[Huch M, Boj SF, Clevers H. Lgr5(+) liver stem cells, hepatic organoids and regenerative medicine. Regen Med. 2013 Jul;8(4):385-7.]
Full Text: http://www.futuremedicine.com/doi/full/10.2217/rme.13.39
The liver is an organ with complex functions. It is responsible for a wide range of vital biological processes, from synthesizing critical plasma proteins like albumin to regulating glucose and ammonia levels. As a result of these diverse and vital functions, loss of liver function results in organ failure and subsequent hypotension, hypoglycemia, encephalopathy and death within days .
Physiological turnover of the liver is largely achieved via de novo replication of its mature cell types, hepatocytes and cholangiocytes (ductal cells), without any direct involvement of dedicated stem/progenitor populations [2–4]. In vertebrates, from fish to humans, the liver also displays a remarkable capacity to regenerate following injury-induced loss of liver mass . This regeneration is achieved in adult liver via two different mechanisms, depending on the type of tissue injury sustained: fully differentiated hepatocytes and ductal cells can re-enter the cell cycle to rapidly compensate for the tissue loss, as seen during hepatectomy or CCl4 chronic damage ; and the so-called ‘oval cell’ response  triggered by hepatotoxins (e.g., 3,5-diethoxycarbonyl-1,4-dihydrocollidine or choline deficient ethionine [CDE]), which cause generalized liver damage with impaired hepatocyte proliferation. Other more controversial homeostasis/regeneration sources in the liver are the liver progenitor cells and ductal cells, which have been proposed to harbor the capacity to generate all functional cell lineages (hepatocytes and ductal cells). Evidence in support of this was obtained from studies employing lineage tracing and CCl4-induced liver damage, when ductal cells were shown to give rise to hepatocytes during normal turnover of the liver and after acute and chronic injury . A separate study by Español-Suñer et al. showed that CDE damage induces the proliferation of ductal cells that contribute to the regeneration of not only ductal cells but also hepatocytes . However, in stark contrast to these studies, via virally-induced Cre tracing, Willenbring’s laboratory showed that liver homeostasis and recovery upon acute damage is almost exclusively achieved by division of hepatocytes with minimal contribution of liver progenitors .
Lgr5 is a stem cell marker in the gut, stomach, hair follicle and mammary gland , but is not expressed in the homeostatic adult liver. However, following both CCl4 acute damage and ‘oval cell response’ damage (3,5-diethoxycarbonyl-1,4-dihydrocollidine and CDE), a population of Lgr5+ stem cells/progenitors has recently been shown to actively contribute to liver regeneration via de novo generation of hepatocytes and ductal cells . This finding has therapeutic implications, but key questions including the origin of the Lgr5+ stem cells still remain to be addressed. Potential sources of these damage-induced Lgr5+ stem cells include Lgr5– progenitor cells from the noninjured liver , recruitment from distant sites (e.g., mesenchymal cells)  or transdifferentiation of hepatocytes into ductal cells, as occurs in tumors of the biliary tree (intrahepatic cholangiocarcinoma) . One can also speculate that the source of the Lgr5+ stem cells is dictated by the type of injury and the specific regeneration requirements of the liver.
The regenerative capacity of the liver becomes exhausted during chronic liver damage, when normal liver tissue is replaced by fibrotic tissue (cirrhosis), ultimately resulting in liver failure . Organ transplantation is the only established life-saving treatment for final-stage liver disease . It is estimated that 20,000 people receive liver transplants each year worldwide . However, its application remains restricted by the limited availability of donor organs, which has prompted efforts to develop alternative treatments ranging from living donor liver transplantation to cell therapy transplantation . Hepatocyte transplantation has been successful in animal models, but its widespread clinical application for transplantation to patients is again restricted by the limited availability of donor hepatocytes from healthy livers.
Despite the enormous replication potential of the liver, there are currently no culture systems available that sustain hepatocyte replication in vitro. Hepatocytes can be maintained in culture for a few days. However, the cells lose their hepatocyte phenotype and function almost immediately , thus precluding its application for cell therapy treatments. To obtain a source of functional hepatocytes, a variety of in vitro systems have been described, ranging from culturing fetal and neonatal liver progenitors  to obtaining hepatocytes by directed differentiation of either embryonic stem cells  or induced pluripotent stem (iPS) cells . However, none of these culture systems generate populations of cells with a self-sustaining capacity that can be expanded into the large amounts of tissue needed for therapeutic cell transplantations.
