Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21(CIP1) and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of beta-catenin localization and HNF3beta expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. CONCLUSION: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors.     

Mature hepatocytes exhibit unexpected plasticity by direct dedifferentiation into liver progenitor cells in culture

Although there have been numerous reports describing the isolation of liver progenitor cells from the adult liver, their exact origin has not been clearly defined; and the role played by mature hepatocytes as direct contributors to the hepatic progenitor cell pool has remained largely unknown. Here, we report strong evidence that mature hepatocytes in culture have the capacity to dedifferentiate into a population of adult liver progenitors without genetic or epigenetic manipulations. By using highly purified mature hepatocytes, which were obtained from untreated, healthy rat liver and labeled with fluorescent dye PKH2, we found that hepatocytes in culture gave rise to a population of PKH2-positive liver progenitor cells. These cells, liver-derived progenitor cells, which share phenotypic similarities with oval cells, were previously reported to be capable of forming mature hepatocytes, both in culture and in animals. Studies done at various time points during the course of dedifferentiation cultures revealed that hepatocytes rapidly transformed into liver progenitors within 1 week through a transient oval cell-like stage. This finding was supported by lineage-tracing studies involving double-transgenic AlbuminCreXRosa26 mice expressing β-galactosidase exclusively in hepatocytes. Cultures set up with hepatocytes obtained from these mice resulted in the generation of β-galactosidase-positive liver progenitor cells, demonstrating that they were a direct dedifferentiation product of mature hepatocytes. Additionally, these progenitors differentiated into hepatocytes in vivo when transplanted into rats that had undergone retrorsine pretreatment and partial hepatectomy. CONCLUSION: Our studies provide strong evidence for the unexpected plasticity of mature hepatocytes to dedifferentiate into progenitor cells in culture, and this may potentially have a significant effect on the treatment of liver diseases requiring liver or hepatocyte transplantation.     

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl(4)-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.     

Progenitor cells in diseased human liver

Hepatic progenitor cells are immature epithelial cells that reside in the smallest ramifications of the biliary tree in human liver. These cells are capable of differentiating toward the biliary and the hepatocytic lineages and represent the human counterpart of the oval cells in murine liver. An increased number of progenitor cells (referred to as "activation") and differentiation of the same toward hepatocytes or bile duct epithelial cells, or both, is a component of virtually all human liver diseases. The extent of progenitor cell activation and the direction of differentiation are correlated with the severity of the disease and the type of mature epithelial cell (hepatocyte or bile duct epithelial cell), respectively, that is damaged. Analogous to findings in animal models of hepatocarcinogenesis, human hepatic progenitor cells most likely can give rise to hepatocellular carcinoma. The factors that govern human hepatic progenitor cell activation and differentiation are beginning to be identified.     

Hepatic stem cells and liver repopulation

Research on hepatic stem cells has entered a new era of controversy, excitement, and great expectations. Although adult liver stem cells have not yet been isolated, an enormous repopulating capacity of transplanted mature hepatocytes under conditions of continuous liver injury has been discovered. Stem/progenitor cells from fetal liver have been successfully isolated and transplanted, repopulating up to 10% of normal liver. However, progenitor cell lines from adult and embryonic liver have not shown significant repopulating activity. Intensive research on embryonic stem cells has revealed the first promising attempts to use these cells as a source of hepatic progenitors. Conditions for their differentiation in vitro, isolation of purified hepatic progenitor cells, and liver repopulation are currently being evaluated. Multilineage adult progenitor cells of mesenchymal origin from bone marrow, muscle, and brain may turn out to be the long-sought primitive potential stem cells remaining in adult tissues.     

Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors, useful for both basic research and clinical applications. Besides their characterization in colony assays, protocols exist for the cultivation of lymphoid, myeloid, and erythroid cells. With the possible exception of mast cells, however, long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here, we describe for the first time an efficient yet easy strategy to generate mass cultures of pure, immature erythroid progenitors from mouse ES cells (ES-EPs), using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived, adult, definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions, ES-EPs differentiated into mature, enucleated erythrocytes. Importantly, ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition, the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells.     

