Long-term culture of genome-stable bipotent stem cells from adult human liver.

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.

Cholangiocarcinoma cell proliferation is enhanced in primary sclerosing cholangitis: A role for IL-17A.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease of the biliary tree and a risk factor for development of cholangiocarcinoma (CCA). The pathogenesis of PSC-related CCA is largely unclear, although it is assumed that chronic inflammatory environment plays a pivotal role. We aimed to investigate the effect of inflammation-related cytokines in PSC on the proliferation rate of cancer cells. For this, the proliferation index in PSC-CCA and sporadic CCA was determined by Ki-67 immunohistochemistry. The percentage of Ki-67 positivity in cancer cells was significantly higher in PSC-CCA than in sporadic CCA (41.3% ± 5.7% vs 25.8% ± 4.1%; P = .038). To assess which cytokines in the inflammatory environment have the potential to stimulate cancer cell proliferation, patient-derived CCA organoids (CCAOs) were exposed to five cytokines related to PSC (Interleukin (IL)-1ß, IL-6, IL-17A, interferon gamma and tumor necrosis factor alpha). Only IL-17A showed a significant stimulatory effect on cell proliferation in CCAOs, increasing organoid size by 45.9% ± 16.4% (P < .01) and proliferation rate by 38% ± 16% (P < .05). IL-17A immunohistochemistry demonstrated that PSC-CCA might express more IL-17A than sporadic CCA. Moreover, correlation analysis in sporadic CCA and PSC-CCA found a significant correlation between IL-17A expression and proliferation. In conclusion, tumor cell proliferation is increased in PSC-CCA cells compared with sporadic CCA cells. IL-17A increases CCA cell proliferation in vitro and may contribute to the high proliferation rate in PSC-CCA in situ. Therefore, IL-17A represents a new potential therapeutic target in (PSC-)CCA, to be tested in future trials.

Modelling immune cytotoxicity for cholangiocarcinoma with tumour-derived organoids and effector T cells.

BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. METHODS: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. RESULTS: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. CONCLUSIONS: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.

The current status of stem cell-based therapies during ex vivo graft perfusion: An integrated review of four organs.

The use of extended criteria donor grafts is a promising strategy to increase the number of organ transplantations and reduce waitlist mortality. However, these organs are often compromised and/or damaged, are more susceptible to preservation injury, and are at risk for developing post-transplant complications. Ex vivo organ perfusion is a novel technology to preserve donor organs while providing oxygen and nutrients at distinct perfusion temperatures. This preservation method allows to resuscitate grafts and optimize function with therapeutic interventions prior to solid organ transplantation. Stem cell-based therapies are increasingly explored for their ability to promote regeneration and reduce the inflammatory response associated with in vivo reperfusion. The aim of this review is to describe the current state of stem cell-based therapies during ex vivo organ perfusion for the kidney, liver, lung, and heart. We discuss different strategies, including type of cells, route of administration, mechanisms of action, efficacy, and safety. The progress made within lung transplantation justifies the initiation of clinical trials, whereas more research is likely required for the kidney, liver, and heart to progress into clinical application. We emphasize the need for standardization of methodology to increase comparability between future (clinical) studies.

Kinome profiling of cholangiocarcinoma organoids reveals potential druggable targets that hold promise for treatment stratification.

BACKGROUND: Cholangiocarcinoma is a rare but lethal cancer of the biliary tract. Its first-line treatment is currently restricted to chemotherapy, which provides limited clinical benefit. Kinase inhibitors targeting oncogenic intracellular signaling have changed the treatment paradigm of cancer over the last decades. However, they are yet to be widely applied in cholangiocarcinoma therapy. Cholangiocarcinoma has marked molecular heterogeneity, which complicates the discovery of new treatments and requires patient stratification. Therefore, we investigated whether a commercial kinome profiling platform could predict druggable targets in cholangiocarcinoma. METHODS: Kinase activity in patient-derived cholangiocarcinoma organoids, non-tumorous adjacent tissue-derived and healthy donor-derived intrahepatic cholangiocyte organoids was determined using the PamChip® phosphotyrosine kinase microarray platform. Kinome profiles were compared and correlated with RNA sequencing and (multi-)kinase inhibitor screening of the cholangiocarcinoma organoids. RESULTS: Kinase activity profiles of individual cholangiocarcinoma organoids are different and do not cluster together. However, growth factor signaling (EGFR, PDGFRß) and downstream effectors (MAPK pathway) are more active in cholangiocarcinoma organoids and could provide potential druggable targets. Screening of 31 kinase inhibitors revealed several promising pan-effective inhibitors and compounds that show patient-specific efficacy. Kinase inhibitor sensitivity correlated to the activity of its target kinases for several inhibitors, signifying them as potential predictors of response. Moreover, we identified correlations between drug response and kinases not directly targeted by those drugs. CONCLUSIONS: In conclusion, kinome profiling is a feasible method to identify druggable targets for cholangiocarcinoma. Future studies should confirm the potential of kinase activity profiles as biomarkers for patient stratification and precision medicine.

