Direct neuronal conversion of microglia/macrophages reinstates neurological function after stroke.

Although generating new neurons in the ischemic injured brain would be an ideal approach to replenish the lost neurons for repairing the damage, the adult mammalian brain retains only limited neurogenic capability. Here, we show that direct conversion of microglia/macrophages into neurons in the brain has great potential as a therapeutic strategy for ischemic brain injury. After transient middle cerebral artery occlusion in adult mice, microglia/macrophages converge at the lesion core of the striatum, where neuronal loss is prominent. Targeted expression of a neurogenic transcription factor, NeuroD1, in microglia/macrophages in the injured striatum enables their conversion into induced neuronal cells that functionally integrate into the existing neuronal circuits. Furthermore, NeuroD1-mediated induced neuronal cell generation significantly improves neurological function in the mouse stroke model, and ablation of these cells abolishes the gained functional recovery. Our findings thus demonstrate that neuronal conversion contributes directly to functional recovery after stroke.

A Deactivation Factor of Fibrogenic Hepatic Stellate Cells Induces Regression of Liver Fibrosis in Mice.

BACKGROUND AND AIMS: Hepatic stellate cells (HSCs), a key player in the progression of liver fibrosis, are activated by various inflammatory stimuli and converted to myofibroblast-like cells with excessive collagen production. Despite many attempts to suppress activation of HSCs or inhibit collagen production in activated HSCs, their clinical applications have not been established yet. Recently, the deactivation of HSCs has been reported as a mechanism underlying the reversibility of experimental liver fibrosis. In the present study, we sought for deactivation factors of HSCs that induce regression of established liver fibrosis. APPROACH AND RESULTS: We identified transcription factor 21 (Tcf21) as one of the transcription factors whose expression was up-regulated in parallel to the differentiation of fetal HSCs. Expression of Tcf21 in HSCs remarkably decreased during culture-induced activation in vitro and in murine and human fibrotic liver tissue in vivo. This reduced Tcf21 expression was recovered during the spontaneous regression of murine liver fibrosis. Tcf21 was also examined for its effects by adeno-associated virus serotype 6-mediated Tcf21 gene transfer into cultured activated HSCs and mice with carbon tetrachloride- or methionine-choline deficient diet-induced liver fibrosis. Overexpression of Tcf21 in activated HSCs not only suppressed fibrogenic gene expression but also restored cells, at least in part, to a quiescent phenotype both in vitro and in vivo. These phenotypic changes of HSCs were accompanied by the regression of steatohepatitis and fibrosis and improved hepatic architecture and function. CONCLUSIONS: Tcf21 has been identified as a deactivation factor of fibrogenic HSCs, providing insight into a treatment strategy for the otherwise intractable liver fibrosis.

Visualization and isolation of zone-specific murine hepatocytes that maintain distinct cytochrome P450 oxidase expression in primary culture.

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/ß-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

Identification of a Novel Bone Marrow Cell-Derived Accelerator of Fibrotic Liver Regeneration Through Mobilization of Hepatic Progenitor Cells in Mice.

Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.

Loss of fibrocystin promotes interleukin-8-dependent proliferation and CTGF production of biliary epithelium.