小鼠胚胎干细胞诱导为肝细胞的研究

胚胎干细胞(ES细胞)是从体外受精胚囊的内细胞团分离建立的、具有发育全能性的细胞系，在特定条件下可向多种细胞分化，是细胞移植治疗很有前途的细胞来源。目的：探明ES细胞是否能被诱导分化为肝细胞，并探索小鼠ES细胞诱导分化为肝细胞的分化条件。方法：在ES细胞培养液中分别加入肝细胞生长因子(HGF)、β-神经细胞生长因子(NGF)和维甲酸(BA)以及与小鼠胎肝细胞共培养，观察分化细胞的形态学变化，逆转录聚合酶链反应(RT—PCR)检测白蛋白和转甲状腺蛋白mRNA水平的表达，免疫组化法检测甲胎蛋白和α1—抗胰蛋白酶蛋白水平的表达。结果：ES细胞培养液中加入HGF和β-NGFl5天后，分化出较多的上皮样细胞，并检测出白蛋白、转甲状腺蛋白mRNA水平的表达和甲胎蛋白、αl-抗胰蛋白酶蛋白水平的表达，表明ES细胞已诱导分化为肝细胞。ES细胞与胎肝细胞共培养2天后，分化出单一形态的上皮细胞，同样有上述肝细胞标志物的阳性表达。RA诱导出的细胞除转甲状腺蛋白mRNA外，无其他肝细胞标志物的阳性表达。结论：在HGF和β—NGF的作用下，有部分小鼠ES细胞被诱导为肝细胞，RA则无此作用。共培养方法也能诱导出肝细胞标志物阳性的细胞，并且细胞形态较单一。小鼠ES细胞有向肝细胞分化的潜能。     

Liver regeneration in acute severe liver impairment: a clinicopathological correlation study.

BACKGROUND: Although normally quiescent, the adult mammalian liver possesses a great capacity to regenerate after different types of injury. Major players in the regeneration process are mature residual cells, including hepatocytes, cholangiocytes and stromal cells. However, if the regenerative capacity of mature cells is impaired, hepatic progenitor cells (HPCs) are activated and expand into the liver parenchyma. Upon transit amplification, the progenitor cells generate new hepatocytes and biliary cells to restore liver homeostasis. AIMS/METHODS: To study the relationship between different histopathological parameters as well as their correlations with clinical parameters and outcome, we examined liver specimens from 74 patients with acute or subacute severe liver impairment by immunohistochemistry for CK7/CK19 (evaluation of HPCs activation/differentiation), Mib1(Ki 67)/P21 (evaluation of proliferative activity/proliferation arrest of hepatocytes) and hematoxylin and eosin (evaluation of hepatocyte loss). RESULTS: Of the 74 patients, 32% survived without transplantation, 14% died without transplantation and 54% were transplanted. Our results show that a threshold of 50% loss of hepatocytes, associated with significant decrease in the proliferative activity of remaining mature hepatocytes, is needed for extensive hepatic progenitor cell activation. Such activation is a sign of disease severity and occurs early (within 1 week) in the disease course. However, development of intermediate hepatocytes, suggesting HPCs differentiation towards mature hepatocytes, takes at least 1 week's time. We found a positive correlation between histopathological parameters (percentage hepatocyte loss, number of proliferating hepatocytes and number of HPCs) and clinical parameters of liver impairment such as model for end stage liver diseases (MELD). Surviving patients compared with those who either died or were transplanted had significantly less hepatocyte loss, less HPCs activation and more mature hepatocyte proliferative activity. Hepatocyte proliferative activity and degree of hepatocyte loss were the most important independent histopathological parameters in predicting outcome. CONCLUSION: Liver biopsy can provide important additional information in a patient with severe acute liver impairment.     

Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells

BACKGROUND/AIMS: Hybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs). METHODS: Colonies of SHs were collected and replated on a collagen sponge. Hepatic functions were examined by ELISA, immunoblotting, and Northern blotting. Cells in the sponge were characterized by immunocytochemistry and transmission electron microscopy. Urea synthesis was measured and metabolization of fluorescein diacetate was examined. RESULTS: SHs could proliferate and expand to form a hepatic organoid in the sponge. Albumin secretion and other hepatic protein production of the cells in the sponge increased with time in culture and the amounts were much larger than for those obtained from cells grown on dishes. Morphologically and functionally differentiated hepatocytes were observed and some CK19-positive cells formed duct-like structures within the sponge. Excretion of fluorescein was observed in bile canaliculi. CONCLUSIONS: Hepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs.     

丁酸钠诱导体外培养的大鼠肝卵圆细胞分化为成熟肝细胞

目的探讨分化刺激剂丁酸钠对体外培养的大鼠肝卵圆细胞分化的影响。方法从喂养含0．1%乙硫氨酸的胆碱缺乏性饮食4～6周的人鼠肝脏中分离出盱卵圆细胞,用免疫细胞化学和逆转录聚合酶链反应(RT-PCR)等方法对其进行鉴定。用0．75mmol/L酸钠处理大鼠肝卵圆细咆后．姬姆萨染色观察细胞表型改变,western blot检测细胞白蛋白的表达水平。结果免疫细胞化学结果显示分离出的细胞既表达成熟肝细咆的标志物白蛋白,也表达胆管细胞的标志物细胞角蛋白19,RT-PCR结果显示这些细胞还表达干细胞的标志物c-kit,但不表达造血干细胞的标志物CD34,表明这些细胞是大鼠肝前体细胞——肝卵圆细胞。0．75mmol/L丁酸钠能诱导大鼠肝卵圆细胞出现明显的表型改变,细胞变大,变圆,核浆比减小,且双核细胞数增多,约占总细胞数的50%左右,同时western blot的结果显示0．75mmol/L丁酸钠能够提高大鼠肝卵圆细胞白蛋白的表达水平。。结论分化刺激剂丁酸钠能诱导体外培养的大鼠肝卵圆细胞向成熟肝细胞分化。     

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.     

Oval cells compensate for damage and replicative senescence of mature hepatocytes in mice with fatty liver disease

Hepatic steatosis may have a generally benign prognosis, either because most hepatocytes are not significantly injured or mechanisms to replace damaged hepatocytes are induced. To determine the relative importance of these mechanisms, we compared hepatocyte damage and replication in ethanol-fed and ob/ob mice with very indolent fatty liver disease to that of healthy control mice and PARP-1(-/-) mice with targeted disruption of the DNA repair enzyme, poly(ADP-ribose) polymerase. Compared to the healthy controls, both groups with fatty livers had significantly higher serum alanine aminotransferase values, hepatic mitochondrial H(2)O(2) production, and hepatocyte oxidative DNA damage. A significantly smaller proportion of the hepatocytes from fatty livers entered S phase when cultured with mitogens. Moreover, this replicative senescence was not reversed by treating cultured hepatocytes with agents (i.e., betaine or leptin) that improve liver disease in intact ethanol-fed or leptin-deficient mice. Hepatocytes from PARP1(-/-) mice also had more DNA damage and reduced DNA synthesis in response to mitogens. However, neither mice with fatty livers nor PARP-1-deficient mice had atrophic livers. All of the mice with senescent mature hepatocytes exhibited hepatic accumulation of liver progenitor (oval) cells and oval cell numbers increased with the demand for hepatocyte replacement. Therefore, although hepatic oxidant production and damage are generally increased in fatty livers, expansion of hepatic progenitor cell populations helps to compensate for the increased turnover of damaged mature hepatocytes. In conclusion, these results demonstrate that induction of mechanisms to replace damaged hepatocytes is important for limiting the progression of fatty liver disease.     