Assessment of human leukocyte antigen matching algorithm PIRCHE-II on liver transplantation outcomes.

For liver transplantations, human leukocyte antigen (HLA) matching is not routinely performed because observed effects have been inconsistent. Nevertheless, long-term liver transplantation outcomes remain suboptimal. The availability of a more precise HLA-matching algorithm, Predicted Indirectly Recognizable HLA Epitopes II (PIRCHE-II), now enables robust assessment of the association between HLA matching and liver transplantation outcomes. We performed a single-center retrospective cohort study of 736 liver transplantation patients. Associations between PIRCHE-II and HLAMatchmaker scores and mortality, graft loss, acute and chronic rejection, ischemic cholangiopathy, and disease recurrence were evaluated with Cox proportional hazards models. Associations between PIRCHE-II with 1-year, 2-year, and 5-year outcomes and severity of acute rejection were assessed with logistic and linear regression analyses, respectively. Subgroup analyses were performed for autoimmune and nonautoimmune indications, and patients aged 30 years and younger, and older than 30 years. PIRCHE-II and HLAMatchmaker scores were not associated with any of the outcomes. However, patients who received transplants for autoimmune disease showed more acute rejection and graft loss, and these risks negatively associated with age. Rhesus mismatch more than doubled the risk of disease recurrence. Moreover, PIRCHE-II was inversely associated with graft loss in the subgroup of patients aged 30 years and younger with autoimmune indications. The absence of associations between PIRCHE-II and HLAMatchmaker scores and the studied outcomes refutes the need for HLA matching for liver (stem cell) transplantations for nonautoimmune disease. For autoimmune disease, the activated immune system seems to increase risks of acute rejection and graft loss. Our results may suggest the benefits of transplantations with rhesus matched but PIRCHE-II mismatched donor livers.

The potential and limitations of intrahepatic cholangiocyte organoids to study inborn errors of metabolism.

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.

Tissue-specific mutation accumulation in human adult stem cells during life.

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Long-term live imaging and multiscale analysis identify heterogeneity and core principles of epithelial organoid morphogenesis.

BACKGROUND: 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. RESULTS: We quantified organoid behaviour at single-cell (microscale), individual-organoid (mesoscale), and entire-culture (macroscale) levels. At single-cell resolution, we monitored formation, monolayer polarisation, and degeneration and identified diverse behaviours, including lumen expansion and decline (size oscillation), migration, rotation, and multi-organoid fusion. Detailed individual organoid quantifications lead to a mechanical 3D agent-based model. A derived scaling law and simulations support the hypotheses that size oscillations depend on organoid properties and cell division dynamics, which is confirmed by bright-field microscopy analysis of entire cultures. CONCLUSION: Our multiscale analysis provides a systematic picture of the diversity of cell organisation in organoids by identifying and quantifying the core regulatory principles of organoid morphogenesis.

Impact of hypoxia and AMPK on CFTR-mediated bicarbonate secretion in human cholangiocyte organoids.

Cholangiocytes express cystic fibrosis transmembrane conductance regulator (CFTR), which is involved in bicarbonate secretion for the protection against bile toxicity. During liver transplantation, prolonged hypoxia of the graft is associated with cholangiocyte loss and biliary complications. Hypoxia is known to diminish CFTR activity in the intestine, but whether it affects CFTR activity in cholangiocytes remains unknown. Thus, the aim of this study is to investigate the effect of hypoxia on CFTR activity in intrahepatic cholangiocyte organoids (ICOs) and test drug interventions to restore bicarbonate secretion. Fifteen different human ICOs were cultured as monolayers and ion channel [CFTR and anoctamin-1 (ANO1)] activity was determined using an Ussing chamber assay with or without AMP kinase (AMPK) inhibitor under hypoxic and oxygenated conditions. Bile toxicity was tested by apical exposure of cells to fresh human bile. Overall gene expression analysis showed a high similarity between ICOs and primary cholangiocytes. Under oxygenated conditions, both CFTR and ANO1 channels were responsible for forskolin and uridine-5'-triphosphate (UTP) UTP-activated anion secretion. Forskolin stimulation in the absence of intracellular chloride showed ion transport, indicating that bicarbonate could be secreted by CFTR. During hypoxia, CFTR activity significantly decreased (P = 0.01). Switching from oxygen to hypoxia during CFTR measurements reduced CFTR activity (P = 0.03). Consequently, cell death increased when ICO monolayers were exposed to bile during hypoxia compared with oxygen (P = 0.04). Importantly, addition of AMPK inhibitor restored CFTR-mediated anion secretion during hypoxia. ICOs provide an excellent model to study cholangiocyte anion channels and drug-related interventions. Here, we demonstrate that hypoxia affects cholangiocyte ion secretion, leaving cholangiocytes vulnerable to bile toxicity. The mechanistic insights from this model maybe relevant for hypoxia-related biliary injury during liver transplantation.NEW & NOTEWORTHY The previously described liver-derived organoids resemble primary cholangiocytes and should be properly named intrahepatic cholangiocyte organoids (ICOs). ICOs have functional cholangiocyte ion channels (CFTR and ANO1). CFTR might be able to secrete bicarbonate directly into the bile duct lumen. Hypoxia inhibits CFTR and ANO1 functionality in ICOs, which can partially be restored by addition of an AMP kinase inhibitor. Hypoxia impairs cholangiocyte resistance against cytotoxic effects of bile, resulting in increased cell death.

