"Small Hepatocytes" in the Liver.

Mature hepatocytes (MHs) in an adult rodent liver are categorized into the following three subpopulations based on their proliferative capability: type I cells (MH-I), which are committed progenitor cells that possess a high growth capability and basal hepatocytic functions; type II cells (MH-II), which possess a limited proliferative capability; and type III cells (MH-III), which lose the ability to divide (replicative senescence) and reach the final differentiated state. These subpopulations may explain the liver's development and growth after birth. Generally, small-sized hepatocytes emerge in mammal livers. The cells are characterized by being morphologically identical to hepatocytes except for their size, which is substantially smaller than that of ordinary MHs. We initially discovered small hepatocytes (SHs) in the primary culture of rat hepatocytes. We believe that SHs are derived from MH-I and play a role as hepatocytic progenitors to supply MHs. The population of MH-I (SHs) is distributed in the whole lobules, a part of which possesses a self-renewal capability, and decreases with age. Conversely, injured livers of experimental models and clinical cases showed the emergence of SHs. Studies demonstrate the involvement of SHs in liver regeneration. SHs that appeared in the injured livers are not a pure population but a mixture of two distinct origins, MH-derived and hepatic-stem-cell-derived cells. The predominant cell-derived SHs depend on the proliferative capability of the remaining MHs after the injury. This review will focus on the SHs that appeared in the liver and discuss the significance of SHs in liver regeneration.

ß-adrenergic receptor agonist promotes ductular expansion during 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced chronic liver injury.

Intrahepatic nerves are involved in the regulation of metabolic reactions and hepatocyte-based regeneration after surgical resection, although their contribution to chronic liver injury remains unknown. Given that intrahepatic nerves are abundant in the periportal tissue, they may be correlated also with cholangiocyte-based regeneration. Here we demonstrate that isoproterenol (ISO), a ß-adrenergic receptor agonist, promoted ductular expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo. Immunofluorescence analysis shows that nerve fibers positive for tyrosine hydroxylase form synaptophysin-positive nerve endings on epithelial cell adhesion molecule-positive (EpCAM+) cholangiocytes as well as on Thy1+ periportal mesenchymal cells (PMCs) that surround bile ducts, suggesting that the intrahepatic biliary tissue are targeted by sympathetic nerves. In vitro analyses indicate that ISO directly increases cAMP levels in cholangiocytes and PMCs. Mechanistically, ISO expands the lumen of cholangiocyte organoids, resulting in promotion of cholangiocyte proliferation, whereas it increases expression of fibroblast growth factor 7, a growth factor for cholangiocytes, in PMCs. Taken together, the results indicate that intrahepatic sympathetic nerves regulate remodeling of bile ducts during DDC-injury by the activation of ß-adrenergic receptors on cholangiocytes and PMCs.

CINC-2 and miR-199a-5p in EVs secreted by transplanted Thy1+ cells activate hepatocytic progenitor cell growth in rat liver regeneration.

