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The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR–Cas9 in both human fetal hepatocyte and adult liver ductal organoid systems. Using a CRISPR–Cas9 and homology-independent organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte organoid cultures takes ~1–2 months. The protocols to genome engineer human liver ductal organoids and human fetal hepatocyte organoids take 2–3 months. Culture conditions are described for long-term expansion of human fetal hepatocytes as 3D organoids. Gene knockin and knockout approaches are also described for organoids derived from human fetal hepatocytes and human adult liver ductal cells.

CRISPR–Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR–Cas9-mediated homology-independent organoid transgenesis (CRISPR–HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR–HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR–HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter—in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin—uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR–HOT simplifies genome editing in human organoids.Artegiani, Hendriks et al. describe a CRISPR–Cas9-based method to efficiently generate human knock-in organoids using non-homologous end joining to study rare intestinal cell types and human hepatocyte division.

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Drug-induced cholestasis (DIC) is poorly understood and its preclinical prediction is mainly limited to assessing the compound’s potential to inhibit the bile salt export pump (BSEP). Here, we evaluated two 3D spheroid models, one from primary human hepatocytes (PHH) and one from HepaRG cells, for the detection of compounds with cholestatic liability. By repeatedly co-exposing both models to a set of compounds with different mechanisms of hepatotoxicity and a non-toxic concentrated bile acid (BA) mixture for 8 days we observed a selective synergistic toxicity of compounds known to cause cholestatic or mixed cholestatic/hepatocellular toxicity and the BA mixture compared to exposure to the compounds alone, a phenomenon that was more pronounced after extending the exposure time to 14 days. In contrast, no such synergism was observed after both 8 and 14 days of exposure to the BA mixture for compounds that cause non-cholestatic hepatotoxicity. Mechanisms behind the toxicity of the cholestatic compound chlorpromazine were accurately detected in both spheroid models, including intracellular BA accumulation, inhibition of ABCB11 expression and disruption of the F-actin cytoskeleton. Furthermore, the observed synergistic toxicity of chlorpromazine and BA was associated with increased oxidative stress and modulation of death receptor signalling. Combined, our results demonstrate that the hepatic spheroid models presented here can be used to detect and study compounds with cholestatic liability.

Recent advances in the development of various culture platforms are promising for achieving more physiologically relevant in vitro hepatic models using primary human hepatocytes (PHHs). Previous studies have shown the value of PHHs three-dimensional (3D) spheroid models, cultured in low cell number (1330–2000 cells/3D spheroid), to study long-term liver function as well as pharmacological drug effects and toxicity. In this study, we report that only plateable PHHs aggregate and form compact 3D spheroids with a success rate of 79%, and 96% reproducibility. Out of 3D spheroid forming PHH lots, 65% were considered stable (<50% ATP decrease) over the subsequent 14 days of culture, with reproducibility of a given PHH lot being 82%. We also report successful coculturing of PHHs with human liver nonparenchymal cells (NPCs). Crude P1 c -NPC fractions were obtained by low centrifugation of the PHH supernatant fraction followed by a few days of culture before harvesting and cryopreservation. At aggregation of PHHs/P1 c -NPCs (2:1 ratio 3D spheroids), liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells were successfully integrated and remained present throughout the subsequent 14-day culture period as revealed by mRNA expression markers and immunostaining. Increased mRNA expression of albumin (ALB), apolipoprotein B (APOB), cytochrome P450 3A4 (CYP3A4), and increased albumin secretion compared to PHH 3D spheroid monocultures highlighted that in a 3D spheroid coculture, configuration with NPCs, PHH functionality is increased. We thus achieved the development of a more integrated coculture model system requiring low cell numbers, of particular interest due to the scarcity of human liver NPCs.

The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders ( APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB −/− and MTTP −/− organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets. Organoid models of early liver disease aid target discovery and drug screening.

Pluripotent stem cell (PSC)-derived human brain organoids enable the study of human brain development in vitro. Typically, the fate of PSCs is guided into subsequent specification steps through static medium switches. In vivo, morphogen gradients are critical for proper brain development and determine cell specification, and associated defects result in neurodevelopmental disorders. Here, we show that initiating neural induction in a temporal stepwise gradient guides the generation of brain organoids composed of a single, self-organized apical-out neuroepithelium, termed ENOs (expanded neuroepithelium organoids). This is at odds with standard brain organoid protocols in which multiple and independent neuroepithelium units (rosettes) are formed. We find that a prolonged, decreasing gradient of TGF-β signaling is a determining factor in ENO formation and allows for an extended phase of neuroepithelium expansion. In-depth characterization reveals that ENOs display improved cellular morphology and tissue architectural features that resemble in vivo human brain development, including expanded germinal zones. Consequently, cortical specification is enhanced in ENOs. ENOs constitute a platform to study the early events of human cortical development and allow interrogation of the complex relationship between tissue architecture and cellular states in shaping the developing human brain. PSC-brain organoids are typically formed by static medium switches. Here, authors show that a temporal morphogen gradient during neural induction allows the formation of well-specified cortical organoids with a self-organized single neuroepithelium.

Recently two novel enzymes were identified in the outer mitochondrial membrane, mARC1 and mARC2. These molybdenum containing enzymes can reduce a variety of N-hydroxylated compounds, such as N-hydroxy-guanidines and sulfohydroxamic acids, as well as convert nitrite into nitric oxide (NO). However, their endogenous functions remain unknown. Here we demonstrate a specific developmental pattern of expression of these enzymes. mARC1, but not mARC2, was found to be expressed in fetal human liver, whereas both, in particular mARC2, are abundant in adult liver and also expressed in omental and subcutaneous fat. Caloric diet restriction of obese patients caused a decreased expression of mARC2 in liver, similar to that seen in the livers of starved rats. Knock down of mARC2 expression by siRNA in murine adipocytes had statistically significant effect on the level of diglycerides and on the fatty acid composition of some triglycerides, concomitantly a clear trend toward the reduced formation of most of triglyceride and phospholipid species was observed. The involvement of mARC2 in the metabolism of the hepatotoxic drug ximelagatran was evaluated in hepatocytes and adipocytes. Ximelagatran was shown to cause oxidative stress and knock down of mARC2 in adipocytes prevented ximelagatran induced inhibition of mitochondrial respiration. In conclusion, our data indicate that mARC1 and mARC2 have different developmental expression profiles, and that mARC2 is involved in lipogenesis, is regulated by nutritional status and responsible for activation of ximelagatran into a mitotoxic metabolite(s).

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood. Human hepatocytes remain hard to grow in vitro. Here, the authors temporally map the early stages of organoid growth initiated from fetal and adult hepatocytes, leveraging this knowledge to design maturation and improved expansion conditions.

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A . Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1 -mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACA fus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC. An in-depth understanding of the molecular pathogenesis of fibrolamellar carcinoma (FLC) is hampered due to limited human preclinical models. Here the authors engineer human organoids to reflect different FLC genetic backgrounds and show that hepatocytes can be a cell-of-origin of FLC that transdifferentiate into ductal/progenitor like cells due to combined BAP1 and PRKAR2A loss.