BACKGROUND & AIMS: Congenital hepatic fibrosis (CHF) is a genetic liver disease resulting in abnormal proliferation of cholangiocytes and progressive hepatic fibrosis. CHF is caused by mutations in the PKHD1 gene and the subsequent dysfunction of the protein it encodes, fibrocystin. However, the underlying molecular mechanism of CHF, which is quite different from liver cirrhosis, remains unclear. This study investigated the molecular mechanism of CHF pathophysiology using a genetically engineered human induced pluripotent stem (iPS) cell model to aid the discovery of novel therapeutic agents for CHF. METHODS: PKHD1-knockout (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using the CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts [CCs]) in a 3D-culture system. RESULTS: The CCs were composed of a monolayer of cholangiocyte-like cells. The proliferation of PKHD1-KO CCs was significantly increased by interleukin-8 (IL-8) secreted in an autocrine manner. IL-8 production was significantly elevated in PKHD1-KO CCs due to mitogen-activated protein kinase pathway activation caused by fibrocystin deficiency. The production of connective tissue growth factor (CTGF) was also increased in PKHD1-KO CCs in an IL-8-dependent manner. Furthermore, validation analysis demonstrated that both the serum IL-8 level and the expression of IL-8 and CTGF in the liver samples were significantly increased in patients with CHF, consistent with our in vitro human iPS-disease model of CHF. CONCLUSIONS: Loss of fibrocystin function promotes IL-8-dependent proliferation of, and CTGF production by, human cholangiocytes, suggesting that IL-8 and CTGF are essential for the pathogenesis of CHF. IL-8 and CTGF are candidate molecular targets for the treatment of CHF. LAY SUMMARY: Congenital hepatic fibrosis (CHF) is a genetic liver disease caused by mutations of the PKHD1 gene. Dysfunction of the protein it encodes, fibrocystin, is closely associated with CHF pathogenesis. Using an in vitro human induced pluripotent stem cell model and patient samples, we showed that the loss of fibrocystin function promotes proliferation of cholangiocytes and the production of connective tissue growth factor (CTGF) in an interleukin 8 (IL-8)-dependent manner. These results suggest that IL-8 and CTGF are essential for the pathogenesis of CHF.

Establishment and analysis of a mouse model that regulates sex-related differences in liver drug metabolism.

The adult liver performs many metabolic functions for maintaining homeostasis. There are several sex differences in liver function and disease pathogenesis. One important function of the liver is drug metabolism, where cytochrome p450s (CYPs) in hepatocytes are the main enzymes involved. The toxicity of various drugs and chemicals differs with sex due to differences in hepatocytic CYP expression. However, the molecular mechanism regulating sex-related differences in drug metabolism remains unknown. In this study, we identified transcriptional regulator B-cell lymphoma 6 (Bcl6) as an important factor in sex-biased differential CYP expression. Microarray analysis of livers derived from liver-specific Bcl6-knockout mice showed that Bcl6 is required for sex-biased CYP expression patterns in the liver. Additionally, quantitative PCR analysis revealed that hepatocytic expression of male-biased genes, such as Cyp2d9, Cyp2u1, Cyp4a12a/12b, and Cyp7b1, in liver-specific Bcl6-knockout male mice significantly decreased to levels similar to those observed in wild-type female mice. Conversely, hepatocytic expression of female-biased genes, such as Cyp2a4/2a5, Cyp2b9, Cyp3a41, and Cyp17a1, significantly increased in liver-specific Bcl6-knockout male mice. Deletion of Bcl6 caused female-like expression of CYPs in male livers. These results suggest that Bcl6 is a key regulator of sex-related differential regulation of drug metabolism. Moreover, serum sex hormone levels and fertility did not change in liver-specific, Bcl6-knockout mice. Hepatocytic Bcl6 regulates sex-related differential CYP expression in the liver without changing the sex of the whole body. Thus, this mouse model is useful for analyzing liver-specific sex-dependent regulation of drug metabolism and pathogenesis.

Liver maturation deficiency in p57(Kip2)-/- mice occurs in a hepatocytic p57(Kip2) expression-independent manner.

Fetal hepatic stem/progenitor cells, hepatoblasts, are highly proliferative cells and the source of both hepatocytes and cholangiocytes. In contrast, mature hepatocytes have a low proliferative potency and high metabolic functions. Cell proliferation is regulated by cell cycle-related molecules. However, the correlation between cell cycle regulation and hepatic maturation are still unknown. To address this issue, we revealed that the cell cycle inhibitor p57(Kip2) was expressed in the hepatoblasts and mesenchymal cells of fetal liver in a spatiotemporal manner. In addition, we found that hepatoblasts in p57(Kip2)-/- mice were highly proliferative and had deficient maturation compared with those in wild-type (WT) mice. However, there were no remarkable differences in the expression levels of cell cycle- and bipotency-related genes except for Ccnd2. Furthermore, p57(Kip2)-/- hepatoblasts could differentiate into mature hepatocytes in p57(Kip2)-/- and WT chimeric mice, suggesting that the intrinsic activity of p57(Kip2) does not simply regulate hepatoblast maturation.

