Liver cells…in 3D

With a breakthrough that led to the first ever in-vitro expansion of liver stem cells, researchers from the Wellcome Trust/Cancer Research UK Gurdon Institute think they are on the brink of a hepatological revolution – and it has won them the 2013 NC3Rs international prize. Here, Meritxell Huch tells us more about the award winning work Despite the enormous replication potential of the liver, functional hepatocytes can be maintained only for very few days in culture, thus precluding their application for cell therapy, toxicological studies or drug screenings. To obtain a source of functional hepatocytes, a variety of in vitro systems have been described, ranging from culturing foetal and neonatal liver progenitors to obtaining hepatocytes by directed differentiation of either Embryonic Stem cells (ES) or induced Pluripotent Stem Cells (iPS). Liver stem cells have the potential to self-renew and differentiate into the functional hepatic lineages. However, it has proven difficult to expand these cells beyond a few days in culture. We have recently described a 3D culture system that allows, for the first time, the expansion of liver stem cells in vitro¹. The expanded cells generate functional hepatocytes in vitro and in vivo, following transplantation into a mouse model of liver disease. This novel liver culture system opens the opportunity to study, for the first time, liver stem cell proliferation and differentiation and sets up a platform for drug screening, toxicological studies and personalised medicine. Because the cells can be expanded into millions of cells even when starting from one single cell, this novel culture system has the potential to reduce the number of animals used in liver research. The huge potential of this culture system for liver research as well as its potential reduction of the number of animals in the field has been recognised by the National Centre for the 3Rs (NC3Rs), which awarded the 2013 NC3Rs international prize to these studies. The liver is the largest organ in the human body. It develops important biological functions, such as synthesising essential serum proteins, regulating metabolism and detoxifying a wide variety of endogenous and exogenous molecules. The architecture of the tissue is quite unique and complex. The primary functional unit of the liver is the hepatic lobule, a perfectly organised structure resulting from the interaction between epithelial (hepatocytes and ductal cells), endothelial (sinusoidal cells), resident macrophages (kupffer cells) and mesenchymal cells (portal fibroblasts and stellate cells)². Deregulation of these harmonic interactions can result in either cancer, or tissue degeneration. Liver diseases range from inherited metabolic diseases to viral hepatitis, liver cancer and obesity-related fatty liver disease. According to Asrani and colleagues, overall these account for the eighth-related cause of death in the United States³. In fact, more than 2,000 people die annually while waiting for a liver transplant⁴. Failure in the management of liver diseases can be attributed as much to the shortage of donor livers as to our poor understanding of the mechanisms behind liver pathology. [caption id="attachment_38436" align="alignright" width="200"] Figure 1: Liver organoids derived from a healthy mouse liver tissue self-renew long term when cultured under defined Expansion Medium. a: scheme of the protocol. b: representative image of a liver organoid culture expanded for >3 months in vitro.[/caption] Stem cells (ES, iPS or adult tissue-specific stem cells) represent an attractive alternative cell source to liver transplant, because of their unlimited self-renew capacity and multipotency. Self-renewal, maintenance and repair of tissues in adult mammals depend on small reservoirs of tissue-specific stem cells. These adult stem cells can be defined by two essential features: self-renewal, that is, maintenance over long periods of time, and differentiation capacity, understood as their ability of the cells to produce differentiated daughter cells of the pertinent tissue⁵. In organs with extensive cellular turnover, the surface receptor LGR5, a Wnt target gene and receptor for the Wnt agonists Rspondins⁶, marks an actively cycling stem cell population in organs with extensive cell renewal such as the gut, hair follicle and stomach⁷.  