BACKGROUND: Small hepatocyte-like progenitor cells (SHPCs) are hepatocytic progenitor cells that transiently form clusters in rat livers treated with retrorsine (Ret) that underwent 70% partial hepatectomy (PH). We previously reported that transplantation of Thy1+ cells obtained from D-galactosamine-treated livers promotes SHPC expansion, thereby accelerating liver regeneration. Extracellular vesicles (EVs) secreted by Thy1+ cells induce sinusoidal endothelial cells (SECs) and Kupffer cells (KCs) to secrete IL17B and IL25, respectively, thereby activating SHPCs through IL17 receptor B (RB) signaling. This study aimed to identify the inducers of IL17RB signaling and growth factors for SHPC proliferation in EVs secreted by Thy1+ cells (Thy1-EVs). METHODS: Thy1+ cells isolated from the livers of rats treated with D-galactosamine were cultured. Although some liver stem/progenitor cells (LSPCs) proliferated to form colonies, others remained as mesenchymal cells (MCs). Thy1-MCs or Thy1-LSPCs were transplanted into Ret/PH-treated livers to examine their effects on SHPCs. EVs were isolated from the conditioned medium (CM) of Thy1-MCs and Thy1-LSPCs. Small hepatocytes (SHs) isolated from adult rat livers were used to identify factors regulating cell growth in Thy1-EVs. RESULTS: The size of SHPC clusters transplanted with Thy1-MCs was significantly larger than that of SHPC clusters transplanted with Thy1-LSPCs (p = 0.02). A comprehensive analysis of Thy1-MC-EVs revealed that miR-199a-5p, cytokine-induced neutrophil chemoattractant-2 (CINC-2), and monocyte chemotactic protein 1 (MCP-1) were candidates for promoting SHPC growth. Additionally, miR-199a-5p mimics promoted the growth of SHs (p = 0.02), whereas CINC-2 and MCP-1 did not. SECs treated with CINC-2 induced Il17b expression. KCs treated with Thy1-EVs induced the expression of CINC-2, Il25, and miR-199a-5p. CM derived from SECs treated with CINC-2 accelerated the growth of SHs (p = 0.03). Similarly, CM derived from KCs treated with Thy1-EVs and miR-199a-5p mimics accelerated the growth of SHs (p = 0.007). In addition, although miR-199a-overexpressing EVs could not enhance SHPC proliferation, transplantation of miR-199a-overexpressing Thy1-MCs could promote the expansion of SHPC clusters. CONCLUSION: Thy1-MC transplantation may accelerate liver regeneration owing to SHPC expansion, which is induced by CINC-2/IL17RB signaling and miR-199a-5p via SEC and KC activation.

Isolation of Small Hepatocyte-Like Progenitor Cells from Retrorsine/Partial Hepatectomy Rat Livers by Laser Microdissection.

Small hepatocyte-like progenitor cells (SHPCs) are known as liver stem/progenitor cells (LSPCs). SHPCs transiently appear and form clusters in rat livers treated with retrorsine (Ret) and a 70% partial hepatectomy (PH). The Ret/PH model has been used widely to analyze the effectiveness of cell transplantation and the mechanisms of LSPC proliferation. Laser microdissection (LMD) is a powerful tool that can excise and collect specific areas of cells from a tissue slice with a laser under a microscope. These cells exhibiting morphological alterations different from the surrounding cells may be analyzed by gene expression profiling. Specific markers of SHPCs have not yet been identified, in part, because it is difficult to isolate SHPCs from the liver using fluorescence or magnetic-activated cell sorting. To examine the underlying mechanism for SHPC growth, we established comprehensive gene expression profiles for SHPCs captured from liver sections using LMD. In this chapter, we introduce a method to isolate SHPCs from liver tissue sections using LMD for gene expression analysis.

Generation of functional liver organoids on combining hepatocytes and cholangiocytes with hepatobiliary connections ex vivo.

In the liver, the bile canaliculi of hepatocytes are connected to intrahepatic bile ducts lined with cholangiocytes, which remove cytotoxic bile from the liver tissue. Although liver organoids have been reported, it is not clear whether the functional connection between hepatocytes and cholangiocytes is recapitulated in those organoids. Here, we report the generation of a hepatobiliary tubular organoid (HBTO) using mouse hepatocyte progenitors and cholangiocytes. Hepatocytes form the bile canalicular network and secrete metabolites into the canaliculi, which are then transported into the biliary tubular structure. Hepatocytes in HBTO acquire and maintain metabolic functions including albumin secretion and cytochrome P450 activities, over the long term. In this study, we establish functional liver tissue incorporating a bile drainage system ex vivo. HBTO enable us to reproduce the transport of hepatocyte metabolites in liver tissue, and to investigate the way in which the two types of epithelial cells establish functional connections.

Extracellular vesicles containing miR-146a-5p secreted by bone marrow mesenchymal cells activate hepatocytic progenitors in regenerating rat livers.