Matrix metalloproteinase-14 mediates formation of bile ducts and hepatic maturation of fetal hepatic progenitor cells.

Fetal hepatic stem/progenitor cells, called hepatoblasts, play central roles in liver development; however, the molecular mechanisms regulating the phenotype of these cells have not been completely elucidated. Matrix metalloproteinase (MMP)-14 is a type I transmembrane proteinase regulating pericellular proteolysis of the extracellular matrix and is essential for the activation of several MMPs and cytokines. However, the physiological functions of MMP-14 in liver development are unknown. Here we describe a functional role for MMP-14 in hepatic and biliary differentiation of mouse hepatoblasts. MMP-14 was upregulated in cells around the portal vein in perinatal stage liver. Formation of bile duct-like structures in MMP-14-deficient livers was significantly delayed compared with wild-type livers in vivo. In vitro biliary differentiation assays showed that formation of cholangiocytic cysts derived from MMP-14-deficient hepatoblasts was completely impaired, and that overexpression of MMP-14 in hepatoblasts promoted the formation of bile duct-like cysts. In contrast, the expression of molecules associated with metabolic functions in hepatocytes, including hepatic nuclear factor 4α and tryptophan 2,3-dioxygenase, were significantly increased in MMP-14-deficient livers. Expression of the epidermal growth factor receptor and phosphorylation of mitogen-activated protein kinases were significantly upregulated in MMP-14-deficient livers. We demonstrate that MMP-14-mediated signaling in fetal hepatic progenitor cells promotes biliary luminal formation around the portal vein and negatively controls the maturation of hepatocytes.

Investigation of bipotent differentiation of hepatoblasts using inducible diphtheria toxin receptor-transgenic mice.

AIM: Hepatic progenitor cells, called hepatoblasts, are highly proliferative and exhibit bipotential differentiation into hepatocytes and cholangiocytes in the fetal liver. Thus, they are the ideal source for transplantation therapy. Although several studies have been performed in vitro, the molecular mechanisms regulating hepatoblast differentiation in vivo following transplantation remain poorly understood. The aim of this study was to investigate an in vivo model to analyze hepatoblast bipotency and proliferative ability. METHODS: Hepatic transplantation model using Cre-inducible diphtheria toxin receptor-transgenic mice (iDTR), and albafpCre mice expressing Cre under the control of albumin and α-fetoprotein (AFP) regulatory elements were established. Fresh hepatoblasts were transplanted into diphtheria toxin (DT)-injected iDTRalbafpCre mice and we analyzed their differentiation and proliferation abilities by immunostaining and gene expression profiles. RESULTS: Fresh hepatoblasts transplanted into DT-injected iDTRalbafpCre mice engrafted and differentiated into both hepatocytes and cholangiocytes. Additionally, the number of engrafted hepatoblast-derived hepatocytes increased following partial hepatectomy and serial DT injections. Expression levels of hepatic functional genes in transplanted hepatoblast-derived hepatocytes were similar to that of normal hepatocytes. CONCLUSION: In our iDTRalbafpCre transplantation model, fresh hepatoblasts could differentiate into hepatocytes and cholangiocytes. In addition, these donor cells were induced to proliferate by the following liver injury stimulation. This result suggests that this model is valuable for investigating hepatoblast differentiation pathways in vivo.

Gene targeting study reveals unexpected expression of brain-expressed X-linked 2 in endocrine and tissue stem/progenitor cells in mice.

