowships (12N5415N LV). This work was supported by the
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HeMiBio consortium funded by the European Commission and Cosmetics Europe as part of the SEURAT-
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1 cluster (N° HEALTH-F5-2010-266777) and by the IWT project HILIM-3D (SBO 140045).
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Furthermore, we thank P. Antony (Université Du Luxembourg) for the possibility to use the XFe96
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Analyser, S. Kalaydjiev and D. Buurman (Seahorse Bioscience) for the help in the performance and
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interpretation of the Mito Stress test, A. P. Batista (ITQB) for the help in the interpretation of the ECAR and
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OCR results.
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REFERENCES
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1.
5.
6. 7. 8.
9.
10. 11. 12. 13.
TE D
4.
EP
3.
Mederacke, I. et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 4, 2823 (2013). Friedman, S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 88, 125-172 (2008). Olsen, A.L. et al. Hepatic stellate cells require a stiff environment for myofibroblastic differentiation. Am J Physiol Gastrointest Liver Physiol 301, G110-118 (2011). Coll, M. et al. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells. Sci Rep 5, 11549 (2015). Dusabineza, A.C. et al. Hepatic stellate cells improve engraftment of human primary hepatocytes: A pre-clinical transplantation study in animal model. Cell Transplant, doi: 10.3727/096368915X096686788 (2015). Thoen, L.F. et al. A role for autophagy during hepatic stellate cell activation. J Hepatol 55, 13531360 (2011). EL Taghdouini, A. et al. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells. Oncotarget (2015). Klein, S., Mueller, D., Schevchenko, V. & Noor, F. Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study. J Appl Toxicol 34, 1078-1086 (2014). Lubberstedt, M. et al. HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. J Pharmacol Toxicol Methods 63, 59-68 (2011). Leite, S.B. et al. Three-dimensional HepaRG model as an attractive tool for toxicity testing. Toxicol Sci 130, 106-116 (2012). Gunness, P. et al. 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci 133, 67-78 (2013). Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249-257 (2000). Folkman, J. & Hochberg, M. Self-regulation of growth in three dimensions. J Exp Med 138, 745753 (1973).
AC C
2.
M AN U
331
14
ACCEPTED MANUSCRIPT
19. 20. 21.
22. 23. 24. 25.
26.
27. 28. 29. 30. 31. 32. 33. 34.
RI PT
18.
SC
17.
M AN U
16.
TE D
15.
Antherieu, S., Rogue, A., Fromenty, B., Guillouzo, A. & Robin, M.A. Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells. Hepatology 53, 1895-1905 (2011). Bogman, K., Erne-Brand, F., Alsenz, J. & Drewe, J. The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins. Journal of pharmaceutical sciences 92, 1250-1261 (2003). Antoine, D.J. et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology 58, 777787 (2013). Lee, S.S., Buters, J.T., Pineau, T., Fernandez-Salguero, P. & Gonzalez, F.J. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 271, 12063-12067 (1996). Nakagawa, H. et al. Deletion of apoptosis signal-regulating kinase 1 attenuates acetaminopheninduced liver injury by inhibiting c-Jun N-terminal kinase activation. Gastroenterology 135, 13111321 (2008). Herrmann, J., Haas, U., Gressner, A.M. & Weiskirchen, R. TGF-beta up-regulates serum response factor in activated hepatic stellate cells. Biochim Biophys Acta 1772, 1250-1257 (2007). Marques, P.E. et al. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology 56, 1971-1982 (2012). Pradere, J.-P. et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 58, 1461-1473 (2013). Aoyama, T., Paik, Y.H. & Seki, E. Toll-like receptor signaling and liver fibrosis. Gastroenterol Res Pract 2010, 1-8 (2010). Mannaerts, I. et al. Chronic administration of valproic acid inhibits activation of mouse hepatic stellate cells in vitro and in vivo. Hepatology 51, 603-614 (2010). Kisseleva, T. et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci U S A 109, 9448-9453 (2012). Chen, C., Krausz, K.W., Idle, J.R. & Gonzalez, F.J. Identification of novel toxicity-associated metabolites by metabolomics and mass isotopomer analysis of acetaminophen metabolism in wild-type and Cyp2e1-null mice. J Biol Chem 283, 4543-4559 (2008). Prill, S. et al. Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450independent mitochondrial toxicity of acetaminophen and amiodarone. Arch Toxicol, doi:10.1007/s00204-00015-01537-00202 (2015). Mitchell, C. et al. Protection against hepatocyte mitochondrial dysfunction delays fibrosis progression in mice. Am J Pathol 175, 1929-1937 (2009). Jung, S.A. et al. Experimental model of hepatic fibrosis following repeated periportal necrosis induced by allylalcohol. Scand J Gastroenterol 35, 969-975 (2000). Lindsay, K. et al. Liver fibrosis in patients with psoriasis and psoriatic arthritis on long-term, high cumulative dose methotrexate therapy. Rheumatology (Oxford) 48, 569-572 (2009). Pomponio, G. et al. In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments. Toxicol In Vitro (2014). Covaci, O.I., Bucur, B. & Radu, G.L. Acrolein detection based on alcohol dehydrogenase inhibition. International Journal of Environmental Analytical Chemistry 93, 325-334 (2013). Auerbach, S.S., Mahler, J., Travlos, G.S. & Irwin, R.D. A comparative 90-day toxicity study of allyl acetate, allyl alcohol and acrolein. Toxicology 253, 79-88 (2008). Kaplowitz, N. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov 4, 489-499 (2005). Vinken, M. The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology 312, 158-165 (2013).
EP
14.
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36. 37. 38. 39.
Leite, S.B. et al. Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications. Toxicol In Vitro 25, 825-832 (2011). Nambotin, S.B., Tomimaru, Y., Merle, P., Wands, J.R. & Kim, M. Functional consequences of WNT3/Frizzled7-mediated signaling in non-transformed hepatic cells. Oncogenesis 1, e31 (2012). Watelet, J. et al. Toxicity of chronic paracetamol ingestion. Aliment Pharmacol Ther 26, 15431544; author reply 1545-1546 (2007). Friedman, S.L., Sheppard, D., Duffield, J.S. & Violette, S. Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med 5, 167sr161 (2013). Henderson, N.C. et al. Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 19, 1617-1624 (2013).
