* joined first author # joined corresponding author

Gilles S van Tienderen, Ling Li, Laura Broutier, Yoshimasa Saito, Patricia Inacio, Meritxell Huch, Florin M Selaru#, Luc J W van der Laan#, Monique Ma Verstegen
Hepatobiliary tumor organoids for personalized medicine: a multicenter view on establishment, limitations, and future directions.
Cancer Cell, 40(3) 226-230 (2022)
Reliable establishment of tumor organoids is paramount to advance applications of organoid technology for personalized medicine. Here, we share our multi-center experience on initiation and tumorigenic confirmation of hepatobiliary cancer organoids. We discuss current concerns, propose potential solutions, and provide future perspectives for improvements in hepatobiliary cancer organoid establishment.

Jelena Krstic, Isabel Reinisch, Katharina Schindlmaier, Markus Galhuber, Zina Riahi, Natascha Berger, Nadja Kupper, Elisabeth Moyschewitz, Martina Auer, Helene Michenthaler, Christoph Nössing, Maria R Depaoli, Jeta Ramadani-Muja, Sinem Usluer, Sarah Stryeck, Martin Pichler, Beate Rinner, Alexander J A Deutsch, Andreas Reinisch, Tobias Madl, Riccardo Zenezini Chiozzi, Albert J R Heck, Meritxell Huch, Roland Malli, Andreas Prokesch
Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism.
Sci Adv, 8(3) Art. No. eabh2635 (2022)
Open Access DOI
Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC.

German Belenguer✳︎, Gianmarco Mastrogiovanni✳︎, Clare Pacini✳︎, Zoe Hall, Anna Dowbaj, Robert Arnes-Benito, Aleksandra Sljukic, Nicole Prior, Sofia Kakava, Charles R. Bradshaw, Susan E Davies, Michele Vacca, Kourosh Saeb-Parsy, Bon-Kyoung Koo, Meritxell Huch
RNF43/ZNRF3 loss predisposes to hepatocellular-carcinoma by impairing liver regeneration and altering the liver lipid metabolic ground-state.
Nat Commun, 13(1) Art. No. 334 (2022)
Open Access DOI
RNF43/ZNRF3 negatively regulate WNT signalling. Both genes are mutated in several types of cancers, however, their contribution to liver disease is unknown. Here we describe that hepatocyte-specific loss of Rnf43/Znrf3 results in steatohepatitis and in increase in unsaturated lipids, in the absence of dietary fat supplementation. Upon injury, Rnf43/Znrf3 deletion results in defective hepatocyte regeneration and liver cancer, caused by an imbalance between differentiation/proliferation. Using hepatocyte-, hepatoblast- and ductal cell-derived organoids we demonstrate that the differentiation defects and lipid alterations are, in part, cell-autonomous. Interestingly, ZNRF3 mutant liver cancer patients present poorer prognosis, altered hepatic lipid metabolism and steatohepatitis/NASH signatures. Our results imply that RNF43/ZNRF3 predispose to liver cancer by controlling the proliferative/differentiation and lipid metabolic state of hepatocytes. Both mechanisms combined facilitate the progression towards malignancy. Our findings might aid on the management of those RNF43/ZNRF3 mutated individuals at risk of developing fatty liver and/or liver cancer.

Lucía Cordero-Espinoza✳︎, Anna Dowbaj✳︎, Timo N Kohler, Bernhard Strauss, Olga Sarlidou, German Belenguer, Clare Pacini, Nuno P Martins, Ross Dobie, John R Wilson-Kanamori, Richard Butler, Nicole Prior, Palle Serup, Florian Jug, Neil C Henderson, Florian Hollfelder, Meritxell Huch
Dynamic cell contacts between periportal mesenchyme and ductal epithelium act as a rheostat for liver cell proliferation.
Cell Stem Cell, 28(11) 1907-1921 (2021)
Open Access DOI
In the liver, ductal cells rarely proliferate during homeostasis but do so transiently after tissue injury. These cells can be expanded as organoids that recapitulate several of the cell-autonomous mechanisms of regeneration but lack the stromal interactions of the native tissue. Here, using organoid co-cultures that recapitulate the ductal-to-mesenchymal cell architecture of the portal tract, we demonstrate that a subpopulation of mouse periportal mesenchymal cells exerts dual control on proliferation of the epithelium. Ductal cell proliferation is either induced and sustained or, conversely, completely abolished, depending on the number of direct mesenchymal cell contacts, through a mechanism mediated, at least in part, by Notch signaling. Our findings expand the concept of the cellular niche in epithelial tissues, whereby not only soluble factors but also cell-cell contacts are the key regulatory cues involved in the control of cellular behaviors, suggesting a critical role for cell-cell contacts during regeneration.