Interestingly, these actively cycling adult stem cells can be cultured and expanded in vitro into 3D structures that we have termed organoids from both, intestine⁸ and stomach⁹. In stark contrast to these highly proliferative organs, the liver is an organ with low physiological turnover (~200-300 days). Under physiological conditions pre-existing mature cells (hepatocytes and ductal cells) are responsible for liver physiological turnover¹⁰, although liver progenitors can also contribute to normal liver homeostasis, albeit at much lower ratio. One of the defining features of the liver is its remarkable regeneration capacity upon damage¹⁰ and its ability to maintain a constant size despite injury². The degree to which liver stem cells mediate liver regeneration depends on the type of repair the adult liver can undergo. Upon partial hepatectomy or CCl₄ damage, fully differentiated hepatocytes in the remaining healthy liver lobes can re-enter the cell cycle to rapidly compensate for the tissue loss¹⁰ while liver progenitors contribute to less extent to the regeneration of the tissue. Alternatively, certain toxic agents (e.g. DDC, CDE) can cause a generalised liver tissue decay, which results in the activation of a liver progenitor cell population that regenerates the tissue by giving rise to hepatocytes and biliary epithelial cells¹¹. We recently demonstrated that upon toxic damage, Wnt signalling becomes highly up-regulated in the areas of active regeneration. The Wnt target and stem cell marker Lgr5 cannot be detected under physiological conditions, however, marks a new population of stem/progenitor cells that becomes activated following damage and actively contribute to liver repair via de-novo generation of hepatocytes and ductal cells¹. This was the first evidence that a Wnt-responsive stem cell contributes to the regeneration of the liver tissue upon damage. [caption id="attachment_38437" align="alignleft" width="200"] Figure 2: Liver organoids acquire hepatocyte fate when cultured into a differentiation medium. a: scheme of the protocol showing the genes expression changes upon differentiation. b: representative image of an expanded liver organoid differentiated into hepatocyte like cells in vitro. Note the expression of Albumin and HNF4a, hepatocyte-specific genes. Arrows indicate binucleated cells, a hallmark of mature hepatocytes.[/caption] The knowledge acquired from the in vivo regeneration studies, allowed us to establish a new culture system that maintains the self-renewal status of liver stem cells in vitro. That is, these stem cells expand unlimitedly in culture, into long-lived 3D structures that we have called “liver organoids”¹ (Figure 1). Liver primary organoid cultures expand in the absence of a mesenchymal niche (>1 year in expansion in culture) even from adult liver single cells. Importantly, isolated adult hepatic cells retain their differentiation potential over time when cultured into organoids. The cultured cells express ductal markers and, when subjected to a differentiation protocol, ~33-50% of the cells differentiate into functional hepatocytes in vitro, thus mimicking “mini-livers” in culture (Figure 2). The huge expansion and differentiation capacity of the liver organoid cultures facilitated the engraftment and repopulation of the livers of mice with inherited metabolic disease (Tyrosinemia type I) and partially restored their hepatic function (Figure 3). Of note, the expanded stem cells maintain their chromosomal numbers over time, and do not form tumours upon transplantation into mice, in contrast to ES or iPS-derived hepatocyes¹², that suffer from inherent genetic stability problems¹³. Therefore, the liver organoid cultures hold promise as a safe clinical source of hepatocytes for transplantation. However, to fully address any safety issue, an in depth analysis of their genetic stability by means of WGS (whole genome sequencing) of cells maintained for long periods of time in culture will be necessary. These findings were summarised in our manuscript entitled In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration, published in Nature in 2013¹. The culture system described in this work provides the stem cell field with a novel in vitro model that allows the expansion of liver stem cells and the study of cellular processes such as proliferation and differentiation in vitro, in culture. Furthermore, cells can be genetically manipulated in culture, by either viral or non-viral methods, which enable the study of individual genes in vitro, thus gaining insights in their function before manipulating these on the mouse. In the future, if the technology is transferred to grow human liver, it would appear to be a simpler and more physiologically relevant system that the classical cell lines used up to now. The advantage of growing tissue directly from the adult liver has the potential to allow us modelling diseases in vitro.  