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FIGURE LEGENDS
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Figure 1: Human 3D Hep/HSC co-culture characterization. (A) Spheroid formation and maintenance
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over 21 days. White scale bar represents 100 µm; upon 100 measurements, co-culture spheroid size was
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determined to be 180 ± 20µm. (B) Merged and separate pictures of CYP3A4, PDGFR-β and DAPI of 3D
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Hep/HSC paraffin sections on day 21; white scale bar represents 50 µm. Pictures are representative of
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the culture spheroids in the 3 experimental repeats. (C-F) Hepatocyte functionality of Hep-containing
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cultures; (C) Confocal image of CMFDA bile accumulation (arrows) in the Hep/HSC spheroids on day 17;
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(D) Day 21 CYP induction upon exposure to prototype CYP inducers (Rifampicin – RIF; β-Naphtoflavone –
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BNF; Phenobarbital – PB) for 48 hours. (E) Albumin secretion rate on days 7 and 21; (F) Gene expression
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of reference hepatocyte markers (Albumin; Phase I – CYP3A4; Phase II –GSTa1; transporter –
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SLCO1B1) on days 7 and 21 of culture. dCt over GAPDH values are displayed. (G) mRNA levels of HSC
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activation markers using HepaRG medium on days 7 and 21. dCt over GAPDH values are displayed.
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Error bars represent standard deviations (N=3 assays, pool of 6 spheroids/time point); *p<0.05 vs 3D
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Hep/HSC. Expression of these markers on 2D and 3D Hep cultures vary from 2.8-6x, 800-14000x and
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1000-2400x lower than in HSCs respectively for ACTA2, COL1A1 and LOX.
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Figure 2: Single exposure of human hepatic organoids to Acetaminophen. (A) Time-line of APAP
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exposure to the 3D cultures. (B) Cell viability in 3D Hep mono- and co-cultures after 24 hours incubation
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with different concentrations of APAP. Each graphs shows 3 independent experiments where each point
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represents the average % ATP over control of n=4-5 spheroids. (C) Relative mRNA gene expression of
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ACTA2, COL1A1, COL3A1 and LOXL2 in the three 3D cultures showing APAP dose-dependent gene
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transcription in 3D Hep/HSC. Each point represents the pool of 5 spheroids. *p<0.05 3D HSC vs 3D
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Hep/HSC; $p<0.05 3D Hep vs 3D Hep/HSC; N≥3 independent assays (4-6 spheroids/condition). (D)
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Procollagen measured in the supernatant 24h after incubation with APAP/solvent. N= 3 assays
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(supernatant
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Immunohistochemistry for Caspase 3, αSMA, Collagen and cross-linked collagen (Sirius-red) on paraffin
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pooled
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spheroids).
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embedded hepatic organoids exposed to 0 and 20 mM APAP. Black bars represent 50 µm. All error bars
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represent standard deviations.
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Figure 3: Hepatic organoids recapitulate several aspects of liver fibrosis. (A, C) Cell viability of the
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hepatic organoids exposed to APAP alone or together with (A) cytokine/inflammatory mixture (CK) or (C)
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Valproic Acid (VPA); (B, D, E) Relative mRNA levels of COL1A1, COL3A1 and LOXL2 from control (0 mM
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APAP) and APAP-exposed Hep/HSC organoids (5-40 mM) and the effect of co-treatment with the (C) CK
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mixture or (D) VPA and (e) APAP wash out. *p<0.05; test vs APAP alone; p<0.05 vs 0 mM APAP; N≥2
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independent assays (n=4-6 spheroids/condition).
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Figure 4: Oxidative phosphorylation and glycolysis rates in 3D human hepatic cultures. (A)
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Seahorse Mito Stress test profile with key parameters of mitochondrial function. (B) Basal respiration, ATP
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production and Spare respiration capacity of the 3D Hep/HSC co-culture and respective mono-cultures
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after 12 and 24h in low buffer capacity assay medium (0 mM APAP). (C) Oxygen consumption rate
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(OCR) and extracellular acidification rate (ECAR) of 3D cultures exposed to 10 mM APAP and respective
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controls at 12h and 24h incubation. Error bars correspond to the standard deviations of measurements of
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3-4 independent spheroids. p<0.05 vs. 12h; p<0.05 control vs 10 mM APAP (12h), *p<0.05 control vs 10
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mM APAP (24h). FCCP - Trifluoromethoxy carbonylcyanide phenylhydrazone, A+R – Antimycin A &
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Rotenone, Oligo – Oligomycin.
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Figure 5: Single- and repeated-exposure of Methotrexate and Allyl alcohol to hepatic organoids.
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(A) Schematic representation of the compound exposure experimental setup. (B, E) Cell viability
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represented by %ATP (EC50(AA-single)= 57.5 ± 8.6 µM); (C, F) mRNA levels of LOXL2 and COL1A1; (D,
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G) Procollagen secretion in culture supernatant (day 14 and 21); Error bars represent the standard
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deviations between independent assays (N=2), each condition represents a pool of 5-6 spheroids. Graph
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inserts in (C) and (F) represent the second repeat of the assay.
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Procollagen levels in supernatant of the hepatic organoids and 3D HSC or Hep mono-cultures upon 24h of
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single exposure at day 20. (I) Brigthfield images of Hep/HSC organoids at day 21 after single or repeated
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exposure to the different compounds. (J) Immunohistochemical detection of cross-linked collagen (Sirius-
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red), Collagen1 and αSMA in sections of day 21 Hep/HSC organoids (single and repeated exposure).
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Black bar represents 100 µm.
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Figure 6: In vivo data obtained from BALB/c mice after single and repeated exposure to APAP. (A)
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ALT blood levels 24h after a single exposure to 300 mg/kg APAP or vehicle control (B) mRNA levels of
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Acta2, Col1a1 and Lox in mouse HSCs isolated 24h after a single injection of 300 mg APAP/kg and the
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vehicle control; (C, D) Sirius-red images and quantification of the percentage of red stained surface in
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mice livers after 4 weeks of repeated exposure to APAP or CCl4.
