Liver progenitor cell-driven liver regeneration

The liver is a highly regenerative organ, but its regenerative capacity is compromised in severe liver diseases. Hepatocyte-driven liver regeneration that involves the proliferation of preexisting hepatocytes is a primary regeneration mode. On the other hand, liver progenitor cell (LPC)-driven liver regeneration that involves dedifferentiation of biliary epithelial cells or hepatocytes into LPCs, LPC proliferation, and subsequent differentiation of LPCs into hepatocytes is a secondary mode. This secondary mode plays a significant role in liver regeneration when the primary mode does not effectively work, as observed in severe liver injury settings. Thus, promoting LPC-driven liver regeneration may be clinically beneficial to patients with severe liver diseases. In this review, we describe the current understanding of LPC-driven liver regeneration by exploring current knowledge on the activation, origin, and roles of LPCs during regeneration. We also describe animal models used to study LPC-driven liver regeneration, given their potential to further deepen our understanding of the regeneration process. This understanding will eventually contribute to developing strategies to promote LPC-driven liver regeneration in patients with severe liver diseases.


Introduction
The liver is an essential and multifunctional organ in vertebrates. It consists of hepatocytes and biliary epithelial cells (BECs) that are differentiated from common progenitor cells called hepatoblasts during development. Hepatocytes, a major cell type in the liver, detoxify various metabolites, regulate glucose and lipid metabolism, synthesize serum proteins, and secrete bile. BECs form the biliary network that transports bile from hepatocytes to the gallbladder 1 . Upon food ingestion, bile is released from the gallbladder into the duodenum and helps absorb fats in the gut.
The liver is also a highly regenerative organ. It is able to restore its mass and function after injury. Depending on the source of regenerating hepatocytes, there are two modes of liver regeneration: hepatocyte-and LPC-driven liver regeneration 2,3 . For instance, upon two-third partial hepatectomy, the remaining hepatocytes proliferate to restore the resected liver mass 4 (hepatocyte-driven regeneration). On the other hand, when hepatocyte proliferation is compromised, BECs are able to dedifferentiate into liver progenitor cells (LPCs), also known as oval cells. Then, these LPCs later differentiate into hepatocytes 2,3 (LPC-driven regeneration). In addition to this BEC-to-LPC dedifferentiation, hepatocytes can dedifferentiate into LPCs and later differentiate back into hepatocytes 5 .
Given that liver diseases have been a major health concern due to their high prevalence and poor long-term clinical outcome, the regenerative potential of the liver is especially important to note. Globally, approximately two million deaths per year are caused by severe liver diseases, including viral hepatitis, liver cirrhosis, and liver cancer 6 . Currently, liver transplantation is the only curative option for these life-threatening diseases; however, the shortage of donor livers limits this option. Thus, patients with severe liver diseases often die while waiting for a donor liver 6,7 . Given that LPC-driven liver regeneration is prevalent in severe liver injury settings, augmenting this regeneration mode should be clinically beneficial to patients with severe liver diseases.
In this review, we focus mainly on LPC-driven liver regeneration by examining recent findings and exploring current knowledge about the activation, origin, and role of LPCs during liver regeneration. We also provide information on the clinical significance and therapeutic potential of LPCs.

Features of LPCs
Hepatocyte-driven liver regeneration is achieved by the proliferation of preexisting hepatocytes 4 , whereas LPCdriven liver regeneration is achieved by the proliferation and subsequent differentiation of LPCs 2,3 . Thus, LPCdriven liver regeneration is important in severe liver injury settings accompanied by impaired hepatocyte proliferation. In LPC-driven liver regeneration, hepatocytes or BECs first dedifferentiate into LPCs following LPC proliferation, and the LPCs then differentiate into hepatocytes 1,8 .