Identification of genes specifically expressed in stem/progenitor cells is an important issue in developmental and stem cell biology. Genome-wide gene expression analyses in liver cells performed in this study have revealed a strong expression of X-linked genes that include members of the brain-expressed X-linked (Bex) gene family in stem/progenitor cells. Bex family genes are expressed abundantly in the neural cells and have been suggested to play important roles in the development of nervous tissues. However, the physiological role of its individual members and the precise expression pattern outside the nervous system remain largely unknown. Here, we focused on Bex2 and examined its role and expression pattern by generating knock-in mice; the enhanced green fluorescence protein (EGFP) was inserted into the Bex2 locus. Bex2-deficient mice were viable and fertile under laboratory growth conditions showing no obvious phenotypic abnormalities. Through an immunohistochemical analysis and flow cytometry-based approach, we observed unique EGFP reporter expression patterns in endocrine and stem/progenitor cells of the liver, pyloric stomach, and hematopoietic system. Although Bex2 seems to play redundant roles in vivo, these results suggest the significance and potential applications of Bex2 in studies of endocrine and stem/progenitor cells.

Human pluripotent stem cell-derived cholangiocytes: current status and future applications.

PURPOSE OF REVIEW: Pluripotent stem cells, such as embryonic stem cells and inducible pluripotent stem (iPS) cells, have high proliferative multipotency for differentiation into mature functional cells that are useful for treatment and basic research on several diseases. Cholangiocytes are differentiated from fetal hepatic progenitor cells (hepatoblasts) and are important for transport of bile acids that are synthesized by mature hepatocytes in the liver. However, the molecular mechanisms of development and function of human cholangiocytes remain unknown. This review mentions the potential of human cholangiocytic culture from pluripotent stem cells to contribute to the analyses of the human bile duct system and diseases. RECENT FINDINGS: Recent studies found that human hepatic cholangiocytic cells can be differentiated from human embryonic stem and iPS cells in a suitable culture condition. Cholangiocytic cysts have epithelial cell polarity formed in a three-dimensional cell culture system using extracellular matrices. SUMMARY: Disease pathogenesis was elucidated in vitro using differentiated cells from disease-related iPS cells. Using genome-editing enzymes, iPS cells with disease-specific gene mutations can be easily and rapidly established. These disease-related iPS cells and cholangiocytic culture system may be useful for analyses and drug screening of human bile duct diseases.

Stem and progenitor cell systems in liver development and regeneration.

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell-specific surface markers including delta-like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13(+) CD133(+) fraction of human iPS-derived cells contained numerous hepatic progenitor-like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.

Wnt5a signaling mediates biliary differentiation of fetal hepatic stem/progenitor cells in mice.

UNLABELLED: The molecular mechanisms regulating differentiation of fetal hepatic stem/progenitor cells, called hepatoblasts, which play pivotal roles in liver development, remain obscure. Wnt signaling pathways regulate the development and differentiation of stem cells in various organs. Although a ß-catenin-independent noncanonical Wnt pathway is essential for cell adhesion and polarity, the physiological functions of noncanonical Wnt pathways in liver development are unknown. Here we describe a functional role for Wnt5a, a noncanonical Wnt ligand, in the differentiation of mouse hepatoblasts. Wnt5a was expressed in mesenchymal cells and other cells of wild-type (WT) midgestational fetal liver. We analyzed fetal liver phenotypes in Wnt5a-deficient mice using a combination of histological and molecular techniques. Expression levels of Sox9 and the number of hepatocyte nuclear factor (HNF)1ß(+) HNF4α(-) biliary precursor cells were significantly higher in Wnt5a-deficient liver relative to WT liver. In Wnt5a-deficient fetal liver, in vivo formation of primitive bile ductal structures was significantly enhanced relative to WT littermates. We also investigated the function of Wnt5a protein and downstream signaling molecules using a three-dimensional culture system that included primary hepatoblasts or a hepatic progenitor cell line. In vitro differentiation assays showed that Wnt5a retarded the formation of bile duct-like structures in hepatoblasts, leading instead to hepatic maturation of such cells. Whereas Wnt5a signaling increased steady-state levels of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in fetal liver, inhibition of CaMKII activity resulted in the formation of significantly more and larger-sized bile duct-like structures in vitro compared with those in vehicle-supplemented controls. CONCLUSION: Wnt5a-mediated signaling in fetal hepatic stem/progenitor cells suppresses biliary differentiation. These findings also suggest that activation of CaMKII by Wnt5a signaling suppresses biliary differentiation. (HEPATOLOGY 2013;).