BACKGROUND: Small hepatocyte-like progenitor cells (SHPCs) appear to form transient clusters in rat livers treated with retrorsine (Ret) and 70% partial hepatectomy (PH). We previously reported that the expansion of SHPCs was amplified in Ret/PH-treated rat livers transplanted with Thy1+ cells derived from D-galactosamine-treated injured livers. Extracellular vesicles (EVs) produced by hepatic Thy1+ donor cells activated SHPCs via interleukin (IL)-17 receptor B signaling. As bone marrow-derived mesenchymal cells (BM-MCs) also express Thy1, we aimed to determine whether BM-MCs could also promote the growth of SHPCs. METHODS: BM-MCs were isolated from dipeptidyl-peptidase IV (DPPIV)-positive rats. BM-MCs or BM-MC-derived EVs were administered to DPPIV-negative Ret/PH rat livers, and the growth and the characteristics of SHPC clusters were evaluated 14 days post-treatment. miRNA microarrays and cytokine arrays examined soluble factors within EVs. Small hepatocytes (SHs) isolated from an adult rat liver were used to identify factors enhancing hepatocytic progenitor cells growth. RESULTS: The recipient's livers were enlarged at 2 weeks post-BM-MC transplantation. The number and the size of SHPCs increased remarkably in livers transplanted with BM-MCs. BM-MC-derived EVs also stimulated SHPC growth. Comprehensive analyses revealed that BM-MC-derived EVs contained miR-146a-5p, interleukin-6, and stem cell factor, which could enhance SHs' proliferation. Administration of EVs derived from the miR-146a-5p-transfected BM-MCs to Ret/PH rat livers remarkably enhanced the expansion of SHPCs. CONCLUSIONS: miR-146a-5p involved in EVs produced by BM-MCs may play a major role in accelerating liver regeneration by activating the intrinsic hepatocytic progenitor cells.

Prolonged oxidative stress and delayed tissue repair exacerbate acetaminophen-induced liver injury in aged mice.

The liver gradually loses its regenerative capabilities with aging. However, it remains unknown whether aging affects drug-induced liver injury. Here, we used acetaminophen induced acute liver injury model to compare tissue injury and regeneration of aged mice (>80 weeks old) with young ones (8-10 weeks old). The mortality of aged mice after acetaminophen injury was higher than that of young mice. Transient increase of serum GOT and decrease of reduced glutathione (GSH) were not returned to original levels in aged mice even at 48 hours. In addition, Foxm1b and its targets Ccnd1 and Cdk1 were upregulated in young but not in aged mice after 48 hours. Moreover, an apoptosis-related gene, Cidea, was upregulated specifically in aged livers, which was consistent with increased number of TUNEL+ hepatocytes. Unexpectedly, damaged hepatocytes were retained in aged liver tissue, which may be caused by impaired recruitment of macrophages to the damaged area, without increases in Ccl2 after acetaminophen injury. Collectively, prolonged oxidative stress due to delayed recovery of GSH and the retention of damaged hepatocytes may suppress tissue repair and hepatocyte proliferation, resulting in exacerbation of acetaminophen injury in aged mice. Thus, aging is a risk factor conferring susceptibility against drug-induced liver injury.

Self-Renewal Capability of Hepatocytic Parental Progenitor Cells Derived From Adult Rat Liver Is Maintained Long Term When Cultured on Laminin 111 in Serum-Free Medium.