Large-Scale Production of LGR5-Positive Bipotential Human Liver Stem Cells.

BACKGROUND AND AIMS: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. APPROACH AND RESULTS: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40-fold cell expansion after 2 weeks, compared with 6-fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver-like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up-regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. CONCLUSIONS: We established a highly efficient method for culturing large numbers of LGR5-positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish.

Biliary disorders can lead to life-threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5+ organoids onto tissue-specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte-markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs.

The biological process of lysine-tRNA charging is therapeutically targetable in liver cancer.

BACKGROUND & AIMS: Mature transfer RNAs (tRNA) charged with amino acids decode mRNA to synthesize proteins. Dysregulation of translational machineries has a fundamental impact on cancer biology. This study aims to map the tRNAome landscape in liver cancer patients and to explore potential therapeutic targets at the interface of charging amino acid with tRNA. METHODS: Resected tumour and paired tumour-free (TFL) tissues from hepatocellular carcinoma (HCC) patients (n = 69), and healthy liver tissues from organ transplant donors (n = 21), HCC cell lines, and cholangiocarcinoma (CC) patient-derived tumour organoids were used. RESULTS: The expression levels of different mature tRNAs were highly correlated and closely clustered within individual tissues, suggesting that different members of the tRNAome function cooperatively in protein translation. Interestingly, high expression of tRNA-Lys-CUU in HCC tumours was associated with more tumour recurrence (HR 1.1; P = .022) and worse patient survival (HR 1.1; P = .0037). The expression of Lysyl-tRNA Synthetase (KARS), the enzyme catalysing the charge of lysine to tRNA-Lys-CUU, was significantly upregulated in HCC tumour tissues compared to tumour-free liver tissues. In HCC cell lines, lysine deprivation, KARS knockdown or treatment with the KARS inhibitor cladosporin effectively inhibited overall cell growth, single cell-based colony formation and cell migration. This was mechanistically mediated by cell cycling arrest and induction of apoptosis. Finally, these inhibitory effects were confirmed in 3D cultured patient-derived CC organoids. CONCLUSIONS: The biological process of charging tRNA-Lys-CUU with lysine sustains liver cancer cell growth and migration, and is clinically relevant in HCC patients. This process can be therapeutically targeted and represents an unexplored territory for developing novel treatment strategies against liver cancer.

Modeling liver cancer and therapy responsiveness using organoids derived from primary mouse liver tumors.

The current understanding of cancer biology and development of effective treatments for cancer remain far from satisfactory. This in turn heavily relies on the availability of easy and robust model systems that resemble the architecture/physiology of the tumors in patients to facilitate research. Cancer research in vitro has mainly been based on the use of immortalized 2D cancer cell lines that deviate in many aspects from the original primary tumors. The recent development of the organoid technology allowing generation of organ-buds in 3D culture from adult stem cells has endowed the possibility of establishing stable culture from primary tumors. Although culturing organoids from liver tumors is thought to be difficult, we now convincingly demonstrate the establishment of organoids from mouse primary liver tumors. We have succeeded in culturing 91 lines from 129 liver tissue/tumors. These organoids can be grown in long-term cultures in vitro. About 20% of these organoids form tumors in immunodeficient mice upon (serial) transplantation, confirming their tumorigenic and self-renewal properties. Interestingly, single cells from the tumor organoids have high efficiency of organoid initiation, and a single organoid derived from a cancer cell is able to initiate a tumor in mice, indicating the enrichment of tumor-initiating cells in the tumor organoids. Furthermore, these organoids recapitulate, to some extent, the heterogeneity of liver cancer in patients, with respect to phenotype, cancer cell composition and treatment response. These model systems shall provide enormous opportunities to advance our research on liver cancer (stem cell) biology, drug development and personalized medicine.