The molecular mechanisms of LPC activation
Extensive LPC activation and expansion are detected in patients with various liver diseases, including alcoholic or nonalcoholic liver disease 25 , chronic viral hepatitis 26,27 , and cholestatic hepatitis 28 , all of which cause a massive loss of hepatocytes in the liver. LPC activation, also referred to as ductular reactions, involves the proliferation and expansion of LPCs in the periportal regions in the liver, thereby increasing the number of bile ductules. Additionally, LPC activation involves macrophage infiltration, extracellular matrix remodeling, and myofibroblast activation 29,30 .
The mechanisms of LPC activation have been extensively investigated using animal models of chronic liver injury. Hepatocyte-specific deletion of the inhibitor kappa B kinase with Alb-Cre mice inhibited hepatocyte proliferation and induced apoptosis 31 . Dying hepatocytes in these mice produced hedgehog ligands that promote the outgrowth of LPCs and myofibroblasts 31 . Hepatocytespecific deletion of survivin with Alb-Cre mice also induced hepatocyte apoptosis and inflammation, eliciting LPC activation 32 . These findings support that the extensive loss of hepatocytes and the impairment of hepatocyte proliferation induce LPC activation. Furthermore, it was recently reported that YAP and mTORC1 signaling are important for LPC activation. YAP and mTORC1 signaling positively regulate the growth of BECderived organoids in vitro and the proliferation of BECs and LPCs in mice 24 . TET1-mediated epigenetic remodeling through YAP signaling was also recently reported to positively control LPC activation 33 .
Additionally, macrophages are known to associate with LPC activation during regeneration. Dying hepatocytes and their debris are engulfed and removed by Kupffer cells, which are the resident macrophages in the liver. These macrophages secrete tumor necrosis factor Fig. 1 The structure of the liver in mammals and zebrafish. The liver consists of hepatocytes, BECs, Kupffer cells, HSCs, and endothelial cells. LPCs are thought to arise near bile ductules, also known as the canals of Hering, which are positioned between the bile duct and hepatocytes. In the zebrafish liver, most BECs make up bile ductules.
(TNF)-like weak inducer of apoptosis (TWEAK), a member of the proinflammatory TNF family, and TWEAK binds to its receptor FGF-inducible 14 (Fn14) expressed on LPCs 30 . Fn14 was increasingly expressed on LPCs in chronic hepatitis C and other human liver diseases 30 . Moreover, overexpression of TWEAK in hepatocytes stimulated LPC proliferation through the nuclear factor-κB (NF-κB) signaling pathway 30,34 .

Origins of LPCs
Despite the absence of LPC-specific markers that are expressed in LPCs but not in BECs, BECs were hypothesized to be the origin of LPCs due to their phenotypical similarity and locational contiguity 48 (Fig. 2). This was later validated by lineage tracing. By genetically labeling nearly all hepatocytes in mice, the Grompe group showed that BECs in mice fed a DDC diet for 2 weeks contributed to LPCs 5 . The same group also showed, by tracing the lineage of Sox9 + BECs with Sox9-CreERT2 mice, that BECs gave rise to LPCs in DDC diet, choline-deficient, ethionine-supplemented diet (CDE), and CCl 4 injury models 49 . By tracing the lineage of BECs with Krt19-CreERT mice, it was also shown that BECs contribute to LPCs in the DDC and CDE models 50,51 . Although these lineage-tracing studies have validated BECs as the origin of LPCs, the activated LPCs in the studies did not differentiate into hepatocytes. Later, the fact that LPCs originate from BECs was also confirmed in liver injury models in which LPCs significantly contribute to hepatocytes 52 . In mice with β1-integrin knocked down in all hepatocytes, thus blocking hepatocyte proliferation, BEC lineage tracing revealed the significant contribution of BECs to hepatocytes in several liver injury models, including DDC 52 . Hepatocyte-specific overexpression of p21 combined with the liver injury models also exhibited a significant contribution of BECs to hepatocytes 52 . BEC-to-LPC dedifferentiation was also observed in zebrafish [53][54][55] . Upon the severe loss of hepatocytes in zebrafish larvae by pharmacogenetic means, BECs dedifferentiated into LPCs and subsequently differentiated into hepatocytes. In this zebrafish model, nearly all hepatocytes in the recovered larvae originated from BECs 53 .
In addition to BECs, lineage-tracing studies revealed hepatocytes as an additional source of LPCs 5,51,56 (Fig. 2). By labeling nearly all hepatocytes in mice, the Grompe group showed that hepatocytes in mice fed a DDC diet for 6, but not 2, weeks contributed to LPCs 5 . Once the liver injury was gone, these hepatocyte-derived LPCs reverted to hepatocytes 5 . These data suggest that hepatocytes can contribute to the LPC population by undergoing metaplasia in chronic liver injury settings. Supporting this hepatocyte origin, overexpression of constitutively active YAP1 (ref. 57 ) or Notch 56 in mature hepatocytes converted the hepatocytes to LPCs. Moreover, inhibiting YAP 23 or Notch 56 signaling in hepatocytes suppressed their conversion to LPCs in the DDC model. Hepatocyteto-LPC dedifferentiation was also observed in zebrafish 58 . In Tg(fabp10a:pt-β-catenin) zebrafish larvae, which overexpress a stable form of β-catenin in hepatocytes, a subset of hepatocytes dedifferentiated into LPCs and later differentiated into hepatocytes 58 , as observed in the mice fed a DDC diet 5 . Although some lineage-tracing studies suggested hepatic stellate cells (HSCs) as an origin of LPCs and regenerated hepatocytes 59-62 , other lineagetracing studies showed no conversion of HSCs to LPCs or hepatocytes in multiple liver injury settings 50,63 , raising uncertainty regarding HSCs as an origin of LPCs (Fig. 2).