In this study, we investigated how the ability of hepatocytic parental progenitor cells (HPPCs) to self-renew can be maintained and how laminin (LN) isoforms play an important role in their self-renewal and maturation. Hepatocytes isolated from adult rat livers were cultured on hyaluronic acid to form colonies consisting of CD44+ small hepatocytes, which could be passaged on dishes coated with Matrigel. When second-passage cells were plated on Matrigel, LN111, or LN511, HPPCs appeared on Matrigel and LN111 but not on LN511. We identified two types of cells among the second-passage cells: Small, round cells and large, flat ones were observed on Matrigel, whereas the former and latter ones were specifically attached on LN111 and LN511, respectively. We hypothesized that small and round cells are the origin of HPPC colonies, and the binding to LN111 could be key to maintaining their self-renewal capability. Among the integrins involved in LN binding, integrins α3 and ß1 were expressed in colonies on LN111 more than in those on LN511, whereas ß4 was more strongly expressed in colonies on LN511. Integrin α3highα6ß1high cells could form HPPC colonies on LN111 but not on LN511, whereas integrin α6ß1low cells could not on either LN111 or LN511. In addition, neutralizing anti-integrin ß1 and anti-LN111 antibodies inhibited the passaged cells' ability to attach and form colonies on LN111 by HPPCs. Matrigel overlay induced second-passage cells growing on LN111 to increase their expression of hepatic functional genes and to form 3-dimensional colonies with bile canalicular networks, whereas such a shift was poorly induced when they were grown onLN511. Conclusion: These results suggest that the self-renewal capability of HPPCs depends on LN111 through integrin ß1 signaling.

Isolation and Expansion of Rat Hepatocytic Progenitor Cells.

This protocol showed procedures to isolate and expand small hepatocytes (SHs), as hepatocytic progenitor cells, from a rat liver. SHs are identified as a subpopulation of mature hepatocytes in a healthy liver. SHs can proliferate to form colonies in serum-free medium on hyaluronic acid-coated dishes, of which cells show CD44 positivity (CD44+ SHs). CD44+ SHs can be separated and purified from colonies by using anti-CD44 antibodies after enzymatic dissociation. CD44+ SHs can proliferate to form colonies on Engelbreth-Holm-Swarm gel (EHS-gel)-coated dishes in the serum-free medium for a long period and subculture for several times. Even after the second passage, the cells possess characteristics of hepatocytes such as expression of albumin and HNF4α. In addition, when the cells are treated with EHS-gel, they can recover highly differentiated functions of hepatocytes such as glycogen production, CYP activity, and bile secretion.

Intrahepatic bile ducts guide establishment of the intrahepatic nerve network in developing and regenerating mouse liver.

Epithelial organs consist of multiple tissue structures, such as epithelial sheets, blood vessels and nerves, which are spatially organized to achieve optimal physiological functions. The hepatic nervous system has been implicated in physiological functions and regeneration of the liver. However, the processes of development and reconstruction of the intrahepatic nerve network and its underlying mechanisms remain unknown. Here, we demonstrate that neural class III ß-tubulin (TUBB3)+ nerve fibers are not distributed in intrahepatic tissue at embryonic day 17.5; instead, they gradually extend along the periportal tissue, including intrahepatic bile ducts (IHBDs), after birth. Nerve growth factor (Ngf) expression increased in biliary epithelial cells (BECs) and mesenchymal cells next to BECs before nerve fiber extension, and Ngf was upregulated by hairy enhancer of slit 1 (Hes family bHLH transcription factor 1; Hes1). Ectopic NGF expression in mature hepatocytes induced nerve fiber extension into the parenchymal region, from where these fibers are normally excluded. Furthermore, after BECs were damaged by the administration of 4,4-diaminodiphenylmethane, the nerve network appeared shrunken; however, it was reconstructed after IHBD regeneration, which depended on the NGF signal. These results suggest that IHBDs guide the extension of nerve fibers by secreting NGF during nerve fiber development and regeneration.

Hepatocytic parental progenitor cells of rat small hepatocytes maintain self-renewal capability after long-term culture.