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S0142-9612(15)00922-9
DOI:
10.1016/j.biomaterials.2015.11.026
Reference:
JBMT 17203
To appear in:
Biomaterials
Received Date: 24 September 2015 Revised Date:
30 October 2015
Accepted Date: 13 November 2015
Please cite this article as: Leite SB, Roosens T, El Taghdouini A, Mannaerts I, Smout AJ, Najimi M, Sokal E, Noor F, Chesne C, van Grunsven LA, Novel human hepatic organoid model enables testing of drug-induced liver fibrosis in vitro, Biomaterials (2015), doi: 10.1016/j.biomaterials.2015.11.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Novel human hepatic organoid model enables testing of drug-induced liver
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fibrosis in vitro
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Sofia B. Leite , Tiffany Roosens , Adil El Taghdouini , Inge Mannaerts , Ayla J. Smout , Mustapha Najimi , 2 3 4 1* Etienne Sokal , Fozia Noor , Christophe Chesne , Leo A. van Grunsven
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*Correspondence to: Prof. Leo van Grunsven ([email protected]) or Dr. Sofia Batista Leite ([email protected]). Faculty of Medicine and Pharmacy, Laarbeeklaan 103, 1090 Brussel, Building D Ground Floor Room D022/025B. TEL (+32) 2 477 44 09;
Liver Cell Biology Laboratory, Vrije Universiteit Brussel (VUB), Belgium
Laboratory of Pediatric Hepatology and Cell Therapy, Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain (UCL), Belgium
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Biochemical Engineering Institute, Saarland University, Germany
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List of abbreviations (by order of appearance): HSC, Hepatic Stellate Cell; Hep, HepaRG; 3D, threedimensional; Hep/HSC, Hep and HSC co-culture, hepatic organoid; PDGFR-β, Platelet derived growth factor receptor β; CYP, cytochrome P450; RIF, Rifampicin; BNF, β-Naphtoflavone; PB, Phenobarbital; MRP2, Multidrug resistance-associated protein 2; CMFDA, 5-chloromethylfluorescein diacetate; 2D, twodimensional; mRNA, messenger Ribonucleic Acid; dCT, deltaCT, gene expression relative to housekeeping gene; TGFβ, Transforming Growth Factor β; LPS, Lipopolysaccharide; APAP, Acetaminophen; ATP, Adenosine Triphosphate; αSMA, alpha Smooth Muscle Actin; HDAC, Histone deacetylase; CK, Cytokine mixture; VPA, Valproic Acid; CCl4, Carbon Tetrachloride; EC50, Half Maximal Effective Concentration; OCR, Oxygen consumption rate; ECAR, Extracellular acidification; Oligo, Oligomycin; FCCP, Trifluoromethoxy carbonylcyanide phenylhydrazone; A+R, Antimycin A and Rotenone; MTX, Methotrexate; ALT, Alanine Aminotransferase; AOP, Adverse Outcome Pathway; DILI, Drug-induced liver injury.
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ABSTRACT
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Current models for in vitro fibrosis consist of simple mono-layer cultures of rodent hepatic
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stellate cells (HSC), ignoring the role of hepatocyte injury. We aimed to develop a method
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allowing the detection of hepatocyte-mediated and drug-induced liver fibrosis. We used HepaRG
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(Hep) and primary human HSCs cultured as 3D spheroids in 96-well plates. These resulting
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scaffold-free organoids were characterized for CYP induction, albumin secretion, and hepatocyte
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and HSC-specific gene expression by qPCR. The metabolic competence of the organoid over 21
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days allows activation of HSCs in the organoid in a drug- and hepatocyte-dependent manner.
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After a single dose or repeated exposure for 14 days to the pro-fibrotic compounds Allyl alcohol
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and Methotrexate, hepatic organoids display fibrotic features such as HSC activation, collagen
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secretion and deposition. Acetaminophen was identified by these organoids as an inducer of
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hepatotoxic-mediated HSC activation which was confirmed in vivo in mice. This novel hepatic
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organoid culture model is the first that can detect hepatocyte-dependent and compound-induced
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HSC activation, thereby representing an important step forward towards in vitro compound
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testing for drug-induced liver fibrosis.
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Keywords: organoid; liver fibrosis; DILI; in vitro; hepatic stellate cell; Hepatocyte; HepaRG; APAP; Methotrexate; Allyl alcohol
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INTRODUCTION
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Hepatic stellate cells (HSCs) are the major collagen producing cells during conditions of
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sustained hepatic injury (either metabolic, cholestatic, viral or toxic), when hepatocyte damage
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triggers a cascade of events leading to activation of quiescent HSCs into a myofibroblastic
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(activated) HSC state1. HSC activation is mediated by a plethora of pathways that finally result in
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increased secretion of extracellular matrix proteins, such as collagens, that accumulate as scar
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tissue (fibrosis) within the liver parenchyma and to liver cirrhosis in a later stage2. To date, the
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best in vitro fibrosis models consist of mono-layer cultures of freshly isolated rodent HSCs in
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regular tissue culture dishes which leads to “spontaneous” HSC activation3. Obvious limitations
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of these cultures are the rodent background and the un-controlled and hepatocyte damage-
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independent activation of the HSCs, making these cultures less suitable for pro- and anti-fibrotic
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compound testing translatable to human.
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We developed a novel three-dimensional (3D) human co-culture model where both hepatocyte
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functionality and HSC quiescence can be maintained for at least 21 days. This novel system
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allows hepatotoxicity testing as well as drug-provoked and hepatocyte-dependent HSC
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activation and fibrosis.
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MATERIAL AND METHODS
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Cell culture
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HepaRG/HSC cell culture. Human liver non-parenchymal fractions isolation were obtained from
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the Agreed Hepatocytes & Hepatic Stem cell Bank (Saint-Luc Hospital and Université Catholique
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de Louvain). This raw material (obtained after written and signed informed consent) was
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processed for Hepatic Stellate Cell (HSC) isolation and prepared for culture as previously
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described4-6. Cells from two different donors were used, both male, healthy and under 13 years
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old. For a thorough characterization of the cells we refer to El Taghdouini et. al.7 and Coll & El
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Taghdouini et. al.4. For the 3D cultures cells were used between passages 5 and 10.
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Differentiated cryopreserved HepaRG® cells were obtained from Biopredic. On day 0 of culture,
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HSCs were trypsinized and HepaRG (Hep) thawed according to the instructions of the provider.
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For the generation of the cell spheroids, 96-well plates treated with cell-repellent (Greiner) were
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used. Mono- and co-culture suspensions were prepared and seeded in the cell densities shown
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below:
Hep seeding 5 (x10 cells/ml)
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After 15-30 min in the incubator, plates were placed on an orbital shaker, where stirring was kept
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at 80 rpm for the entire culture period. The outer wells were filled with 100 µl of PBS to minimize
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liquid evaporation. Culture medium was refreshed (90%) every second day. 3D Hep/HSC co-
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cultures and control mono-cultures were cultured for 21 days. Cross diameter of the spheroids
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never exceeded 200 µm.
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HepaRG/HSC culture medium. Day 0 cells were resuspended in HepaRG thawing medium and
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from day 1 to 21 cells were kept in the HepaRG culture medium 0% DMSO (Biopredic), unless
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otherwise stated. In the APAP proof-of-concept studies, medium was replaced by HepaRG
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Induction/Toxicity medium with 0.1% (v/v) DMSO (Sigma-Aldrich, St. Louis, MO, USA) during
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compound incubation. In the repeated versus single compound exposure assays, on day 8, the
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HepaRG culture medium 0% DMSO was replaced by the Serum-free HepaRG maintenance
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medium described in Klein et. al. 20138 and kept in this medium until the end of culture
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The remaining material and methods are available in supplementary information.