Liver stem cells have the potential to self-renew and differentiate into the functional hepatic lineages [5,8]. However, it has proven difficult to expand these cells beyond a few days in culture [5,9,16]. Recently, we have described a novel liver stem cell culture system that allows indefinite in vitro expansion (for >1 year) of liver progenitors  into ‘liver organoids’, in a defined culture medium in the absence of a mesenchymal niche. Importantly, isolated adult hepatic cells retain their differentiation potential over time in this culture system. The cultured cells express ductal markers and, when subjected to a differentiation protocol based on removing the proliferation signals and inhibiting ductal fate, approximately 33–50% of the cells differentiate into functional hepatocytes in vitro. The huge expansion and differentiation capacity of the liver organoid cultures facilitated the engraftment and repopulation of the livers of mice with congenital metabolic liver disease and partially restored its hepatic function. Selecting for hepatocyte-specific populations prior to transplantation, optimizing the transplantation protocols and differentiation protocols towards hepatocyte fate (by converting 100% of the cells to hepatocytes), is expected to markedly enhance transplantation efficiency and achieve a complete rescue of the liver function in these mice. It will be interesting to test whether this culture system is not only applicable to liver metabolic diseases but also to diseases in which biliary function is impaired, such as biliary atresia. Patients with this condition exhibit defective differentiation of biliary tracts in the liver, causing atresia and preventing the secretion of bile to the intestine . Transplantation of stem cells harboring the capacity to differentiate into functional biliary duct cells has not been attempted yet, but the liver organoid cultures seem predisposed to adopt
a biliary fate and could be an excellent source of biliary cells to treat cholangiopathies.
Another important feature of the liver organoid cultures is the long-term maintenance of genomic integrity in cultured cells. In contrast to iPS cell-derived hepatocyes  or hepatocyes obtained via direct viral conversion of fibroblasts [18,19], the cultured cells in the organoid system are obtained directly from adult tissue without the need for genetic modifications or introduction of reprogramming factors. Furthermore, no malignant transformation is observed upon transplantation [Huch M, Clevers H, Unpublished Data]. Collectively, this highlights an advantage of the liver organoid culture system over existing cellular reprogramming technologies that suffer from inherent genetic stability problems . The liver organoid cultures therefore hold promise as a safe clinical source of hepatocytes for transplantation. Although, to fully address any safety issue, whole-genome sequencing of the cells maintained for long periods of time in culture will be necessary.
We have recently established intestinal organoids from cystic fibrosis patients and demonstrated that these can be used directly to demonstrate the defective chloride channel and its restoration by decreased temperatures or experimental corrector compounds . Along these lines, liver organoid cultures hold potential not only for evaluating autologous stem cell transplantation but also for modeling diseases in vitro. Establishing an organoid culture system from human liver patients will facilitate the modeling of inherited liver disorders, such as α1 antitrypsin deficiency, familial hypercholesterolemia or biliary atresia, among others. By growing liver organoids from patients with inherited liver diseases, it will be possible to determine the specific differentiation defects in their liver epithelial stem cells and consequently to define specific treatment regimens for each patient (personalized medicine). iPS cell lines have been generated to model liver metabolic disorders , but again, the effects of the Yamanaka reprogramming factors on the phenotype of the disease itself are still unknown and unpredictable, and may vary between patients. In this regard, growing tissue directly from the damaged liver would appear to be a simpler and more standardized approach. However, in order to fully model hepatocyte-related diseases, it will be necessary to optimize differentiation protocols that allow 100% conversion of the liver organoid-derived cells into hepatocytes.