The liver has a variety of functions for maintaining homeostasis, and hepatocytes play a major role. In contrast with the high regenerative capacity of mature hepatocytes (MHs) in vivo, they have not been successfully expanded ex vivo. Here we demonstrate that CD44-positive cells sorted from small hepatocyte (SH) colonies derived from a healthy adult rat liver can proliferate on a Matrigel-coated dish in serum-free chemically defined medium; in addition, a subpopulation of the cells can divide more than 50 times in a period of 17 weeks every 4-week-passage. The passage cells retained the capability to recover highly differentiated functions, such as glycogen storage, CYP activity and bile secretion. When Matrigel-treated cells from the third passage were transplanted into retrorsine/partial hepatectomy-treated rat livers, the cells engrafted to differentiate into MHs and cholangiocytes. These results suggest that long-term cultured CD44+ SHs retain hepatocytic characteristics in vitro and the capability to differentiate into hepatocytes and cholangiocytes in vivo. Thus, a newly identified subpopulation of MHs possessing the attributes of hepatocytic stem/progenitor cells can be passaged several times without losing hepatocytic characteristics.

Progressive induction of hepatocyte progenitor cells in chronically injured liver.

Differentiated epithelial cells show substantial lineage plasticity upon severe tissue injuries. In chronically injured mouse livers, part of hepatocytes become Sry-HMG box containing 9 (Sox9) (+) epithelial cell adhesion molecule (-) hepatocyte nuclear factor 4 α (+) biphenotypic hepatocytes. However, it is not clear whether all Sox9+ hepatocytes uniformly possess cellular properties as hepatocyte progenitors. Here, we examined the microarray data comparing Sox9+ hepatocytes with mature hepatocytes and identified CD24 as a novel marker for biphenotypic hepatocytes. Immunohistochemical analyses showed that part of Sox9+ hepatocytes near expanded ductular structures expressed CD24 in the liver injured by 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet and by bile duct ligation. Indeed, Sox9+ hepatocytes could be separated into CD24- and CD24+ cells by fluorescence activated cell sorting. The ratio of CD24+ cells against CD24- ones in Sox9+ hepatocytes gradually increased while DDC-injury progressed and colony-forming capability mostly attributed to CD24+ cells. Although hepatocyte markers were remarkably downregulated in of Sox9+ CD24+ hepatocytes, they re-differentiated into mature hepatocytes in vitro and in vivo. Our current results demonstrate that the emergence of biphenotypic hepatocytes is a sequential event including the transition from CD24- and CD24+ status, which may be a crucial step for hepatocytes to acquire progenitor properties.

Transplantation of Thy1+ Cells Accelerates Liver Regeneration by Enhancing the Growth of Small Hepatocyte-Like Progenitor Cells via IL17RB Signaling.

Small hepatocyte-like progenitor cells (SHPCs) transiently form clusters in rat livers treated with retrorsine (Ret)/70% partial hepatectomy (PH). When Thy1+ cells isolated from d-galactosamine-treated rat livers were transplanted into the livers of Ret/PH-treated rats, the mass of the recipient liver transiently increased during the first 30 days after transplantation, suggesting that liver regeneration was enhanced. Here we addressed how Thy1+ cell transplantation stimulates liver regeneration. We found that the number and size of SHPC clusters increased in the liver at 14 days after transplantation. GeneChip analysis revealed that interleukin 17 receptor b (IL17rb) expression significantly increased in SHPCs from livers transplanted with Thy1+ cells. We subsequently searched for ligand-expressing cells and found that sinusoidal endothelial cells (SECs) and Kupffer cells expressed Il17b and Il25, respectively. Moreover, extracellular vesicles (EVs) separated from the conditioned medium of Thy1+ cell culture induced IL17b and IL25 expression in SECs and Kupffer cells, respectively. Furthermore, EVs enhanced IL17rb expression in small hepatocytes (SHs), which are hepatocytic progenitor cells; in culture, IL17B stimulated the growth of SHs. These results suggest that Thy1-EVs coordinate IL17RB signaling to enhance liver regeneration by targeting SECs, Kupffer cells, and SHPCs. Indeed, the administration of Thy1-EVs increased the number and size of SHPC clusters in Ret/PH-treated rat livers. Sixty days post-transplantation, most expanded SHPCs entered cellular senescence, and the enlarged liver returned to its normal size. In conclusion, Thy1+ cell transplantation enhanced liver regeneration by promoting the proliferation of intrinsic hepatic progenitor cells via IL17RB signaling. Stem Cells 2017;35:920-931.