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92 RESULTS
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Characterization of human liver organoid cultures
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We use cryopreserved differentiated HepaRGs (Heps) as functional hepatocyte-like cells and
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primary human HSCs for the development of the human 3D hepatic organoids. Differentiated
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HepaRG, widely characterized in the last decade, show a close correlation with human
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hepatocyte functions9 which are improved when cultured in 3D10, 11. HSCs are isolated from the
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non-parenchymal fraction of human livers, expanded in culture and frozen down as previously
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described4, 6. Liver organoids are generated by mixing the two single-cell suspensions in a ratio
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of 1Heps:2HSCs in non-attachment round-bottom 96 well-plate wells with orbital stirring. This
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allows for quick generation of spheroids with an optimal diameter of ≤200 µm, representing the
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maximum physiological distance between a cell and a blood vessel12 and precludes the
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formation of necrotic cores13 (Fig. 1A). The diameter variation at day 7 and 21 among the co-
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culture spheroids is 190µm ± 10 µm (n=100) while only 4% of cells incorporate EdU during a 48h
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exposure (Suppl. Fig. 1A), illustrating the non-proliferative nature of the cells. The 3D Hep/HSC
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spheroids demonstrate a segregated organization with a concentration of HSCs in the core of
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the spheroid (PDGFR-β positive cells, with a small elongated nucleus, Fig. 1B) while
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hepatocyte-like cells (CYP3A4 positive cells, with large round nuclei, Fig. 1B) accumulate
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preferentially in the periphery. Phase I hepatocyte metabolic capacity is demonstrated by
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exposing the cells to prototype cytochrome P450 inducers for CYP3A, CYP1A2 and CYP2C9.
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Even though there are less hepatocyte-like cells in the co-culture spheroids, the inducibility of
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the cells shows little variation between mono- and co-cultures of Heps (Fig. 1D). Likewise, CYP-
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induction values are in agreement with CYP-inducing capacities of Heps maintained in similar
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conditions (Suppl. Table 4). Additionally, CYP3A4 staining of 3D Hep/HSC spheroids indicates
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the presence of mature hepatocytes (± 20% of all spheroid cells is CYP3A4 positive, n=4). Note
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that differentiated HepaRGs are 50% hepatocyte-like while the other 50% are cholangiocyte-like
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cells14, thus only 1/6 of the spheroid can be hepatocyte-like cells (Fig. 1B). Excretion of the
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MRP2 substrate CMFDA15 and its accumulation in canulicular structures is only observed after
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fixation, underlining the good elimination capacity of the spheroids (Fig. 1C, arrows). Albumin
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secretion is similar in 3D Hep and 3D Hep/HSC cultures, but is manifold higher than in 2D Hep
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cultures (Fig. 1E). Finally, mRNA levels of hepatocyte markers such as ALB, CY3A4, GSTA1
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and SLCO1B1 is maintained in 3D Hep/HSC cultures at comparable levels to Hep mono-
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cultures and in some cases higher than 2D Hep (Fig. 1F) despite the lower amount of
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hepatocyte-like cells. In the presence of HepaRG culture medium, human 3D HSCs still activate
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from day 7 onwards (Fig. 1G) while in 3D Hep/HSC co-cultures they do not, as can be
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evidenced by the low mRNA levels of ACTA2, COL1A1 and LOX on day 21. Due to the
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mesenchymal origin HepaRG still express these markers, but in much lower extent when
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compared with the HSC-containing cultures (Fig. 1G). Nevertheless the HSCs in the co-culture
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spheroids are still capable of responding to pro-fibrogenic stimuli such as TGFβ and LPS
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(Suppl. Fig. 1B and C), known to have direct effects on HSCs.
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Taken together, these experiments demonstrate functionality of both cell types in 3D Hep/HSC
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cultures and we therefore refer to these cultures as human hepatic organoids. Despite the good
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cellular profile of these organoids after 7 days in culture, we mainly focus on 21 day culture
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characterization which perhaps allows long term repeated dose toxicity testing.
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Drug-induced hepatic injury by acetaminophen
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- Single exposure of human hepatic organoids to Acetaminophen
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To show the drug responsiveness of the organoids we used Acetaminophen (APAP) and
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compared it to the respective mono-cultures for specificity of the response. APAP was selected
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for its kinetic stability, known effective in vitro concentrations, specific toxicity in hepatocytes and
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high human relevance16. After a 24h APAP exposure at day 20, we observe only small
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differences in toxicity between the 3D organoids and the 3D Hep cultures (Fig. 2A,B),
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suggesting a good CYP2E1 metabolism in both cultures17. Nevertheless, there is a tendency for
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higher toxicity in the organoids, more pronounced on day 21 than on day 7 (Suppl. Table 5).
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Upon APAP exposure, Heps disappear from the periphery of the organoids, probably by
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necrosis-mediated cell death, while the ones remaining in a ring surrounding the HSC core are
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caspase 3-positive apoptotic cells (Fig. 2E), representing the second path of APAP-induced
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hepatocyte injury18. Although not known as a pro-fibrotic agent, metabolization of APAP by
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functional hepatocytes in the organoid leads to toxicity mainly in hepatocytes and not HSCs, but
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might lead to hepatocyte-injury dependent HSC activation. Indeed, we detect a strong dose-
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dependent up-regulation of HSC activation-associated mRNAs COL1A1, COL3A1 and LOXL2
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only in the hepatic organoids (Fig. 2C), suggesting HSC activation in response to hepatocyte
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injury. In Hep mono-cultures, APAP exposure also leads to a 2-5 fold increase of collagen
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mRNA levels, however the basal levels of collagen expression are 10-100 fold lower in 3D Hep
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cultures when compared to the organoids (Suppl. Fig. 2A), highlighting the activation state of
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the HSCs, the main producers of collagen in the liver1. Measurements of C-terminal pro-collagen
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I peptide in the culture supernatant show an APAP-dependent increase of collagen protein only
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in the organoid cultures (Fig. 2D and Suppl. Fig. 2B). ACTA2 mRNA levels do not change upon
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APAP exposure in any of the cultures suggesting that the indirect APAP-induced HSC activation
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is independent of TGFβ, since ACTA2 transcription depends largely on TGFβ signaling19. Yet,
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we can detect a slight increase in αSMA protein levels in the center of the organoids after APAP
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exposure (Fig. 2E). Additionally, we confirm collagen production, by immuno-staining and by
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detection of cross-linked collagens (Sirius-red) in sections of hepatic organoids (Fig. 2E).