In summary, a better understanding of the regeneration mechanisms of the liver has facilitated the development of a near-physiologicalex vivo culture system that, for the first time, allows researchers to grow sufficient quantities of functional tissue from stem/progenitor populations to transplant and engraft mice with liver disorders . Since genetic manipulation of growing stem cells is now possible , autologous stem cell transplantation of genetically modified cells should soon become a reality. Modeling liver diseases and generating ‘biobanks’ of healthy and diseased cells for use in drug screening programs should be feasible in the near future.Acknowledgements
The authors are very thankful to N Barker and C Dorrell for helpful insights and proofreading.Financial & competing interests disclosure
This work was supported by grants to M Huch (EU/236954) and SF Boj (EU/232814). H Clevers is supported by Koningin Wilhelmina Fonds (KWF) program grant PF-HUBR-2007-3956. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.Open Access
This work is licensed under the Creative Commons Attribution-NonCommercial 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/
References ChooseTop of pageReferences <<
1 . Michalopoulos GK. Liver regeneration. J. Cell Physiol.213,286–300 (2007). [CrossRef][Medline][CAS] 2 . Español-Suñer R, Carpentier R, Van Hul N et al. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice. Gastroenterology143,1564–1575 (2012). [CrossRef][Medline] 3 . Furuyama K, Kawaguchi Y, Akiyama H et al. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat. Genet.43,34–41 (2011). [CrossRef][Medline][CAS] 4 . Ponder KP. Analysis of liver development, regeneration, and carcinogenesis by genetic marking studies. FASEB J.10,673–682 (1996).[Medline][CAS] 5 . Duncan AW, Dorrell C, Grompe M. Stem cells and liver regeneration. Gastroenterology137,466–481 (2009). [CrossRef][Medline] 6 . Malato Y, Naqvi S, Schürmann N et al. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration. J. Clin. Invest.121,4850–4860 (2011). [CrossRef][Medline][CAS] 7 . Barker N, Bartfeld S, Clevers H. Tissue-resident adult stem cell populations of rapidly self-renewing organs. Cell Stem Cell7,656–670 (2010). [CrossRef][Medline][CAS] 8 . Huch M, Dorrell C, Boj SF et al.In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.Nature494,247–250 (2013). [CrossRef][Medline][CAS] 9 . Dorrell C, Erker L, Schug J et al. Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice. Genes Dev.25,1193–1203 (2011). [CrossRef][Medline][CAS] 10 . Fan B, Malato Y, Calvisi DF et al. Cholangiocarcinomas can originate from hepatocytes in mice. J. Clin. Invest.122,2911–2915 (2012). [CrossRef][Medline][CAS] 11 . Vilarinho S, Lifton RP. Liver transplantation: from inception to clinical practice. Cell150,1096–1099 (2012). [CrossRef][Medline][CAS] 12 . Sigal SH, Brill S, Fiorino AS, Reid LM. The liver as a stem cell and lineage system. Am. J. Physiol.263,G139–G148 (1992). [Medline][CAS] 13 . Michalopoulos GK, Bowen WC, Mule K, Stolz DB. Histological organization in hepatocyte organoid cultures. Am. J. Pathol.159,1877–1887 (2001). [CrossRef][Medline][CAS] 14 . Hannan NR, Segeritz CP, Touboul T, Vallier L. Production of hepatocyte-like cells from human pluripotent stem cells. Nat. Protoc.8,430–437 (2013). [CrossRef][Medline][CAS] 15 . Si-Tayeb K, Noto FK, Nagaoka M et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology51,297–305 (2010). [CrossRef][Medline][CAS] 16 . Shin S, Walton G, Aoki R et al. Foxl1-Cre-marked adult hepatic progenitors have clonogenic and bilineage differentiation potential.Genes Dev.25,1185–1192 (2011). [CrossRef][Medline][CAS] 17 . Mack CL, Sokol RJ. Unraveling the pathogenesis and etiology of biliary atresia. Pediatr. Res.57,87R–94R (2005). [CrossRef][Medline] 18 . Huang P, He Z, Ji S et al. Induction of functional hepatocyte-like cells from mouse fibroblasts by defined factors. Nature475,386–389 (2011). [CrossRef][Medline][CAS] 19 . Sekiya S, Suzuki A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature475,390–393 (2011).[CrossRef][Medline][CAS] 20 . Pera MF. Stem cells: the dark side of induced pluripotency. Nature471,46–47 (2011). [CrossRef][Medline][CAS] 21 . Dekkers JF, Wiegerinck CL, de Jonge HR et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat. Med.doi:10.1038/nm.3201 (2013) (Epub ahead of print). [Medline] 22 . Rashid ST, Corbineau S, Hannan N et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J. Clin. Invest.120,3127–3136 (2010). [CrossRef][Medline][CAS] 23 . Koo BK, Stange DE, Sato T et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods9,81–83 (2011).[CrossRef][Medline]