Lara Campana, Hannah Esser, Meritxell Huch, Stuart J Forbes
Liver regeneration and inflammation: from fundamental science to clinical applications.
Nat Rev Mol Cell Biol, 22(9) 608-624 (2021)
Liver regeneration is a complex process involving the crosstalk of multiple cell types, including hepatocytes, hepatic stellate cells, endothelial cells and inflammatory cells. The healthy liver is mitotically quiescent, but following toxic damage or resection the cells can rapidly enter the cell cycle to restore liver mass and function. During this process of regeneration, epithelial and non-parenchymal cells respond in a tightly coordinated fashion. Recent studies have described the interaction between inflammatory cells and a number of other cell types in the liver. In particular, macrophages can support biliary regeneration, contribute to fibrosis remodelling by repressing hepatic stellate cell activation and improve liver regeneration by scavenging dead or dying cells in situ. In this Review, we describe the mechanisms of tissue repair following damage, highlighting the close relationship between inflammation and liver regeneration, and discuss how recent findings can help design novel therapeutic approaches.

Elisa De Crignis, Tanvir Hossain, Shahla Romal, Fabrizia Carofiglio, Panagiotis Moulos, Mir Mubashir Khalid, Shringar Rao, Ameneh Bazrafshan, Monique Ma Verstegen, Farzin Pourfarzad, Christina Koutsothanassis, Helmuth Gehart, Tsung Wai Kan, Robert-Jan Palstra, Charles Boucher, Jan N M IJzermans, Meritxell Huch, Sylvia F Boj, Robert R G Vries, Hans Clevers, Luc J W van der Laan, Pantelis Hatzis, Tokameh Mahmoudi
Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma.
Elife, 10 Art. No. e60747 (2021)
Open Access DOI
The molecular events that drive Hepatitis B virus (HBV)-mediated transformation and tumorigenesis have remained largely unclear, due to the absence of a relevant primary model system. Here we propose the use of human liver organoids as a platform for modeling HBV infection and related tumorigenesis. We first describe a primary ex vivo HBV-infection model derived from healthy donor liver organoids after challenge with recombinant virus or HBV-infected patient serum. HBV infected organoids produced cccDNA, HBeAg, expressed intracellular HBV RNA and proteins, and produced infectious HBV. This ex vivo HBV infected primary differentiated hepatocyte organoid platform was amenable to drug screening for both anti-HBV activity as well as for drug-induced toxicity. We also studied HBV replication in transgenically modified organoids; liver organoids exogenously overexpressing the HBV receptor NTCP after lentiviral transduction were not more susceptible to HBV, suggesting the necessity for additional host factors for efficient infection. We also generated transgenic organoids harboring integrated HBV, representing a long-term culture system also suitable for viral production and the study of HBV transcription. Finally, we generated HBV-infected patient-derived liver organoids from non-tumor cirrhotic tissue of explants from liver transplant patients. Interestingly, transcriptomic analysis of patient-derived liver organoids indicated the presence of an aberrant early cancer gene signature, which clustered with the HCC cohort on the TCGA LIHC dataset and away from healthy liver tissue, and may provide invaluable novel biomarkers for the development of HCC and surveillance in HBV infected patients.

Leticia Colyn, Marina Bárcena-Varela, Gloria Álvarez-Sola, M Ujue Latasa, Iker Uriarte, Eva Santamaría, Jose M Herranz, Alvaro Santos-Laso, Maria Arechederra, Mikel Ruiz de Gauna, Patricia Aspichueta, Matteo Canale, Andrea Casadei-Gardini, Maria Francesconi, Simone Carotti, Sergio Morini, Leonard J Nelson, Maria J Iraburu, Chaobo Chen, Bruno Sangro, Jose Jg Marin, Maria L Martinez-Chantar, Jesus M Banales, Robert Arnes-Benito, Meritxell Huch, John Patino, Altaf A Dar, Mehdi Nosrati, Julen Oyarzábal, Felipe Prósper, Jesus Urman, Francisco Javier Cubero, Christian Trautwein, Carmen Berasain#, Maite G Fernandez-Barrena#, Matias A Avila#
Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.
Hepatology, 73(6) 2380-2396 (2021)
Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors.