In fact, establishing organoid culture systems from human liver biopsies will facilitate growing tissues from human liver disorders, ranging from liver cancer to inherited metabolic diseases such as A1ATD (alpha 1 antitrypsin deficiency), Tyrosinemia type I or biliary atresia, among others. [caption id="attachment_38438" align="alignright" width="200"] Figure 3: Transplantation and engraftment of liver organoid derived cells into FAH-/-mutant mice. Liver organoids were expanded, differentiated and transplantated into a mouse model of Tyrosinemia type I human liver disease (Fah–/– mice). a: scheme showing the transplantation protocol. b: Representative positive graft within the liver parenchyma. c: Kaplan-Meier survival curve of transplanted mice with positive engraftment (brown curve, graft+), transplanted mice without evidence of engraftment (blue curve, graft-) and non-transplanted control mice (black curve, not transp.). Plot displays the cumulative survival on a linear scale. Kaplan-Meier survival analysis compares overall survival rates between two groups. Log-rank test is used to compare differences in survival. *, log-rank=0.02 (graft+ vs graft-), log-rank= 0.007 (graft+ vs not transp.).[/caption] Of note, liver metabolic diseases are rare/orphan diseases (less than 1 in 2000 births), often abandoned by the scientific community, which are in desperate need of finding good therapeutic strategies. The in-depth study of the mechanisms behind these diseases is hampered by the lack of suitable models that faithfully recapitulate the disease. By growing liver organoids from patients with these diseases, it will be possible to better understand the specific defects in their liver epithelial stem cells and consequently to define specific treatments for each patient (personalized medicine). Furthermore, liver organoid cultures hold potential not only for evaluating autologous stem cell transplantation or disease modelling but also for toxicological and drug screenings, which at present are only possible on animals. Along these lines, this work has an immediate impact on the use of animals in research and on the application the 3Rs in the liver regeneration field. It enables replacing animal models to study liver stem cell proliferation, maintenance and differentiation, in a physiologically relevant ex-vivo model and, consequently, allows the reduction of the number of animals used for liver stem cell studies. It not only replaces and reduces the number of animals but also improves animal welfare (refine), as less animals suffering from liver failure might be used. The potential of the culture system has recently been recognised by the prestigious NC3Rs council, which awarded the international NC3Rs 3Rs prize 2013 for the development of this “mini-livers” in vitro. In summary, a better understanding of the regeneration mechanisms of the liver has facilitated the development of an ex-vivo, near-physiologically relevant culture system that allows, for the first time, growing enough functional mouse liver tissue to transplant into mice with liver disorders¹. Thanks to the novel and remarkable CRISPR/Cas9 technology, that facilitates modifying the genome of single stem cells and subsequently selecting clones with the desired genetic modification, it is now possible to genetically correct intestinal stem cells¹⁴. Therefore, transferring this technology to the human liver tissue would open up the avenue to liver autologous stem cell transplantation of genetically corrected liver cells. Modelling liver diseases and generating biobanks of healthy and diseased cells for use in drug and toxicology screening programs should be feasible in the near future. Author Meritxell Huch of the Wellcome Trust/Cancer Research UK, Gurdon Institute in Cambridge Acknowledgements Meritxell Huch is supported by a Wellcome Trust Recruitment Enhancement award grant. I am thankful to Hans Clevers and my colleagues at the Hubrecht Institute (Utrecht, The Netherlands) and OHSU (Portland, Oregon-USA) where this worked was carried out. Also, I am grateful to the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs) for awarding the international 2013 prize to that research and GlaxoSmithKline for sponsoring the NC3Rs prize. References  1. Huch M, Dorrell C, Boj SF, van Es JH, Li VS, van de Wetering M, Sato T, Hamer K, Sasaki N, Finegold MJ, Haft A, Vries RG, Grompe M, Clevers H. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 2013; 494(7436): 247-50.Duncan, A. 2. W., Dorrell, C. & Grompe, M. Stem cells and liver regeneration. Gastroenterology 2009; 137, 466-481. 3. Asrani SK, Larson JJ, Yawn B, Therneau TM, Kim WR. Underestimation of liver-related mortality in the United States. Gastroenterology 2013; 145: 375–82. 4. Vilarinho S, Lifton RP. Liver transplantation: from inception to clinical practice. Cell 2012; 150: 1096-9. 5. Li L, Clevers H. Coexistence of quiescent and active adult stem cells in mammals. 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