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- Hepatic organoids recapitulate several aspects of liver fibrosis
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Our results with APAP exposed organoids suggest that besides the known hepatotoxicity, APAP
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has the capacity to induce HSC activation through hepatocyte injury. This prompted us to
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investigate drug-dependent HSC activation further by addressing in these organoids three in
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vivo features of HSC activation: (i) enhanced HSC activation due to inflammation20
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inhibition of HSC activation by histone deacetylase (HDAC) inhibition; and (iii) recovery from
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HSC activation. We mimic the cytokine release from immune cells22 by performing the APAP
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exposure in the presence of a cytokine mixture (CK: TNFα, IL1β, IFNα and IFNγ). While CK
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alone (0 mM APAP+CK) has no effect on the culture read-outs, when combined with APAP, the
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toxic effect is potentiated and the up-regulation of COL1A1, COL3A1 and LOXL2 mRNA is
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increased (Fig. 3A, B). We then used valproic acid (VPA), an HDAC inhibitor that inhibits HSC
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activation induced by CCl4 in mice23. Similar to in vivo, VPA does not affect APAP toxicity in the
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organoids while up-regulation of the activation markers at the highest APAP concentrations was
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inhibited (Fig. 3C, D). Finally, organoids cultured for 3 additional days after APAP washout,
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show a decrease in HSC activation markers (Fig. 3E) indicative of HSC recovery24.
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APAP seems to affect HSC mono-cultures only at the highest concentrations (EC50 = 31.4
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±11mM). Instead of the CYP-mediated metabolization of APAP into NAPQI which is the main
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toxic pathway in hepatocytes, this effect could also be linked to mitochondrial oxidative stress25,
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26
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assessed the mitochondrial functions and glycolytic activity by measuring the oxygen
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consumption rate (OCR) and the extracellular acidification rates (ECAR) respectively (Fig. 4A).
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Comparison of the basal respiration, ATP production and spare respiratory capacity in the
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untreated control samples (Fig. 4B), suggests that the hepatic organoid cultures are more stable
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than the equivalent mono-cultures. Furthermore, when comparing Hep-containing cultures with
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the HSC mono-cultures, all parameters are roughly ten times higher. Especially in the case of
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ATP, this seems to indicate that Heps are the major production source in the organoids and
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suggests that the observed ATP-drop after APAP incubation in the hepatic organoids (Fig. 2B)
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is mainly a consequence of Hep toxicity. Additionally, at 24h, a shift towards a more glycolytic
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metabolism (higher ECAR and lower OCR) is only seen in 3D Hep mono-cultures, which in the
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HSC monocultures was already high, but not in the organoids. In the hepatic organoids, a
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general OCR decrease is observed, only after 24 hours of APAP exposure (Fig. 4C), while in the
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respective mono-cultures this is observed already after 12 hours. Together, this shows that there
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is little influence of APAP on mitochondrial functions in the hepatic organoids in contrast to the
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respective mono-cultures. These findings are in line with the mitochondrial resistance observed
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in hepatocytes during early stages of fibrosis in vivo27.
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Single and repeated exposure of hepatic organoids to Methotrexate and Allyl alcohol.
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To further explore the hepatic organoids as a read-out for drug-induced liver fibrosis we exposed
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the cultures to the reference pro-fibrotic compounds Allyl alcohol, known to induce fibrosis in
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rat28, and Methotrexate (MTX) which is classified to induce liver fibrosis in human subjects after
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prolonged exposure29. Since the fibrotic effect of these compounds in vivo is only observed upon
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long-term exposure, both compounds were tested in single and repeated doses (and compared
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to APAP for HSC activation (Suppl. Fig. 4A-C)). Serum-free culture conditions were established
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to avoid interference of the serum during repeated compound exposure30,8 (Suppl. Fig. 5A). The
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two exposure profiles are compared by assessing the cells on day 21, 24h after the last
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compound dose (Fig. 5A). Single MTX exposure does not affect cell viability at any of the tested
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concentrations (Fig. 5B). However, upon repeated exposure, the percentage of ATP production,
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even at the lowest concentration, decreases to less than 20%. Although repeated exposure to
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1.9 µM MTX leads to smaller spheroids, the amount of disaggregating cells around the spheroid
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is less than in organoids repeatedly exposed to 5 mM APAP (Fig. 5I and Suppl. Fig. 4D),
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suggesting that the lower ATP levels not necessarily represent cell death. While single exposure
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to MTX does not lead to an upregulation of activation markers at the mRNA level in the
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organoids, repeated exposure to MTX clearly induces HSC activation (Fig. 5C and Suppl. Fig.
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6B). Positive Collagen1 and αSMA staining in cross sections of the hepatic organoid however
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demonstrates that HSCs in the core of the spheroids are activated after both single and
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repeated exposures (Fig. 5J). Sirius-red stainings are more in line with the mRNA levels of
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activation showing a much stronger deposition of cross-linked collagen in the repeated dose
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MTX setting than after a single exposure.
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As for the case of Allyl alcohol, the clear dose-response observed at single exposure almost
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disappears upon repeated exposure, letting us to presume induced toxicity resistance of the
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cells (Fig. 5E). This can be explained by the inhibition of its metabolism to acrolein31 observed
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during rat and mice exposures32. In single exposure, despite the drop in ATP at high
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concentrations it does not affect the spheroid integrity (Fig. 5I,J). Both operational exposures
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show an increased tendency of mRNA levels of HSC activation (Fig. 5F and Suppl. Fig. 6C).
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While at the mRNA level the difference between single and repeated exposure to Allyl alcohol is
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not as outspoken as for MTX, at the protein level, there is a strong fibrotic response in the
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repeated exposures leading to a ∼100 fold increase of secreted collagen at day 14 (Fig. 5G) and
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a strong accumulation and cross-linked collagen at the end of the experiment, shown by Sirius-
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red and collagen stainings (Fig. 5J).
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For both Allyl alcohol and MTX, repeated exposure shows high collagen secretion at day 14, 48h
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after the 3rd exposure (Fig. 5D,G). At the end of the culture, the 24h pro-collagen secretion is
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comparable to the single exposure in the case of Allyl alcohol, but lower in the case of MTX (Fig.
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5D). For both compounds, nearly no collagen secretion is detected in the 3D mono-cultures (Fig.
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5H).
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Finally, APAP caused toxicity and HSC activation both in single and repeated-exposures, with a
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shift to lower APAP concentrations for the repeated dose (EC50 from 10.4 ±2.3 mM to 2.25 ±0.35
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mM APAP; Suppl. Fig. 4A). HSC activation followed with a peak at around 5-10 mM for the
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repeated exposure instead of 20-40 mM (Suppl. Fig. 4B and Suppl. Fig. 6A).