Ary Marsee✳︎, Floris J M Roos✳︎, Monique Ma Verstegen, Monique M A null, Helmuth Gehart, Eelco de Koning, Frédéric Lemaigre, Stuart J Forbes, Weng Chuan Peng, Meritxell Huch, Takanori Takebe, Ludovic Vallier, Hans Clevers, Luc J W van der Laan, Bart Spee
Building consensus on definition and nomenclature of hepatic, pancreatic, and biliary organoids.
Cell Stem Cell, 28(5) 816-832 (2021)
Hepatic, pancreatic, and biliary (HPB) organoids are powerful tools for studying development, disease, and regeneration. As organoid research expands, the need for clear definitions and nomenclature describing these systems also grows. To facilitate scientific communication and consistent interpretation, we revisit the concept of an organoid and introduce an intuitive classification system and nomenclature for describing these 3D structures through the consensus of experts in the field. To promote the standardization and validation of HPB organoids, we propose guidelines for establishing, characterizing, and benchmarking future systems. Finally, we address some of the major challenges to the clinical application of organoids.

Flaminia Kaluthantrige Don, Meritxell Huch
Organoids, Where We Stand and Where We Go.
Trends Mol Med, 27(5) 416-418 (2021)
Organoid cultures hold the promise of transforming basic and clinical research. Here, we provide a storyline of the highlights and breakthroughs that have an impact in present clinical research. We also discuss the bottlenecks that delay their full exploitation for the next generation of biomedical research.

Nasim Annabi, Matthew Baker, Alistair Boettiger, Debojyoti Chakraborty, Yvonne Chen, Kizzmekia S Corbett, Bruno Correia, James Dahlman, Tulio de Oliveira, Ali Ertuerk, Mehmet Fatih Yanik, Elizabeth Henaff, Meritxell Huch, Iliyan D Iliev, Thomas Jacobs, Howard Junca, Albert Keung, Ilana Kolodkin-Gal, Smita Krishnaswamy, Madeline Lancaster, Evan Macosko, Mario Alberto Martínez-Núñez, Kyoko Miura, Jenny Molloy, Alfredo Cruz, Randall J Platt, Avery D Posey, Huilin Shao, Mijo Simunovic, Nikolai Slavov, Takanori Takebe, Luk H Vandenberghe, Rajeev K Varshney, Jianbin Wang
Voices of biotech research.
Nat Biotechnol, 39(3) 281-286 (2021)

Lotta Hof, Till Moreth, Michael Koch, Tim Liebisch, Marina Kurtz, Julia Tarnick, Susanna M Lissek, Monique Ma Verstegen, Luc J W van der Laan, Meritxell Huch, Franziska Matthäus, Ernst H K Stelzer, Francesco Pampaloni
Long-term live imaging and multiscale analysis identify heterogeneity and core principles of epithelial organoid morphogenesis.
BMC Biol, 19(1) Art. No. 37 (2021)
Open Access DOI
Organoids are morphologically heterogeneous three-dimensional cell culture systems and serve as an ideal model for understanding the principles of collective cell behaviour in mammalian organs during development, homeostasis, regeneration, and pathogenesis. To investigate the underlying cell organisation principles of organoids, we imaged hundreds of pancreas and cholangiocarcinoma organoids in parallel using light sheet and bright-field microscopy for up to 7 days.

Nikolaus Rajewsky, Geneviève Almouzni, Stanislaw A Gorski, Stein Aerts, Ido Amit, Michela G Bertero, Christoph Bock, Annelien L Bredenoord, Giacomo Cavalli, Susanna Chiocca, Hans Clevers, Bart De Strooper, Angelika Eggert, Jan Ellenberg, Xosé M Fernández, Marek Figlerowicz, Susan M Gasser, Norbert Hubner, Jørgen Kjems, Jürgen A. Knoblich, Grietje Krabbe, Peter Lichter, Sten Linnarsson, Jean-Christophe Marine, John C Marioni, Marc A Marti-Renom, Mihai G Netea, Dörthe Nickel, Marcelo Nollmann, Halina R Novak, Helen Parkinson, Stefano Piccolo, Inês Pinheiro, Ana Pombo, Christian Popp, Wolf Reik, Sergio Roman-Roman, Philip Rosenstiel, Joachim L Schultze, Oliver Stegle, Amos Tanay, Giuseppe Testa, Dimitris Thanos, Fabian J Theis, Maria-Elena Torres-Padilla, Alfonso Valencia, Céline Vallot, Alexander van Oudenaarden, Marie Vidal, Thierry Voet
LifeTime and improving European healthcare through cell-based interceptive medicine.
Nature, 587(7834) 377-386 (2020)
Open Access DOI
Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.