Sal-like protein 4 (SALL4), a stem cell biomarker in liver cancers.

UNLABELLED: Liver cancers, including hepatocellular carcinomas (HCCs), cholangiocarcinomas (CCs), and fibrolamellar HCCs (FL-HCCs) are among the most common cancers worldwide and are associated with a poor prognosis. Investigations of genes important in liver cancers have focused on Sal-like protein 4 (SALL4), a member of a family of zinc finger transcription factors. It is a regulator of embryogenesis, organogenesis, pluripotency, can elicit reprogramming of somatic cells, and is a marker of stem cells. We found it expressed in normal murine hepatoblasts, normal human hepatic stem cells, hepatoblasts and biliary tree stem cells, but not in mature parenchymal cells of liver or biliary tree. It was strongly expressed in surgical specimens of human HCCs, CCs, a combined hepatocellular and cholangiocarcinoma, a FL-HCC, and in derivative, transplantable tumor lines in immune-compromised hosts. Bioinformatics analyses indicated that elevated expression of SALL4 in tumors is associated with poor survival of HCC patients. Experimental manipulation of SALL4's expression results in changes in proliferation versus differentiation in human HCC cell lines in vitro and in vivo in immune-compromised hosts. Virus-mediated gene transfer of SALL4 was used for gain- and loss-of-function analyses in the cell lines. Significant growth inhibition in vitro and in vivo, accompanied by an increase in differentiation occurred with down-regulation of SALL4. Overexpression of SALL4 resulted in increased cell proliferation in vitro, correlating with an increase in expression of cytokeratin19 (CK19), epithelial cell adhesion molecules (EpCAM), and adenosine triphosphate (ATP)-binding cassette-G2 (ABCG2). CONCLUSION: SALL4's expression is an indicator of stem cells, a prognostic marker in liver cancers, correlates with cell and tumor growth, with resistance to 5-FU, and its suppression results in differentiation and slowed tumor growth. SALL4 is a novel therapeutic target for liver cancers.

In vivo imaging visualizes discoid platelet aggregations without endothelium disruption and implicates contribution of inflammatory cytokine and integrin signaling.

The mechanism by which thrombotic vessel occlusion occurs independently of plaque development or endothelial cell (EC) disruption remains unclear, largely because of an inability to visualize the formation of thrombus, especially at the single-platelet level in real time. Here we demonstrate that rapidly developing thrombi composed of discoid platelets can be induced in the mesenteric capillaries, arterioles, and large-sized arteries of living mice, enabling characterization of the kinetics of thrombosis initiation and the multicellular interrelationships during thrombus development. Platelet aggregation without EC disruption was triggered by reactive oxygen species (ROS) photochemically induced by moderate power laser irradiation. The inflammatory cytokines TNF-α and IL-1 could be key components of the EC response, acting through regulation of VWF mobilization to the cell surface. Thrombus formation was then initiated by the binding of platelet GPIbα to endothelial VWF in our model, and this effect was inhibited by the ROS scavenger N-acetylcysteine. Actin linker talin-dependent activation of alphaIIb-beta3 integrin or Rac1 in platelets was required for late-phase thrombus stability. Our novel imaging technology illustrates the molecular mechanism underlying inflammation-based thrombus formation by discoid platelets on undisrupted ECs and suggests control of ROS could be a useful therapeutic target for the prevention of thrombotic diseases.