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Once again, the capacity of the cultures to recover from the insult was analyzed 3 days after
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compound washout, however recovery was only observed in APAP single exposures and 63 µM
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MTX repeated exposures (Suppl. Fig. 7) which most likely reflects the mechanism by which
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these compounds induce HSC activation.
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Pro-fibrotic effect of APAP in vivo
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Acute liver failure as a consequence of exposure to high doses of APAP has been widely
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reported in animals and humans33, but so far there are no reports describing a fibrotic response
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of APAP. Since our results with hepatic organoids indicated that APAP can indirectly induce
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HSC activation, we next investigated the in vivo fibrotic potential of APAP in mice. Blood
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analysis 24h post-injection demonstrates increased alanine transaminase (ALT) levels indicating
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hepatocyte toxicity upon exposure of regular chow-fed mice to 300-500 mg/kg APAP (Fig. 6A).
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300mg/kg APAP is already sufficient for HSC activation within 24 hours, demonstrated by
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increased mRNA levels of Acta2, Col1a1 and Lox in freshly isolated HSCs (Fig. 6B). Bi-weekly
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injections of APAP for 4 and 8 weeks (300 mg/Kg), results in the formation of scar tissue and
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increased αSMA expression in BALB/c and C57BL/6 mice, but has the tendency to decline over
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time (Fig. 6C and Suppl. Fig. 8). Indeed, when compared to CCl4-induced liver injury, APAP
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treatment appears to be less fibrogenic (Fig. 6C, D).
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DISCUSSION
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The microenvironmental factors that can affect a cell’s performance in culture are countless,
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varying from chemical medium composition, mechanical factors or even diffusion rates. In the
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case of HSCs, rigidity of the matrix to which they are attached at least partly determines their
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activation state3. We report, for the first time, metabolically active human hepatic organoids in
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which HSCs maintain a quiescent-like state for 21 days while retaining their capacity to respond
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to pro-fibrotic compounds directly and in a hepatocyte-dependent manner.
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In the last years, simplified models for the use in chemical risk assessment have been
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developed, the so-called Adverse Outcome Pathways - AOP (https://aopkb.org) which should
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finally lead to an integration of all AOPs into one large network34. A good predictive in vitro
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method should at least recapitulate some of the key events of such AOP (Suppl. Fig. 9). The
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developed human hepatic organoid consisting of differentiated HepaRGs and primary HSCs is to
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our knowledge the first in vitro method that mimics drug-induced liver fibrosis and could be used
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to further optimize the AOP of liver fibrosis.
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The HepaRG to HSC cell-ratio of 1:2 and the hepatocyte-specific medium guarantees HSC
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quiescense and keeps the hepatocytes functional. Although the used ratio is far from the
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physiological ratio, we believe that in these organoids the HSCs work as feeder cells to support
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hepatocyte functions35. The central accumulation of HSCs in the organoids is most likely due to
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their stronger capacity to aggregate at the time of spheroid formation; HSCs are cultured in 2D
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beforehand while the HepaRG cells are freshly thawn and immediately put in 3D co-culture. We
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did question the use of primary HSCs and differentiated HepaRG cells by culturing organoids
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consisting of the widely used HepG2 and LX-2 cells, hepatocyte and HSC lines respectively.
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Besides the fact that cells in these organoids keep proliferating, the LX-2 cells activate over time
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without injury and the organoids are much less sensitive to APAP, probably reflecting the low
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hepatocyte functionality of the HepG2 cells (Suppl. Fig. 10).
284
The use of Allyl alcohol and MTX confirms the applicability of the method by showing HSC
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activation only in the organoids and not in 3D mono-cultures. The upregulation of some of the
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activated HSC-associated mRNAs in 3D Hep mono-cultures (albeit remaining at relatively low
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levels) might be due to an EMT-like process due to the treatment with MTX or Allyl alcohol.
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While EMT-like differentiation of hepatocytes is readily observed in vitro36, this does not
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contribute to liver fibrosis formation in vivo1.
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In contrast, repeated-dose exposure of the 3 tested compounds resulted in a significantly
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higher, and sometimes unique up-regulation of COL1A1, COL1A3 and LOXL2 in the organoids
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(Suppl. Fig. 11). These results demonstrate that only the hepatic organoids, and not their
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respective 3D mono-cultures, have the capacity to respond positively to the reference fibrotic
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compounds Methotrexate and Allyl alcohol.
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Even the hepatotoxic-independent HSC activation observed after repeated exposure to Allyl
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alcohol is hepatocyte-dependent since the effect is not observed in the HSC mono-cultures
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(Suppl. Fig. 11C). This highlights the potential of the hepatic organoids to not only “just” identify
298
hepatotoxic compounds that might cause indirect HSC activation (i.e. APAP), but also by
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compounds that are not hepatotoxic but need the presence of functional hepatocytes to
300
indirectly activate HSCs (i.e. Allyl alcohol at the tested concentrations). The in vivo confirmation
301
of the unexpected fibrotic potential of APAP identified in the organoid cultures, highlights the
302
potential to identify thus far unknown fibrotic compounds in vitro that can actually cause fibrosis
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in vivo. The lack of literature describing this fibrotic potential of APAP could be due to the much
304
lower profibrotic capacity than for instance CCl4 and the decline of this effect over time. The
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report from Watelet and co-workers on preliminary APAP fibrosis evidence in humans37
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suggests that this assessment should perhaps be pursued.
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In conclusion, we have developed functional human hepatic organoids that can identify
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compounds that induce fibrosis, a drug-induced liver injury (DILI) rarely addressed in vitro unlike
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steatosis, choleastatis and phospholipidosis. This is a big step forward from the regular 2D HSC
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cultures that are generally used. The hepatic organoids are suitable for repeated dosage and are
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sensitive to the nature of the compounds by displaying differential toxicity and HSC activation
312
profiles. These hepatic organoids represent a substantial improvement when screening for drug-
313
induced liver fibrosis in terms of cost, animal use and prediction of liver fibrosis in human.
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Furthermore, this technology could stimulate the development of culture models representative
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of fibrosis in other organs such as lung and kidney, since these share common mechanisms38, 39.
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ACKNOWLEDGMENTS
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First we would like to acknowledge the finantial support from different entities: T. Roosens and A. El
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Taghdouini are funded by the Institute for the Promotion of Innovation through Science and Technology in
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Flanders (IWT-Vlaanderen; SB/13170 and SB/111008). I. Mannaerts is supported by a Fund of Scientific
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Research Flanders FWO-V post-doctoral fellowships (12N5415N LV). This work was supported by the
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HeMiBio consortium funded by the European Commission and Cosmetics Europe as part of the SEURAT-
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1 cluster (N° HEALTH-F5-2010-266777) and by the IWT project HILIM-3D (SBO 140045).