Nikitas Georgakopoulos, Nicole Prior, Brigitte Angres, Gianmarco Mastrogiovanni, Alex Cagan, Daisy Harrison, Christopher J Hindley, Robert Arnes-Benito, Siong-Seng Liau, Abbie Curd, Natasha Ivory, Benjamin D Simons, Inigo Martincorena, Helmut Wurst, Kourosh Saeb-Parsy, Meritxell Huch
Long-term expansion, genomic stability and in vivo safety of adult human pancreas organoids.
BMC Dev Biol, 20(1) Art. No. 4 (2020)
Open Access DOI
Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus.

Gema Gómez-Mariano, Nerea Matamala, Selene Martínez, Iago Justo, Alberto Marcacuzco, Carlos Jimenez, Sara Monzón, Isabel Cuesta, Cristina Garfia, María Teresa Martínez, Meritxell Huch, Ignacio Pérez de Castro, Manuel Posada, Sabina Janciauskiene, Beatriz Martínez-Delgado
Liver organoids reproduce alpha-1 antitrypsin deficiency-related liver disease.
Hepatol Int, 14(1) 127-137 (2020)
Open Access DOI
Alpha-1 antitrypsin (AAT) is a product of SERPINA1 gene mainly expressed by hepatocytes. Clinically relevant mutations in the SERPINA1 gene, such as Z (Glu342Lys), results in an expression of misfolded AAT protein having high propensity to polymerize, accumulate in hepatocytes and thus to enhance a risk for hepatocyte damage and subsequent liver disease. So far, the relationship between the Z-AAT accumulation and liver cell damage remains not completely understood. We present three-dimensional organoid culture systems, as a novel tool for modeling Z-AAT-related liver diseases.

Nicole Prior, Patricia Inacio, Meritxell Huch
Liver organoids: from basic research to therapeutic applications.
Gut, 68(12) 2228-2237 (2019)
Open Access DOI
Organoid cultures have emerged as an alternative in vitro system to recapitulate tissues in a dish. While mouse models and cell lines have furthered our understanding of liver biology and associated diseases, they suffer in replicating key aspects of human liver tissue, in particular its complex architecture and metabolic functions. Liver organoids have now been established for multiple species from induced pluripotent stem cells, embryonic stem cells, hepatoblasts and adult tissue-derived cells. These represent a promising addition to our toolbox to gain a deeper understanding of this complex organ. In this perspective we will review the advances in the liver organoid field, its limitations and potential for biomedical applications.

Luigi Aloia, Mikel Alexander McKie, Grégoire Vernaz, Lucía Cordero-Espinoza, Niya Aleksieva, Jelle van den Ameele, Francesco Antonica, Berta Font-Cunill, Alexander Raven, Riccardo Aiese Cigliano, German Belenguer, Richard Lester Mort, Andrea H Brand, Magdalena Zernicka-Goetz, Stuart J Forbes, Eric A Miska, Meritxell Huch
Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regeneration.
Nat Cell Biol, 21(11) 1321-1333 (2019)
Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.

Madeline Lancaster, Meritxell Huch
Disease modelling in human organoids.
Dis Model Mech, 12(7) Art. No. dmm039347 (2019)
Open Access DOI
The past decade has seen an explosion in the field of in vitro disease modelling, in particular the development of organoids. These self-organizing tissues derived from stem cells provide a unique system to examine mechanisms ranging from organ development to homeostasis and disease. Because organoids develop according to intrinsic developmental programmes, the resultant tissue morphology recapitulates organ architecture with remarkable fidelity. Furthermore, the fact that these tissues can be derived from human progenitors allows for the study of uniquely human processes and disorders. This article and accompanying poster highlight the currently available methods, particularly those aimed at modelling human biology, and provide an overview of their capabilities and limitations. We also speculate on possible future technological advances that have the potential for great strides in both disease modelling and future regenerative strategies.