Necroptotic Cell Death in Liver Transplantation and Underlying Diseases: Mechanisms and Clinical Perspective.

Cell death is a natural process for the turnover of aged cells, but it can also arise as a result of pathological conditions. Cell death is recognized as a key feature in both acute and chronic hepatobiliary diseases caused by drug, alcohol, and fat uptake; by viral infection; or after surgical intervention. In the case of chronic disease, cell death can lead to (chronic) secondary inflammation, cirrhosis, and the progression to liver cancer. In liver transplantation, graft preservation and ischemia/reperfusion injury are associated with acute cell death. In both cases, so-called programmed cell death modalities are involved. Several distinct types of programmed cell death have been described of which apoptosis and necroptosis are the most well known. Parenchymal liver cells, including hepatocytes and cholangiocytes, are susceptible to both apoptosis and necroptosis, which are triggered by distinct signal transduction pathways. Apoptosis is dependent on a proteolytic cascade of caspase enzymes, whereas necroptosis induction is caspase-independent. Moreover, different from the "silent" apoptotic cell death, necroptosis can cause a secondary inflammatory cascade, so-called necroinflammation, triggered by the release of various damage-associated molecular patterns (DAMPs). These DAMPs activate the innate immune system, leading to both local and systemic inflammatory responses, which can even cause remote organ failure. Therapeutic targeting of necroptosis by pharmacological inhibitors, such as necrostatin-1, shows variable effects in different disease models.

Experimental models to unravel the molecular pathogenesis, cell of origin and stem cell properties of cholangiocarcinoma.

Human cholangiocarcinoma (CCA) is an aggressive tumour entity arising from the biliary tree, whose molecular pathogenesis remains largely undeciphered. Over the last decade, the advent of high-throughput and cell-based techniques has significantly increased our knowledge on the molecular mechanisms underlying this disease while, at the same time, unravelling CCA complexity. In particular, it becomes clear that CCA displays pronounced inter- and intratumoural heterogeneity, which is presumably the consequence of the interplay between distinct tissues and cells of origin, the underlying diseases, and the associated molecular alterations. To better characterize these events and to design novel and more effective therapeutic strategies, a number of CCA experimental and preclinical models have been developed and are currently generated. This review summarizes the current knowledge and understanding of these models, critically underlining their translational usefulness and limitations. Furthermore, this review aims to provide a comprehensive overview on cells of origin, cancers stem cells and their dynamic interplay within CCA tissue.

Dynamics of Proliferative and Quiescent Stem Cells in Liver Homeostasis and Injury.

BACKGROUND & AIMS: Adult liver stem cells are usually maintained in a quiescent/slow-cycling state. However, a proliferative population, marked by leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), was recently identified as an important liver stem cell population. We aimed to investigate the dynamics and functions of proliferative and quiescent stem cells in healthy and injured livers. METHODS: We studied LGR5-positive stem cells using diphtheria toxin receptor and green fluorescent protein (GFP) knock-in mice. In these mice, LGR5-positive cells specifically coexpress diphtheria toxin receptor and the GFP reporter. Lineage-tracing experiments were performed in mice in which LGR5-positive stem cells and their daughter cells expressed a yellow fluorescent protein/mTmG reporter. Slow-cycling stem cells were investigated using GFP-based, Tet-on controlled transgenic mice. We studied the dynamics of both stem cell populations during liver homeostasis and injury induced by carbon tetrachloride. Stem cells were isolated from mouse liver and organoid formation assays were performed. We analyzed hepatocyte and cholangiocyte lineage differentiation in cultured organoids. RESULTS: We did not detect LGR5-expressing stem cells in livers of mice at any stage of a lifespan, but only following liver injury induced by carbon tetrachloride. In the liver stem cell niche, where the proliferating LGR5+ cells are located, we identified a quiescent/slow-cycling cell population, called label-retaining cells (LRCs). These cells were present in the homeostatic liver, capable of retaining the GFP label over 1 year, and expressed a panel of progenitor/stem cell markers. Isolated single LRCs were capable of forming organoids that could be carried in culture, expanded for months, and differentiated into hepatocyte and cholangiocyte lineages in vitro, demonstrating their bona fide stem cell properties. More interestingly, LRCs responded to liver injury and gave rise to LGR5-expressing stem cells, as well as other potential progenitor/stem cell populations, including SOX9- and CD44-positive cells. CONCLUSIONS: Proliferative LGR5 cells are an intermediate stem cell population in the liver that emerge only during tissue injury. In contrast, LRCs are quiescent stem cells that are present in homeostatic liver, respond to tissue injury, and can give rise to LGR5 stem cells, as well as SOX9- and CD44-positive cells.