The beneficial role of LPCs: their differentiation into hepatocytes
Studies with diseased human livers have suggested that BECs dedifferentiate into LPCs and that LPCs can differentiate into hepatocytes 11,64-67 (Fig.  3). Supporting these human studies, lineage-tracing studies in mice 52,68-71 and zebrafish [53][54][55] have demonstrated that in severe liver injury settings, BECs first dedifferentiate into LPCs and subsequently differentiate into hepatocytes. For the initial mouse lineage-tracing studies, several BECspecific, inducible Cre lines, such as Opn-CreERT2, Krt19-CreERT, Hnf1b-CreERT2, and Sox9-CreERT2, were used to trace the fate of BECs in DDC-and CDEmediated liver injury models. While no contribution of BECs to hepatocytes was observed in the DDC model, a few hepatocytes (<2.5%) originated from BECs in the CDE model 5,50,[72][73][74] . This low percentage of contribution of BECs to hepatocytes raised a question about the significance of LPC-driven regeneration in liver regeneration. However, several groups recently showed a significant contribution of BECs to hepatocytes in severe liver injury settings in which hepatocyte proliferation is greatly compromised. To block hepatocyte proliferation, Mdm2 (ref. 70 ), β1-integrin 52 , or β-catenin 68 was deleted specifically in hepatocytes, or p21 was overexpressed in hepatocytes 52 . In addition to these genetic blocks of hepatocyte proliferation, long-term chronic liver injury elicited the natural impairment of hepatocyte proliferation, thereby inducing the differentiation of BEC-derived LPCs into hepatocytes 69,71 . As previously mentioned in the section on LPC origins, hepatocytes as well as BECs can give rise to LPCs, and hepatocyte-derived LPCs appear to revert to hepatocytes during recovery 5 . For the zebrafish lineage-tracing studies, the BEC-specific, inducible Cre line, Tg(Tp1:CreERT2), was used in the complete hepatocyte-ablation model. Upon severe hepatocyte loss, BECs dedifferentiate into LPCs, and subsequently, LPCs differentiate into either hepatocytes or BECs 53-55 . Intriguingly, suppressing LPC differentiation in the zebrafish model impaired liver regeneration and recovery 75,76 , suggesting the beneficial effect of LPC differentiation on liver recovery.
Given the beneficial effect of promoting LPC differentiation, elucidation of the molecular mechanisms of LPC differentiation has been attempted. Liver-specific c-Met knockout mice fed a DDC diet exhibited a significant reduction in the number of A6 + /EpCAM − hepatocytelike cells compared to c-Met wild-type mice, suggesting that HGF/c-Met signaling may regulate the differentiation of LPCs into hepatocytes during regeneration 43 . In addition, treatment with iloprost, a laminin signaling inhibitor, Fig. 3 The beneficial and negative roles of LPCs. LPCs play a beneficial role in liver regeneration/recovery by differentiating themselves into hepatocytes, whereas they play negative roles by contributing to liver fibrosis and tumor formation, such as HCC and ICC.
increased a LPC-derived hepatocyte population in the CDE model, suggesting that LPC microenvironment may control its differentiation 72 . Notch and Wnt/β-catenin signaling are also involved in LPC differentiation 29 . Wnt3a secreted from macrophages induces the expression of Numb, which inhibits Notch signaling, in LPCs, thereby promoting the differentiation of LPCs into hepatocytes. Jag1 expressed in myofibroblasts increases Notch signaling in LPCs, thereby promoting the differentiation of LPCs into BECs 29 . In addition to these mouse studies, zebrafish studies revealed additional players that control LPC differentiation. Suppressing BMP signaling inhibited the differentiation of LPCs into hepatocytes, and this LPC differentiation defect was also observed in smad5 and tbx2b zebrafish mutants 75,76 . Suppressing Hdac1 activity derepressed sox9b expression, thereby inhibiting the differentiation of LPCs into hepatocytes 75,76 .