Nicole Prior, Christopher J Hindley, Fabian Rost, Elena Meléndez, Winnie W Y Lau, Berthold Göttgens, Steffen Rulands, Benjamin D Simons, Meritxell Huch
Lgr5+ stem and progenitor cells reside at the apex of a heterogeneous embryonic hepatoblast pool.
Development, 146(12) Art. No. dev174557 (2019)
Open Access DOI
During mouse embryogenesis, progenitors within the liver known as hepatoblasts give rise to adult hepatocytes and cholangiocytes. Hepatoblasts, which are specified at E8.5-E9.0, have been regarded as a homogeneous progenitor population that initiate differentiation from E13.5. Recently, scRNA-seq analysis has identified sub-populations of transcriptionally distinct hepatoblasts at E11.5. Here, we show that hepatoblasts are not only transcriptionally but also functionally heterogeneous, and that a subpopulation of E9.5-E10.0 hepatoblasts exhibit a previously unidentified early commitment to cholangiocyte fate. Importantly, we also identify a subpopulation constituting 2% of E9.5-E10.0 hepatoblasts that express the adult stem cell marker Lgr5, and generate both hepatocyte and cholangiocyte progeny that persist for the lifespan of the mouse. Combining lineage tracing and scRNA-seq, we show that Lgr5 marks E9.5-E10.0 bipotent liver progenitors residing at the apex of a hepatoblast hierarchy. Furthermore, isolated Lgr5+ hepatoblasts can be clonally expanded in vitro into embryonic liver organoids, which can commit to either hepatocyte or cholangiocyte fates. Our study demonstrates functional heterogeneity within E9.5 hepatoblasts and identifies Lgr5 as a marker for a subpopulation of bipotent liver progenitors.

Pura Muñoz-Cánoves#, Meritxell Huch#
Definitions for adult stem cells debated.
Nature, 563(7731) 328-329 (2018)

Steffen Rulands, Fabienne Lescroart, Samira Chabab, Christopher J Hindley, Nicole Prior, Magdalena K Sznurkowska, Meritxell Huch, Anna Philpott, Cedric Blanpain, Benjamin D Simons
Universality of clone dynamics during tissue development.
Nat Phys, 14(5) 469-474 (2018)
The emergence of complex organs is driven by the coordinated proliferation, migration and differentiation of precursor cells. The fate behaviour of these cells is reflected in the time evolution their progeny, termed clones, which serve as a key experimental observable. In adult tissues, where cell dynamics is constrained by the condition of homeostasis, clonal tracing studies based on transgenic animal models have advanced our understanding of cell fate behaviour and its dysregulation in disease (1, 2). But what can be learned from clonal dynamics in development, where the spatial cohesiveness of clones is impaired by tissue deformations during tissue growth? Drawing on the results of clonal tracing studies, we show that, despite the complexity of organ development, clonal dynamics may converge to a critical state characterized by universal scaling behaviour of clone sizes. By mapping clonal dynamics onto a generalization of the classical theory of aerosols, we elucidate the origin and range of scaling behaviours and show how the identification of universal scaling dependences may allow lineage-specific information to be distilled from experiments. Our study shows the emergence of core concepts of statistical physics in an unexpected context, identifying cellular systems as a laboratory to study non-equilibrium statistical physics.

Lucía Cordero-Espinoza, Meritxell Huch
The balancing act of the liver: tissue regeneration versus fibrosis.
J Clin Invest, 128(1) 85-96 (2018)
Epithelial cell loss alters a tissue's optimal function and awakens evolutionarily adapted healing mechanisms to reestablish homeostasis. Although adult mammalian organs have a limited regeneration potential, the liver stands out as one remarkable exception. Following injury, the liver mounts a dynamic multicellular response wherein stromal cells are activated in situ and/or recruited from the bloodstream, the extracellular matrix (ECM) is remodeled, and epithelial cells expand to replenish their lost numbers. Chronic damage makes this response persistent instead of transient, tipping the system into an abnormal steady state known as fibrosis, in which ECM accumulates excessively and tissue function degenerates. Here we explore the cellular and molecular switches that balance hepatic regeneration and fibrosis, with a focus on uncovering avenues of disease modeling and therapeutic intervention.

Laura Broutier, Gianmarco Mastrogiovanni, Monique Ma Verstegen, Hayley E Francies, Lena Morrill Gavarró, Charles R. Bradshaw, George E Allen, Robert Arnes-Benito, Olga Sidorova, Marcia P Gaspersz, Nikitas Georgakopoulos, Bon-Kyoung Koo, Sabine Dietmann, Susan E Davies, Raaj K Praseedom, Ruby Lieshout, Jan N M IJzermans, Stephen J Wigmore, Kourosh Saeb-Parsy, Mathew J Garnett, Luc J W van der Laan, Meritxell Huch
Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.
Nat Med, 23(12) 1424-1435 (2017)
Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Meritxell Huch#, Emma L Rawlins#
Cancer: Tumours build their niche.
Nature, 545(7654) 292-293 (2017)