Liver Progenitors Isolated from Adult Healthy Mouse Liver Efficiently Differentiate to Functional Hepatocytes In Vitro and Repopulate Liver Tissue.

It has been proposed that tissue stem cells supply multiple epithelial cells in mature tissues and organs. However, it is unclear whether tissue stem cells generally contribute to cellular turnover in normal healthy organs. Here, we show that liver progenitors distinct from bipotent liver stem/progenitor cells (LPCs) persistently exist in mouse livers and potentially contribute to tissue maintenance. We found that, in addition to LPCs isolated as EpCAM+ cells, liver progenitors were enriched in CD45- TER119- CD31- EpCAM- ICAM-1+ fraction isolated from late-fetal and postnatal livers. ICAM-1+ liver progenitors were abundant by 4 weeks (4W) after birth. Although their number decreased with age, ICAM-1+ liver progenitors existed in livers beyond that stage. We established liver progenitor clones derived from ICAM-1+ cells between 1 and 20W and found that those clones efficiently differentiated into mature hepatocytes (MHs), which secreted albumin, eliminated ammonium ion, stored glycogen, and showed cytochrome P450 activity. Even after long-term culture, those clones kept potential to differentiate to MHs. When ICAM-1+ clones were transplanted into nude mice after retrorsine treatment and 70% partial hepatectomy, donor cells were incorporated into liver plates and expressed hepatocyte nuclear factor 4α, CCAAT/enhancer binding protein α, and carbamoylphosphate synthetase I. Moreover, after short-term treatment with oncostatin M, ICAM-1+ clones could efficiently repopulate the recipient liver tissues. Our results indicate that liver progenitors that can efficiently differentiate to MHs exist in normal adult livers. Those liver progenitors could be an important source of new MHs for tissue maintenance and repair in vivo, and for regenerative medicine ex vivo. Stem Cells 2016;34:2889-2901.

Intrahepatic bile ducts are developed through formation of homogeneous continuous luminal network and its dynamic rearrangement in mice.

UNLABELLED: The intrahepatic bile duct (IHBD) is a highly organized tubular structure consisting of cholangiocytes, biliary epithelial cells, which drains bile produced by hepatocytes into the duodenum. Although several models have been proposed, it remains unclear how the three-dimensional (3D) IHBD network develops during liver organogenesis. Using 3D imaging techniques, we demonstrate that the continuous luminal network of IHBDs is established by 1 week after birth. Beyond this stage, the IHBD network consists of large ducts running along portal veins (PVs) and small ductules forming a mesh-like network around PVs. By analyzing embryonic and neonatal livers, we found that newly differentiated cholangiocytes progressively form a continuous and homogeneous luminal network. Elongation of this continuous network toward the liver periphery was attenuated by a potent Notch-signaling inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. Subsequent to this first step, the fine homogenous network is reorganized into the mature hierarchical network consisting of large ducts and small ductules. Between E17 and E18, when the homogenous network is radically reorganized into the mature hierarchical network, bile canaliculi rapidly extend and bile flow into IHBDs may increase. When formation of bile canaliculi was blocked between E16 and E18 by a multidrug resistance protein 2 inhibitor (benzbromarone), the structural rearrangement of IHBDs was significantly suppressed. CONCLUSION: Establishment of the mature IHBD network consists of two sequential events: (1) formation of the continuous luminal network regulated by the Notch-signaling pathway and (2) dynamic rearrangement of the homogeneous network into the hierarchical network induced by increased bile flow resulting from the establishment of hepatobiliary connections. (Hepatology 2016;64:175-188).