The negative roles of LPCs: fibrosis and liver cancer
Hepatic fibrosis, the formation of an abnormally large amount of scar tissue in the liver, occurs in most chronic liver diseases. As a response to liver injury, quiescent HSCs are activated to become profibrogenic myofibroblasts 77 . This HSC activation is associated with LPC activation 78 . A correlation between the number of LPCs and the severity of fibrosis in chronic liver diseases 25,27,[78][79][80] suggests that LPCs may promote hepatic fibrosis (Fig. 3). T helper type 1 (Th1) cells produce IFNγ, which regulates LPC proliferation 81 . BALB/c mice deficient in Th1 signaling fed a CDE diet exhibited reduced LPC proliferation and fibrosis compared to C57Bl/6 mice, which have normal Th1 signaling. Supplementation of IFNγ increased both LPC number and fibrosis in the CDE model 36 . In addition, TWEAK, which induces LPC activation, was associated with liver fibrosis 82 . Administration of TWEAK induced both LPC activation and collagen expression 83 , whereas Fn14 knockout mice fed a CDE diet exhibited reduced LPC proliferation and collagen expression 34 . These results together suggest that LPCs could drive liver fibrosis (Fig. 3).
Liver cancer is the sixth most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide in 2018, accounting for approximately 841,000 new cases and 782,000 deaths annually 6 . Of primary liver cancer cases, 75-85% are hepatocellular carcinoma (HCC), and 10-15% are intrahepatic cholangiocarcinoma (ICC). LPC activation is known to be associated with pathological scarring processes and appears to contribute to liver tumor formation 84 . LPC markers, such as KRT7, KRT19, OV6, and EpCAM, are detected in HCCs 85,86 . More importantly, inhibition of LPC proliferation in chronically injured mouse livers reduced tumor development 38,87,88 . Additionally, functional genomics analysis of human HCCs revealed a Notch-associated signature in one-third of human HCCs 89 . Persistent Notch signaling activation in mouse livers induced features of human hepatocarcinogenesis, including dysplasia and HCC 89 . Given that Notch signaling regulates cancer stem cells during hepatocarcinogenesis 90 , these results together suggest a role for LPCs in HCC formation (Fig. 3).
Given its biliary features, ICC was thought to originate from BECs. Indeed, lineage-tracing studies confirmed the BEC origin 91 . However, ectopic and persistent activation of Notch and AKT signaling in hepatocytes generated ICC, revealing hepatocytes as an additional origin of ICC 92,93 . Moreover, neomorphic mutations of IDH1 or IDH2 that acquire abnormal activity to convert α-ketoglutarate to 2-hydroxyglutarate lead to ICC formation by inhibiting the differentiation of LPCs into hepatocytes 94 . An additional type of primary liver carcinoma that is of interest is combined or mixed hepatocellular cholangiocarcinoma (cHCC-CCA). It has been considered that cHCC-CCA originates from LPCs, which can differentiate into both hepatocytes and BECs 95 . cHCC-CCA also exhibits stem or progenitor features with downregulation of the hepatocyte differentiation program and a commitment to the biliary lineage 96 . These findings together suggest that LPCs contribute to ICC formation regardless of their origin.