Meritxell Huch, Jürgen A. Knoblich, Matthias Lutolf, Alfonso Martinez-Arias
The hope and the hype of organoid research.
Development, 144(6) 938-941 (2017)
The recent increase in organoid research has been met with great enthusiasm, as well as expectation, from the scientific community and the public alike. There is no doubt that this technology opens up a world of possibilities for scientific discovery in developmental biology as well as in translational research, but whether organoids can truly live up to this challenge is, for some, still an open question. In this Spotlight article, Meritxell Huch and Juergen Knoblich begin by discussing the exciting promise of organoid technology and give concrete examples of how this promise is starting to be realised. In the second part, Matthias Lutolf and Alfonso Martinez-Arias offer a careful and considered view of the state of the organoid field and its current limitations, and lay out the approach they feel is necessary to maximise the potential of organoid technology.

Christopher J Hindley, Lucía Cordero-Espinoza, Meritxell Huch
Organoids from adult liver and pancreas: Stem cell biology and biomedical utility.
Dev Biol, 420(2) 251-261 (2016)
Open Access DOI
The liver and pancreas are critical organs maintaining whole body metabolism. Historically, the expansion of adult-derived cells from these organs in vitro has proven challenging and this in turn has hampered studies of liver and pancreas stem cell biology, as well as being a roadblock to disease modelling and cell replacement therapies for pathologies in these organs. Recently, defined culture conditions have been described which allow the in vitro culture and manipulation of adult-derived liver and pancreatic material. Here we review these systems and assess their physiological relevance, as well as their potential utility in biomedicine.

Laura Broutier, Amanda Andersson-Rolf, Christopher J Hindley, Sylvia F Boj, Hans Clevers, Bon-Kyoung Koo, Meritxell Huch
Culture and establishment of self-renewing human and mouse adult liver and pancreas 3D organoids and their genetic manipulation.
Nat Protoc, 11(9) 1724-1743 (2016)
Adult somatic tissues have proven difficult to expand in vitro, largely because of the complexity of recreating appropriate environmental signals in culture. We have overcome this problem recently and developed culture conditions for adult stem cells that allow the long-term expansion of adult primary tissues from small intestine, stomach, liver and pancreas into self-assembling 3D structures that we have termed 'organoids'. We provide a detailed protocol that describes how to grow adult mouse and human liver and pancreas organoids, from cell isolation and long-term expansion to genetic manipulation in vitro. Liver and pancreas cells grow in a gel-based extracellular matrix (ECM) and a defined medium. The cells can self-organize into organoids that self-renew in vitro while retaining their tissue-of-origin commitment, genetic stability and potential to differentiate into functional cells in vitro (hepatocytes) and in vivo (hepatocytes and endocrine cells). Genetic modification of these organoids opens up avenues for the manipulation of adult stem cells in vitro, which could facilitate the study of human biology and allow gene correction for regenerative medicine purposes. The complete protocol takes 1-4 weeks to generate self-renewing 3D organoids and to perform genetic manipulation experiments. Personnel with basic scientific training can conduct this protocol.

Meritxell Huch#, Laurent Dollé#
The plastic cellular states of liver cells: Are EpCAM and Lgr5 fit for purpose?
Hepatology, 64(2) 652-662 (2016)
Open Access DOI
Adult liver cells have been considered restricted regarding their fate and lineage potential. That is, hepatocytes have been thought able only to generate hepatocytes and duct cells, only duct cells. While this may be the case for the majority of scenarios in a state of quiescence or homeostasis, evidence suggests that liver cells are capable of interconverting between cellular states of distinct phenotypic traits. This interconversion or plasticity had been suggested by classical studies using cellular markers, but recently lineage tracing approaches have proven that cells are highly plastic and retain an extraordinary ability to respond differently to normal tissue homeostasis, to tissue repair, or when challenged to expand ex vivo or to differentiate upon transplantation. Stemness, as "self-renewal and multipotency," seems not to be limited to a particular cell type but rather to a cellular state in which cells exhibit a high degree of plasticity and can move back and forth in different phenotypic states. For instance, upon damage cells can dedifferentiate to acquire stem cell potential that allows them to self-renew, repopulate a damaged tissue, and then undergo differentiation. In this review, we will discuss the evidence on cellular plasticity in the liver, focusing our attention on two markers, epithelial cell adhesion molecule and leucine-rich repeat-containing G protein-coupled receptor 5, which identify cells with stem cell potential. (Hepatology 2016;64:652-662).