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Furthermore, we thank P. Antony (Université Du Luxembourg) for the possibility to use the XFe96
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Analyser, S. Kalaydjiev and D. Buurman (Seahorse Bioscience) for the help in the performance and
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interpretation of the Mito Stress test, A. P. Batista (ITQB) for the help in the interpretation of the ECAR and
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OCR results.
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REFERENCES
332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359
1.
5.
6. 7. 8.
9.
10. 11. 12. 13.
TE D
4.
EP
3.
Mederacke, I. et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 4, 2823 (2013). Friedman, S.L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 88, 125-172 (2008). Olsen, A.L. et al. Hepatic stellate cells require a stiff environment for myofibroblastic differentiation. Am J Physiol Gastrointest Liver Physiol 301, G110-118 (2011). Coll, M. et al. Integrative miRNA and Gene Expression Profiling Analysis of Human Quiescent Hepatic Stellate Cells. Sci Rep 5, 11549 (2015). Dusabineza, A.C. et al. Hepatic stellate cells improve engraftment of human primary hepatocytes: A pre-clinical transplantation study in animal model. Cell Transplant, doi: 10.3727/096368915X096686788 (2015). Thoen, L.F. et al. A role for autophagy during hepatic stellate cell activation. J Hepatol 55, 13531360 (2011). EL Taghdouini, A. et al. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells. Oncotarget (2015). Klein, S., Mueller, D., Schevchenko, V. & Noor, F. Long-term maintenance of HepaRG cells in serum-free conditions and application in a repeated dose study. J Appl Toxicol 34, 1078-1086 (2014). Lubberstedt, M. et al. HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro. J Pharmacol Toxicol Methods 63, 59-68 (2011). Leite, S.B. et al. Three-dimensional HepaRG model as an attractive tool for toxicity testing. Toxicol Sci 130, 106-116 (2012). Gunness, P. et al. 3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. Toxicol Sci 133, 67-78 (2013). Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249-257 (2000). Folkman, J. & Hochberg, M. Self-regulation of growth in three dimensions. J Exp Med 138, 745753 (1973).
AC C
2.
M AN U
331
14
ACCEPTED MANUSCRIPT
19. 20. 21.
22. 23. 24. 25.
26.
27. 28. 29. 30. 31. 32. 33. 34.
RI PT
18.
SC
17.
M AN U
16.
TE D
15.
Antherieu, S., Rogue, A., Fromenty, B., Guillouzo, A. & Robin, M.A. Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells. Hepatology 53, 1895-1905 (2011). Bogman, K., Erne-Brand, F., Alsenz, J. & Drewe, J. The role of surfactants in the reversal of active transport mediated by multidrug resistance proteins. Journal of pharmaceutical sciences 92, 1250-1261 (2003). Antoine, D.J. et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology 58, 777787 (2013). Lee, S.S., Buters, J.T., Pineau, T., Fernandez-Salguero, P. & Gonzalez, F.J. Role of CYP2E1 in the hepatotoxicity of acetaminophen. J Biol Chem 271, 12063-12067 (1996). Nakagawa, H. et al. Deletion of apoptosis signal-regulating kinase 1 attenuates acetaminopheninduced liver injury by inhibiting c-Jun N-terminal kinase activation. Gastroenterology 135, 13111321 (2008). Herrmann, J., Haas, U., Gressner, A.M. & Weiskirchen, R. TGF-beta up-regulates serum response factor in activated hepatic stellate cells. Biochim Biophys Acta 1772, 1250-1257 (2007). Marques, P.E. et al. Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Hepatology 56, 1971-1982 (2012). Pradere, J.-P. et al. Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice. Hepatology 58, 1461-1473 (2013). Aoyama, T., Paik, Y.H. & Seki, E. Toll-like receptor signaling and liver fibrosis. Gastroenterol Res Pract 2010, 1-8 (2010). Mannaerts, I. et al. Chronic administration of valproic acid inhibits activation of mouse hepatic stellate cells in vitro and in vivo. Hepatology 51, 603-614 (2010). Kisseleva, T. et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci U S A 109, 9448-9453 (2012). Chen, C., Krausz, K.W., Idle, J.R. & Gonzalez, F.J. Identification of novel toxicity-associated metabolites by metabolomics and mass isotopomer analysis of acetaminophen metabolism in wild-type and Cyp2e1-null mice. J Biol Chem 283, 4543-4559 (2008). Prill, S. et al. Real-time monitoring of oxygen uptake in hepatic bioreactor shows CYP450independent mitochondrial toxicity of acetaminophen and amiodarone. Arch Toxicol, doi:10.1007/s00204-00015-01537-00202 (2015). Mitchell, C. et al. Protection against hepatocyte mitochondrial dysfunction delays fibrosis progression in mice. Am J Pathol 175, 1929-1937 (2009). Jung, S.A. et al. Experimental model of hepatic fibrosis following repeated periportal necrosis induced by allylalcohol. Scand J Gastroenterol 35, 969-975 (2000). Lindsay, K. et al. Liver fibrosis in patients with psoriasis and psoriatic arthritis on long-term, high cumulative dose methotrexate therapy. Rheumatology (Oxford) 48, 569-572 (2009). Pomponio, G. et al. In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments. Toxicol In Vitro (2014). Covaci, O.I., Bucur, B. & Radu, G.L. Acrolein detection based on alcohol dehydrogenase inhibition. International Journal of Environmental Analytical Chemistry 93, 325-334 (2013). Auerbach, S.S., Mahler, J., Travlos, G.S. & Irwin, R.D. A comparative 90-day toxicity study of allyl acetate, allyl alcohol and acrolein. Toxicology 253, 79-88 (2008). Kaplowitz, N. Idiosyncratic drug hepatotoxicity. Nat Rev Drug Discov 4, 489-499 (2005). Vinken, M. The adverse outcome pathway concept: a pragmatic tool in toxicology. Toxicology 312, 158-165 (2013).
EP
14.
AC C
360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407
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ACCEPTED MANUSCRIPT
35.
36. 37. 38. 39.