Pancreatic regeneration: basic research and gene regulation.

Pancreatic regeneration (PR) is an interesting phenomenon that could provide clues as to how the control of diabetes mellitus might be achieved. Due to the different regenerative abilities of the pancreas and liver, the molecular mechanism responsible for PR is largely unknown. In this review, we describe five representative murine models of PR and thirteen humoral mitogens that stimulate ß-cell proliferation. We also describe pancreatic ontogenesis, including the molecular transcriptional differences between α-cells and ß-cells. Furthermore, we review 14 murine models which carry defects in genes related to key transcription factors for pancreatic ontogenesis to gain further insight into pancreatic development.

Downregulation of miR122 by grainyhead-like 2 restricts the hepatocytic differentiation potential of adult liver progenitor cells.

Late fetal and adult livers are reported to contain bipotential liver stem/progenitor cells (LPCs), which share surface markers, including epithelial cell adhesion molecule (EpCAM), with cholangiocytes and differentiate into both hepatocytes and cholangiocytes. However, recent results do not necessarily support the idea that LPCs contribute significantly to cellular turnover and regeneration by supplying new hepatocytes. Here, we examined the colony-forming capability of EpCAM(+) cells isolated from mouse livers between E17 and 11 weeks of age. We found that the number of bipotential colonies was greatly reduced between 1 and 6 weeks, indicating that the number of LPCs decreases during postnatal development. Moreover, bipotential colonies derived from adult LPCs contained substantially fewer albumin(+) cells than those from neonatal LPCs. We further examined the differentiation potential of neonatal and adult LPCs by transplantation and found that neonatal cells differentiated into mature hepatocytes in recipient livers more frequently than adult LPCs. Since we previously reported that the transcription factor grainyhead-like 2 (GRHL2) expressed in EpCAM(+) cells inhibits hepatocytic differentiation, we examined whether targets of GRHL2 might block hepatocytic differentiation. DNA and microRNA microarrays revealed that miR122, the expression of which correlates with hepatocytic differentiation, was greatly reduced in adult as compared with neonatal EpCAM(+) cells. Indeed, GRHL2 negatively regulates the promoter/enhancer activity of the Mir122 gene. Our results indicate that neonatal but not adult EpCAM(+) LPCs have great potential to produce albumin(+) hepatocytes. GRHL2 suppresses transcription of miR122 and thereby restricts the differentiation potential of adult LPCs.

Preoperative hepatocyte transplantation improves the survival of rats with nonalcoholic steatohepatitis-related cirrhosis after partial hepatectomy.

Liver failure after liver resection for cirrhosis is a critical problem, and no effective therapy except liver transplantation is currently available. The objective of this study was to examine whether hepatocyte transplantation (HT) reduces the poststandard liver resection mortality rate of rats with nonalcoholic steatohepatitis (NASH)-related cirrhosis. Liver resection for hepatocellular carcinoma (HCC) combined with NASH-related cirrhosis has become increasingly common. We developed a rat model of acute liver failure after two-thirds partial hepatectomy (PH) for NASH-related cirrhosis. The mechanism by which HT improved the survival of the model rats was examined in short- and long-term investigations. Female DPPIV(-) recipient F344 rats were fed the choline-deficient l-amino acid (CDAA)-defined diet for 12 weeks. Some of the rats were transplanted with male F344 DPPIV(+) rat hepatocytes 24 h before undergoing PH. The overall post-PH survival of each group was evaluated, and short- and long-term pathological and molecular biological evaluations were also performed. Overall survival was significantly longer in the HT group than the non-HT group (7-day survival rates: 46.7% and 7.7%, respectively). Compared with the recipient livers of the non-HT group, numerous Ki-67(+) hepatocytes and few TUNEL(+) hepatocytes were observed in the livers of the HT group. At 6 months after the HT, the DPPIV(+) hepatocytes had partially replaced the recipient liver and formed hepatocyte clusters in the spleen. Preoperative HT might improve the survival of rats with NASH-related cirrhosis after PH by preventing the host hepatocytes from accelerating their growth and falling into apoptosis.