Meritxell Huch, Bon-Kyoung Koo
Modeling mouse and human development using organoid cultures.
Development, 142(18) 3113-3125 (2015)
In vitro three-dimensional (3D) cultures are emerging as novel systems with which to study tissue development, organogenesis and stem cell behavior ex vivo. When grown in a 3D environment, embryonic stem cells (ESCs) self-organize into organoids and acquire the right tissue patterning to develop into several endoderm- and ectoderm-derived tissues, mimicking their in vivo counterparts. Tissue-resident adult stem cells (AdSCs) also form organoids when grown in 3D and can be propagated in vitro for long periods of time. In this Review, we discuss recent advances in the generation of pluripotent stem cell- and AdSC-derived organoids, highlighting their potential for enhancing our understanding of human development. We will also explore how this new culture system allows disease modeling and gene repair for a personalized regenerative medicine approach.

Meritxell Huch, Helmuth Gehart, Ruben van Boxtel, Karien Hamer, Francis Blokzijl, Monique Ma Verstegen, Ewa Ellis, Martien van Wenum, Sabine A Fuchs, Joep de Ligt, Marc van de Wetering, Nobuo Sasaki, Susanne J Boers, Hans Kemperman, Jeroen de Jonge, Jan N M Ijzermans, Edward E S Nieuwenhuis, Ruurdtje Hoekstra, Stephen Strom, Robert R G Vries, Luc J W van der Laan, Edwin Cuppen, Hans Clevers
Long-term culture of genome-stable bipotent stem cells from adult human liver.
Cell, 160(1-2) 299-312 (2015)
Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.

Meritxell Huch
Regenerative biology: The versatile and plastic liver.
Nature, 517(7533) 155-156 (2015)

Christopher J Hindley✳︎, Gianmarco Mastrogiovanni✳︎, Meritxell Huch
The plastic liver: differentiated cells, stem cells, every cell?
J Clin Invest, 124(12) 5099-5102 (2014)
The liver is capable of full regeneration following several types and rounds of injury, ranging from hepatectomy to toxin-mediated damage. The source of this regenerative capacity has long been a hotly debated topic. The damage response that occurs when hepatocyte proliferation is impaired is thought to be mediated by oval/dedifferentiated progenitor cells, which replenish the hepatocyte and ductal compartments of the liver. Recently, reports have questioned whether these oval/progenitor cells truly serve as the facultative stem cell of the liver following toxin-mediated damage. In this issue of the JCI, Kordes and colleagues use lineage tracing to follow transplanted rat hepatic stellate cells, a resident liver mesenchymal cell population, in hosts that have suffered liver damage. Transplanted stellate cells repopulated the damaged rat liver by contributing to the oval cell response. These data establish yet another cell type of mesenchymal origin as the progenitor for the oval/ductular response in the rat. The lack of uniformity between different damage models, the extent of the injury to the liver parenchyma, and potential species-specific differences might be at the core of the discrepancy between different studies. Taken together, these data imply a considerable degree of plasticity in the liver, whereby several cell types can contribute to regeneration.

Sam Behjati, Meritxell Huch, Ruben van Boxtel, Wouter Karthaus, David C Wedge, Asif U Tamuri, Inigo Martincorena, Mia Petljak, Ludmil B Alexandrov, Gunes Gundem, Patrick S Tarpey, Sophie Roerink, Joyce Blokker, Mark Maddison, Laura Mudie, Ben Robinson, Serena Nik-Zainal, Peter Campbell, Nick Goldman, Marc van de Wetering, Edwin Cuppen, Hans Clevers, Michael R Stratton
Genome sequencing of normal cells reveals developmental lineages and mutational processes.
Nature, 513(7518) 422-425 (2014)
The somatic mutations present in the genome of a cell accumulate over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree, the number of divisions that each cell has undergone and the mutational processes that have been operative. Here we describe whole genomes of clonal lines derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal, demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible.

Meritxell Huch✳︎, Paola Bonfanti✳︎, Sylvia F Boj✳︎, Toshiro Sato✳︎, Cindy J M Loomans, Marc van de Wetering, Mozhdeh Sojoodi, Vivian S W Li, Jurian Schuijers, Ana Gracanin, Femke Ringnalda, Harry Begthel, Karien Hamer, Joyce Mulder, Johan H van Es, Eelco de Koning, Robert R G Vries, Harry Heimberg✳︎#, Hans Clevers✳︎#
Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.
EMBO J, 32(20) 2708-2721 (2013)
Open Access DOI
Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