Leite, S.B. et al. Merging bioreactor technology with 3D hepatocyte-fibroblast culturing approaches: Improved in vitro models for toxicological applications. Toxicol In Vitro 25, 825-832 (2011). Nambotin, S.B., Tomimaru, Y., Merle, P., Wands, J.R. & Kim, M. Functional consequences of WNT3/Frizzled7-mediated signaling in non-transformed hepatic cells. Oncogenesis 1, e31 (2012). Watelet, J. et al. Toxicity of chronic paracetamol ingestion. Aliment Pharmacol Ther 26, 15431544; author reply 1545-1546 (2007). Friedman, S.L., Sheppard, D., Duffield, J.S. & Violette, S. Therapy for fibrotic diseases: nearing the starting line. Sci Transl Med 5, 167sr161 (2013). Henderson, N.C. et al. Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med 19, 1617-1624 (2013).
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FIGURE LEGENDS
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Figure 1: Human 3D Hep/HSC co-culture characterization. (A) Spheroid formation and maintenance
422
over 21 days. White scale bar represents 100 µm; upon 100 measurements, co-culture spheroid size was
423
determined to be 180 ± 20µm. (B) Merged and separate pictures of CYP3A4, PDGFR-β and DAPI of 3D
424
Hep/HSC paraffin sections on day 21; white scale bar represents 50 µm. Pictures are representative of
425
the culture spheroids in the 3 experimental repeats. (C-F) Hepatocyte functionality of Hep-containing
426
cultures; (C) Confocal image of CMFDA bile accumulation (arrows) in the Hep/HSC spheroids on day 17;
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(D) Day 21 CYP induction upon exposure to prototype CYP inducers (Rifampicin – RIF; β-Naphtoflavone –
428
BNF; Phenobarbital – PB) for 48 hours. (E) Albumin secretion rate on days 7 and 21; (F) Gene expression
429
of reference hepatocyte markers (Albumin; Phase I – CYP3A4; Phase II –GSTa1; transporter –
430
SLCO1B1) on days 7 and 21 of culture. dCt over GAPDH values are displayed. (G) mRNA levels of HSC
431
activation markers using HepaRG medium on days 7 and 21. dCt over GAPDH values are displayed.
432
Error bars represent standard deviations (N=3 assays, pool of 6 spheroids/time point); *p<0.05 vs 3D
433
Hep/HSC. Expression of these markers on 2D and 3D Hep cultures vary from 2.8-6x, 800-14000x and
434
1000-2400x lower than in HSCs respectively for ACTA2, COL1A1 and LOX.
435
Figure 2: Single exposure of human hepatic organoids to Acetaminophen. (A) Time-line of APAP
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exposure to the 3D cultures. (B) Cell viability in 3D Hep mono- and co-cultures after 24 hours incubation
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with different concentrations of APAP. Each graphs shows 3 independent experiments where each point
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represents the average % ATP over control of n=4-5 spheroids. (C) Relative mRNA gene expression of
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ACTA2, COL1A1, COL3A1 and LOXL2 in the three 3D cultures showing APAP dose-dependent gene
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transcription in 3D Hep/HSC. Each point represents the pool of 5 spheroids. *p<0.05 3D HSC vs 3D
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Hep/HSC; $p<0.05 3D Hep vs 3D Hep/HSC; N≥3 independent assays (4-6 spheroids/condition). (D)
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Procollagen measured in the supernatant 24h after incubation with APAP/solvent. N= 3 assays
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(supernatant
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Immunohistochemistry for Caspase 3, αSMA, Collagen and cross-linked collagen (Sirius-red) on paraffin
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embedded hepatic organoids exposed to 0 and 20 mM APAP. Black bars represent 50 µm. All error bars
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represent standard deviations.
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Figure 3: Hepatic organoids recapitulate several aspects of liver fibrosis. (A, C) Cell viability of the
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hepatic organoids exposed to APAP alone or together with (A) cytokine/inflammatory mixture (CK) or (C)
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Valproic Acid (VPA); (B, D, E) Relative mRNA levels of COL1A1, COL3A1 and LOXL2 from control (0 mM
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APAP) and APAP-exposed Hep/HSC organoids (5-40 mM) and the effect of co-treatment with the (C) CK
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mixture or (D) VPA and (e) APAP wash out. *p<0.05; test vs APAP alone; p<0.05 vs 0 mM APAP; N≥2
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independent assays (n=4-6 spheroids/condition).
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Figure 4: Oxidative phosphorylation and glycolysis rates in 3D human hepatic cultures. (A)
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Seahorse Mito Stress test profile with key parameters of mitochondrial function. (B) Basal respiration, ATP
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production and Spare respiration capacity of the 3D Hep/HSC co-culture and respective mono-cultures
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after 12 and 24h in low buffer capacity assay medium (0 mM APAP). (C) Oxygen consumption rate
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(OCR) and extracellular acidification rate (ECAR) of 3D cultures exposed to 10 mM APAP and respective
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controls at 12h and 24h incubation. Error bars correspond to the standard deviations of measurements of
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3-4 independent spheroids. p<0.05 vs. 12h; p<0.05 control vs 10 mM APAP (12h), *p<0.05 control vs 10
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mM APAP (24h). FCCP - Trifluoromethoxy carbonylcyanide phenylhydrazone, A+R – Antimycin A &
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Rotenone, Oligo – Oligomycin.
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Figure 5: Single- and repeated-exposure of Methotrexate and Allyl alcohol to hepatic organoids.
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(A) Schematic representation of the compound exposure experimental setup. (B, E) Cell viability
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represented by %ATP (EC50(AA-single)= 57.5 ± 8.6 µM); (C, F) mRNA levels of LOXL2 and COL1A1; (D,
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G) Procollagen secretion in culture supernatant (day 14 and 21); Error bars represent the standard
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deviations between independent assays (N=2), each condition represents a pool of 5-6 spheroids. Graph
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inserts in (C) and (F) represent the second repeat of the assay.
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Procollagen levels in supernatant of the hepatic organoids and 3D HSC or Hep mono-cultures upon 24h of
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single exposure at day 20. (I) Brigthfield images of Hep/HSC organoids at day 21 after single or repeated
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exposure to the different compounds. (J) Immunohistochemical detection of cross-linked collagen (Sirius-
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red), Collagen1 and αSMA in sections of day 21 Hep/HSC organoids (single and repeated exposure).
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Black bar represents 100 µm.
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Figure 6: In vivo data obtained from BALB/c mice after single and repeated exposure to APAP. (A)
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ALT blood levels 24h after a single exposure to 300 mg/kg APAP or vehicle control (B) mRNA levels of
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Acta2, Col1a1 and Lox in mouse HSCs isolated 24h after a single injection of 300 mg APAP/kg and the
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vehicle control; (C, D) Sirius-red images and quantification of the percentage of red stained surface in
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mice livers after 4 weeks of repeated exposure to APAP or CCl4.
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