Sry HMG box protein 9-positive (Sox9+) epithelial cell adhesion molecule-negative (EpCAM-) biphenotypic cells derived from hepatocytes are involved in mouse liver regeneration.

It has been shown that mature hepatocytes compensate tissue damages not only by proliferation and/or hypertrophy but also by conversion into cholangiocyte-like cells. We found that Sry HMG box protein 9-positive (Sox9(+)) epithelial cell adhesion molecule-negative (EpCAM(-)) hepatocyte nuclear factor 4α-positive (HNF4α(+)) biphenotypic cells showing hepatocytic morphology appeared near EpCAM(+) ductular structures in the livers of mice fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. When Mx1-Cre:ROSA mice, which were injected with poly(I:C) to label mature hepatocytes, were fed with the DDC diet, we found LacZ(+)Sox9(+) cells near ductular structures. Although Sox9(+)EpCAM(-) cells adjacent to expanding ducts likely further converted into ductular cells, the incidence was rare. To know the cellular characteristics of Sox9(+)EpCAM(-) cells, we isolated them as GFP(+)EpCAM(-) cells from DDC-injured livers of Sox9-EGFP mice. Sox9(+)EpCAM(-) cells proliferated and could differentiate to functional hepatocytes in vitro. In addition, Sox9(+)EpCAM(-) cells formed cysts with a small central lumen in collagen gels containing Matrigel® without expressing EpCAM. These results suggest that Sox9(+)EpCAM(-) cells maintaining biphenotypic status can establish cholangiocyte-type polarity. Interestingly, we found that some of the Sox9(+) cells surrounded luminal spaces in DDC-injured liver while they expressed HNF4α. Taken together, we consider that in addition to converting to cholangiocyte-like cells, Sox9(+)EpCAM(-) cells provide luminal space near expanded ductular structures to prevent deterioration of the injuries and potentially supply new hepatocytes to repair damaged tissues.

Hepatic biliary epithelial cells acquire epithelial integrity but lose plasticity to differentiate into hepatocytes in vitro during development.

In developing organs, epithelial tissue structures are mostly developed by the perinatal period. However, it is unknown whether epithelial cells are already functionally mature and whether they are fixed in their lineage. Here we show that epithelial cells alter their plasticity during postnatal development by examining the differentiation potential of epithelial cell adhesion molecule (EpCAM)(+) cholangiocytes (biliary epithelial cells) isolated from neonatal and adult mouse livers. We found that neonatal cholangiocytes isolated from 1-week-old liver converted into functional hepatocytes in the presence of oncostatin M and Matrigel®. In contrast, neither morphological changes nor expression of hepatocyte markers were induced in adult cholangiocytes. The transcription factors hepatocyte nuclear factor 4α and CCAAT/enhancer binding protein α (C/EBPα), which are necessary for hepatocytic differentiation, were induced in neonatal cholangiocytes but not in adult cells, whereas grainyhead-like 2 (Grhl2) and hairy-enhance of slit 1 (Hes1), which are implicated in cholangiocyte differentiation, were continuously expressed in adult cells. Overexpression of C/EBPα and Grhl2 promoted and inhibited hepatocytic differentiation, respectively. Furthermore, adult cholangiocytes formed a monolayer with higher barrier function than neonatal ones did, suggesting that cholangiocytes are still in the process of epithelial maturation even after forming tubular structures during the neonatal period. Taken together, these results suggest that cholangiocytes lose plasticity to convert into hepatocytes during epithelial maturation. They lose competency to upregulate hepatocytic transcription factors and downregulate cholagiocytic ones under conditions inducing hepatocytic differentiation. Our results suggest that a molecular machinery augmenting epithelial integrity limits lineage plasticity of epithelial cells.