Meritxell Huch, Craig Dorrell, Sylvia F Boj, Johan H van Es, Vivian S W Li, Marc van de Wetering, Toshiro Sato, Karien Hamer, Nobuo Sasaki, Milton J Finegold, Annelise Haft, Robert R G Vries, Markus Grompe, Hans Clevers
In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
Nature, 494(7436) 247-250 (2013)
The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

Nick Barker✳︎, Maarten B Rookmaaker✳︎, Pekka Kujala, Annie Ng, Marc Leushacke, Hugo J Snippert, Marc van de Wetering, Shawna Tan, Johan H van Es, Meritxell Huch, Richard Poulsom, Marianne C Verhaar, Peter J Peters, Hans Clevers
Lgr5(+ve) stem/progenitor cells contribute to nephron formation during kidney development.
Cell Rep, 2(3) 540-552 (2012)
Open Access DOI
Multipotent stem cells and their lineage-restricted progeny drive nephron formation within the developing kidney. Here, we document expression of the adult stem cell marker Lgr5 in the developing kidney and assess the stem/progenitor identity of Lgr5(+ve) cells via in vivo lineage tracing. The appearance and localization of Lgr5(+ve) cells coincided with that of the S-shaped body around embryonic day 14. Lgr5 expression remained restricted to cell clusters within developing nephrons in the cortex until postnatal day 7, when expression was permanently silenced. In vivo lineage tracing identified Lgr5 as a marker of a stem/progenitor population within nascent nephrons dedicated to generating the thick ascending limb of Henle's loop and distal convoluted tubule. The Lgr5 surface marker and experimental models described here will be invaluable for deciphering the contribution of early nephron stem cells to developmental defects and for isolating human nephron progenitors as a prerequisite to evaluating their therapeutic potential.

Bon-Kyoung Koo✳︎, Daniel E Stange✳︎, Toshiro Sato, Wouter Karthaus, Henner F Farin, Meritxell Huch, Johan H van Es, Hans Clevers
Controlled gene expression in primary Lgr5 organoid cultures.
Nat Methods, 9(1) 81-83 (2011)
The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

Meritxell Huch, Hans Clevers
Sox9 marks adult organ progenitors.
Nat Genet, 43(1) 9-10 (2011)

Robert R G Vries✳︎, Meritxell Huch✳︎, Hans Clevers
Stem cells and cancer of the stomach and intestine.
Mol Oncol, 4(5) 373-384 (2010)
Cancer in the 21st century has become the number one cause of death in developed countries. Although much progress has been made in improving patient survival, tumour relapse is one of the important causes of cancer treatment failure. An early observation in the study of cancer was the heterogeneity of tumours. Traditionally, this was explained by a combination of genomic instability of tumours and micro environmental factors leading to diverse phenotypical characteristics. It was assumed that cells in a tumour have an equal capacity to propagate the cancer. This model is currently known as the stochastic model. Recently, the Cancer stem cell model has been proposed to explain the heterogeneity of a tumour and its progression. According to this model, the heterogeneity of tumours is the result of aberrant differentiation of tumour cells into the cells of the tissue the tumour originated from. Tumours were suggested to contain stem cell-like cells, the cancer stem cells or tumour-initiating cells, which are uniquely capable of propagating a tumour much like normal stem cells fuel proliferation and differentiation in normal tissue. In this review we discuss the normal stem cell biology of the stomach and intestine followed by both the stochastic and cancer stem cell models in light of recent findings in the gastric and intestinal systems. The molecular pathways underlying normal and tumourigenic growth have been well studied, and recently the stem cells of the stomach and intestine have been identified. Furthermore, intestinal stem cells were identified as the cells-of-origin of colon cancer upon loss of the tumour suppressor APC. Lastly, several studies have proposed the positive identification of a cancer stem cell of human colon cancer. At the end we compare the cancer stem cell model and the stochastic model. We conclude that clonal evolution of tumour cells resulting from genetic mutations underlies tumour initiation and progression in both cancer models. This implies that at any point during tumour development any tumour cell can revert to a cancer stem cell after having gained a clonal advantage over the original cancer stem cell. Therefore, these models represent two sides of the same coin.

Nick Barker, Meritxell Huch, Pekka Kujala, Marc van de Wetering, Hugo J Snippert, Johan H van Es, Toshiro Sato, Daniel E Stange, Harry Begthel, Maaike van den Born, Esther Danenberg, Stieneke van den Brink, Jeroen Korving, Arie Abo, Peter J Peters, Nick Wright, Richard Poulsom, Hans Clevers
Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.
Cell Stem Cell, 6(1) 25-36 (2010)
The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.