Animal models for LPC research
Given the high cost and ethical issues of human studies, animal models have been used to study human liver diseases. Among them, rodents are widely used because of their remarkable genetic similarity to humans 97 . LPCs were first identified in rats 98 , and their activation was primarily investigated in rat models of chronic liver injury in which hepatocyte proliferation was compromised 99 . In this model, hepatocyte necrosis was induced by injecting D-galactosamine, and AFP + LPCs were observed in the periportal area 99 . LPC activation and proliferation were also observed in the Solt-Farber liver injury model in which 2-acetylaminofluorene was administered to rats followed by two-third partial hepatectomy 100,101 .
Given the advantage of genetic manipulation in mice, mouse models have been widely used for LPC research 97 . Mice fed a DDC diet are used to study metabolic liver diseases, sclerosing cholangitis, and biliary fibrosis 102 . Mice fed a CDE diet exhibit steatosis, inflammation, LPC activation and expansion, portal fibrosis, and HCC 103 . CCl 4 is also widely used as a hepatic toxin that induces chronic liver injury. Repeated injection of CCl 4 causes centrilobular necrosis followed by a wound-healing process 97 . It also induces LPC activation and expansion, fibrosis, and cirrhosis 69,97,104 . Additionally, administration of thioacetamide induces chronic inflammation, LPC activation and expansion, fibrosis, cirrhosis, and liver cancer 71,105,106 . Since all these liver toxins induce LPC activation and expansion, these mouse models have been used to investigate the mechanisms of LPC activation and expansion.
As previously mentioned in the section on the beneficial role of LPCs, additional mouse models for BEC/LPCdriven liver regeneration in which BECs significantly contribute to regenerating hepatocytes have recently been established. In these mouse models, liver toxins, such as CDE and DDC, were used to induce liver injury, and hepatocyte proliferation was additionally suppressed 52,68 . Using the hepatocyte-specific p21-overexpressing model, the positive role of TET1 in LPC-driven liver regeneration was recently reported 33 . These mouse models will allow one to better understand the molecular mechanisms of LPC-driven liver regeneration, particularly LPC differentiation.
In addition to rodent models, zebrafish have relatively recently been used for LPC research due to their small size, which allows for simple chemical treatment. Various zebrafish liver injury models, including one-third partial hepatectomy 107,108 , ethanol treatment 55,109 , oncogene-induced liver cancers [110][111][112] , and hepatocyte ablation [53][54][55] , have been established. In particular, the hepatocyte-ablation model has been used to study LPCdriven liver regeneration. Our group developed the Tg (fabp10a:CFP-NTR) zebrafish line that expresses nitroreductase (NTR) under the hepatocyte-specific fabp10a promoter. Since NTR converts the nontoxic prodrug metronidazole (Mtz) into a cytotoxic drug, Mtz treatment specifically ablates hepatocytes in Tg(fabp10a:CFP-NTR) fish. Upon extensive hepatocyte loss, BECs dedifferentiate into LPCs, and LPCs subsequently differentiate into hepatocytes, thereby leading to full liver recovery 53 . Using this model, we recently reported that BMP signaling regulates LPC-driven liver regeneration through Tbx2a and Id2a 75 and that bromodomain and extraterminal proteins (BET), Hdac1, Kdm1a, Sox9b, and Notch3 regulate LPC-driven liver regeneration 76,113,114 . Using the same zebrafish model, two other groups also reported that mTORC1 signaling regulates LPC-driven liver regeneration 115,116 .
In the Tg(fabp10a:CFP-NTR) model, LPC-driven liver regeneration occurs robustly and rapidly. Although this rapid regeneration has allowed us to identify small molecules that impair liver regeneration 53 , it is not suitable for identifying compounds that promote LPC-driven liver regeneration. Our group has recently established a new zebrafish liver injury model for LPC-driven liver regeneration in which LPCs slowly differentiate into hepatocytes 58 . In this model, Tg(fabp10a:pt-β-catenin) zebrafish larvae, which overexpress a stable form of β-catenin in hepatocytes, exhibited hepatocyte damage by oncogene-induced senescence and apoptosis, LPC activation, fibrosis, and differentiation of LPCs into hepatocytes, leading to the recovery of the liver. The activated LPCs persist for several days and gradually differentiate into hepatocytes. This slow progression of LPC differentiation allows the identification of small molecules that can promote the differentiation of LPCs into hepatocytes. Indeed, using this model, we discovered that treatment with EGFR inhibitors promoted LPC-driven liver regeneration, particularly the differentiation of LPCs into hepatocytes 58 . These zebrafish models will not only further help to better understand the molecular mechanisms of LPC-driven liver regeneration but also provide significant insights into promoting LPC-driven regeneration in patients with chronic liver diseases.

Conclusions
Although LPC-driven liver regeneration occurs to restore liver parenchyma in chronic liver diseases, it does not appear to occur effectively in patients with advanced liver disease 117 . A correlation between LPC numbers and disease severity in patients with chronic liver diseases 27 implies not only that LPCs are activated in the patients but also that the LPCs ineffectively differentiate into hepatocytes. Persistent LPCs induce inflammation and subsequent fibrosis by secreting proinflammatory cytokines 118 . Since promoting the differentiation of LPCs into hepatocytes can generate more functional hepatocytes and concomitantly reduce fibrosis, a strategy to promote the differentiation is an attractive therapeutic option for patients with advanced liver disease. To establish such a strategy, it is crucial to identify appropriate target molecules of which manipulation promotes the differentiation of LPCs into hepatocytes. Research using the animal models of chronic and severe liver injury will help to discover such molecules and eventually make the strategy feasible.