Mesenchymal progenitor cells in mouse foetal liver regulate differentiation and proliferation of hepatoblasts.

BACKGROUND & AIMS: Hepatoblasts are somatic progenitor cells of the foetal liver that possess high proliferative capacity and bi-potency for differentiation into both hepatocytes and cholangiocytes. Although mesenchymal cells are known to be important for liver ontogeny, current understanding of their interaction with hepatoblasts remains obscure. Mesenchymal cell populations in the developing liver were purified and their potential to support proliferation and differentiation of hepatoblasts was examined. METHODS: Foetal liver cells were fractionated with a flow cytometer using antibodies against cell surface markers. Gene expression of mesenchymal-specific transcripts and morphological characteristics were analysed. The ability of the mesenchymal cells to support hepatoblast function was analysed using a transwell and direct coculture system. RESULTS: CD45(-) Ter119(-) CD71(-) Dlk1(mid) PDGFRα(+) cells from the mid-foetal stage liver expressed the mesenchymal cell-specific transcription factors H2.0-like homeobox 1 and LIM homeobox 2 at high levels. Foetal mesenchymal cells make contact with hepatoblasts in vivo and possess the potential to differentiate into chondrocytes, osteocytes and adipocytes under appropriate cell culture conditions, indicating that these cells are possible candidates for mesenchymal stem/progenitor cells. Foetal mesenchymal cells expressed pleiotrophin, hepatocyte growth factor and midkine 1, which are involved in the growth of hepatoblasts. Using the coculture system with hepatoblasts and foetal mesenchymal cells, these cells were shown to support proliferation and maturation of hepatoblasts through indirect and direct interactions respectively. CONCLUSIONS: Dlk1(mid) PDGFRα(+) cells in non-haematopoetic fraction derived from the foetal liver exhibit mesenchymal stem/progenitor cell characteristics and have abilities to support proliferation and differentiation of hepatoblasts.

Drug metabolic activity as a selection factor for pluripotent stem cell-derived hepatic progenitor cells.

As a metabolic organ, the liver plays a variety of roles, including detoxification. It has been difficult to obtain stable supplies of hepatocytes for transplantation and for accurate hepatotoxicity determination in drug discovery research. Human pluripotent stem cells, capable of unlimited self-renewal, may be a promising source of hepatocytes. In order to develop a stable supply of embryonic stem cell (ESC)-derived hepatocytes, we have purified human ESC-derived hepatic progenitor cells with exposure to cytocidal puromycin by using their ability to metabolize drugs. Hepatic progenitor cells stably proliferated at least 220-fold over 120 days, maintaining hepatic progenitor cell-like properties. High drug-metabolizing hepatic progenitor cells can be matured into liver cells by suppressing hepatic proliferative signals. The method we developed enables the isolation and proliferation of functional hepatic progenitors from human ESCs, thereby providing a stable supply of high-quality cell resources at high efficiency. Cells produced by this method may facilitate cell therapy for hepatic diseases and reliable drug discovery research.

In vitro expansion and functional recovery of mature hepatocytes from mouse adult liver.