New insights into liver stem cells

Hepatic progenitor cells are bi-potential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic and the cholangiocytic lineages. In adult livers, hepatic progenitor cells are quiescent stem cells with a low proliferating rate, representing a reserve compartment that is activated only when the mature epithelial cells of the liver are continuously damaged or inhibited in their replication, or in cases of severe cell loss.Hepatic progenitor cell activation has been described in various acute and chronic liver diseases. Their niche is composed by numerous cells such as Hepatic Stellate Cells, endothelial cells, hepatocytes, cholangiocytes, Kupffer cells, pit cells and inflammatory cells. All these cells, numerous hormones and growth factors could interact and cross-talk with progenitor cells influencing their proliferative and differentiative processes. Hepatic progenitor cells and their niche could represent, in the near future, a target for therapeutic approaches to liver disease based on cell-specific drug delivery systems.Isolation and transplantation of hepatic progenitor cells could represent a new approach for therapy of end-stage chronic liver diseases, as they offer many advantages to transplantation of mature hepatocytes. The possibility of applying stem cell therapy to liver diseases will represent a major goal in this field.     

Enrichment of hepatic progenitor cells from adult mouse liver

Hepatic progenitor cells (HPCs) have been characterized in several drug-treated rodent models and in the fetal liver; however, their properties have not been fully clarified in the normal adult liver, presumably because of their relatively small population and the existence of mature hepatocytes. In an attempt to resolve this issue, we developed a new enrichment system for HPCs using their cell aggregate formation properties. Nonparenchymal cells (NPCs) derived from enzymatically digested liver cells in normal adult mouse liver were treated in a hypoxic 2-hour suspension culture under constant shaking. This procedure resulted in cell aggregate formation and almost complete elimination of mature hepatocytes. Cell aggregates were formed only in Ca(2+)-containing medium, suggesting cadherin-dependent cell-cell adhesion. In these cell aggregates, 95% consisted of vascular endothelial cells that expressed VE-cadherin. The remaining 5% consisted of rapidly proliferating, small epithelial cells that expressed alpha-fetoprotein (AFP), E-cadherin, and albumin but not cytokeratin 19 (CK19), alpha-smooth muscle actin, or VE-cadherin. These results are consistent with an immature hepatic cell phenotype. When these immature hepatic cells were cultured with 10(-7) mol/L dexamethasone and 1% dimethyl sulfoxide, the de novo expression of mature hepatocyte markers such as tryptophan-2,3-dioxygenase (TO) was induced concomitantly with the induction of morphologic characteristics such as mitochondria- and peroxisome-rich cytoplasm and bile canaliculi formation. In conclusion, our methodology allows the enrichment of immature hepatic cells from the normal adult mouse. These cells are capable of growth and maturation along the hepatocyte lineage, indicating that these cells are HPCs.     

Identification of expandable human hepatic progenitors which differentiate into mature hepatic cells in vivo

BACKGROUND: Liver diseases include a wide spectrum of both acute and chronic conditions which are associated with significant morbidity and mortality worldwide. Hepatocyte transplantation has therapeutic potential in the treatment of liver diseases, but its clinical use is hampered by the lack of donor tissue. Generation of hepatocytes in vitro from adult or fetal liver cell progenitors or, alternatively, identification of a progenitor population which in vivo can generate mature liver cells could solve this problem. METHODS: CD117+/CD34+/Lin- human fetal liver cells were isolated by magnetic cell sorting and expanded in culture. Both freshly isolated and in vitro expanded cells in various passages were studied for their ability to be functional in hepatic parenchyma following d-galactosamine (GalN) induced injury in nude C57 black mice. RESULTS: Freshly isolated and in vitro expanded CD117+/CD34+/Lin- cells, when transplanted intrasplenically into GalN treated mice, morphologically and functionally differentiated into hepatocytes and cholangiocytes. Human specific albumin, alpha fetoprotein, cytokeratin 19, and antitrypsin mRNA were expressed in mouse liver. In addition, the human progenitor cells expressed glucose-6-phosphatase, glycogen, albumin, gamma glutamyl transpeptidase, and dipeptidyl peptidase IV after transplantation. Expanded cells in various passages maintained their capacity to differentiate into functional liver cells. CONCLUSIONS: Fetal liver CD117+/CD34+/Lin- progenitors and their progeny proliferated in vitro and also functionally differentiated into mature hepatic cells in an acute liver injury model. Successful in vitro expansion of liver progenitor cells provides a basis for developing cell therapy strategies, metabolic and toxicity testing systems, and may serve as a vehicle for gene therapy.