BACKGROUND: Mature hepatocytes retain the ability to regenerate the liver lobule fully in vivo following injury. Several cytokines and soluble factors (hepatocyte growth factors, epidermal growth factors, insulin and nicotinamide) are known to be important for proliferation of mature hepatocytes in vitro. However, hepatocytes monolayer-cultured on extracellular matrices have gradually lost their specific functions, particularly those in drug metabolism. AIM: We have explored and established a new culture system for expansion of functional hepatocytes. METHODS: We evaluated two approaches for efficient expansion of mature hepatocytes: (i) Co-culture with mouse embryonic fibroblasts (MEF); (ii) Addition to culture of inhibitors of cell signals involved in liver regeneration. After expansion steps, 3-dimensional spheroid-forming culture was used to re-induce mature hepatocellular function. RESULTS: The addition of inhibitors for tumour growth factor (TGF) ß and glycogen synthase kinase (GSK) 3ß efficiently induced in vitro expansion of mature hepatocytes. Although expression of hepatocellular functional genes decreased after expansion in monolayer culture, their expression and the activity of cytochrome P450 enzymes significantly increased with spheroid formation. Furthermore, when hepatocytes were co-cultured with MEF, addition of a MAPK/ERK kinase (MEK) inhibitor at the spheroid formation step enhanced drug-metabolism-related gene expression. CONCLUSION: Combination of the MEF co-culture system with the addition of inhibitors of TGFß and GSK3ß induced in vitro expansion of hepatocytes. Moreover, expression of mature hepatocellular genes and the activity of drug-metabolism enzymes in expanded hepatocytes were re-induced after spheroid culture.

Liver Injury and Cell Survival in Non-Alcoholic Steatohepatitis Regulated by Sex-Based Difference through B Cell Lymphoma 6.

The liver is a crucial organ for maintaining homeostasis in living organisms and is the center of various metabolic functions. Therefore, abnormal metabolic activity, as in metabolic syndrome, leads to pathological conditions, such as abnormal accumulation of lipids in the liver. Inflammation and cell death are induced by several stresses in the fatty liver, namely steatohepatitis. In recent years, an increase in non-alcoholic steatohepatitis (NASH), which is not dependent on excessive alcohol intake, has become an issue as a major cause of liver cirrhosis and liver cancer. There are several recent findings on functional sex-based differences, NASH, and cell stress and death in the liver. In particular, NASH-induced liver injury and tumorigeneses were suppressed by B cell lymphoma 6, the transcriptional factor regulating sex-based liver functional gene expression. In this review, we discuss cell response to stress and lipotoxicity in NASH and its regulatory mechanisms.

Drug metabolic activity is a critical cell-intrinsic determinant for selection of hepatocytes during long-term culture.

BACKGROUND: The liver plays an important role in various metabolic processes, including protein synthesis, lipid and drug metabolisms and detoxifications. Primary culture of hepatocytes is used for the understanding of liver physiology as well as for the drug development. Hepatocytes are, however, hardly expandable in vitro making it difficult to secure large numbers of cells from one donor. Alternatively, systems using animal models and hepatocellular carcinoma cells have been established, but interspecies differences, variation between human cell sources and limited hepatic functions are among the challenges faced when using these models. Therefore, there is still a need for a highly stable method to purify human hepatocytes with functional sufficiency. In this study, we aimed to establish an in vitro long-term culture system that enables stable proliferation and maintenance of human hepatocytes to ensure a constant supply. METHODS: We first established a growth culture system for hepatocytes derived from patients with drug-induced liver injury using fetal mouse fibroblasts and EMUKK-05 medium. We then evaluated the morphology, proliferative capacity, chromosome stability, gene and protein expression profiles, and drug metabolic capacity of hepatocytes in early, middle and late passages with and without puromycin. In addition, hepatic maturation in 3D culture was evaluated from morphological and functional aspects. RESULTS: In our culture system, the stable proliferation of human hepatocytes was achieved by co-culturing with mouse fetal fibroblasts, resulting in dedifferentiation into hepatic progenitor-like cells. We purified human hepatocytes by selection with cytocidal puromycin and cultured them for more than 60 population doublings over a span of more than 350 days. Hepatocytes with high expression of cytochrome P450 genes survived after exposure to cytocidal antibiotics because of enhanced drug-metabolizing activity. CONCLUSIONS: These results show that this simple culture system with usage of the cytocidal antibiotics enables efficient hepatocyte proliferation and is an effective method for generating a stable supply of hepatocytes for drug discovery research at a significant cost reduction.