Hepatocyte-like Phenotype Research Articles

Molecular mechanisms underlying human adipose tissue-derived stromal cells differentiation into a hepatocyte-like phenotype

Background Adipose tissue-derived stromal cells (ATSCs) hold great promises in regenerative medicine. In the last decade, several studies have reported the plasticity of ATSCs toward a hepatocyte-like phenotype. Nonetheless, the molecular mechanisms underlying the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. Aim In this study, we compared the full genome expression profiles of ATSCs cultured for 4 weeks under pro-hepatogenic conditions to undifferentiated ATSCs, in order to depict the molecular events involved in ATSC hepatic transdifferentiation. Methods Analysis was performed using the Affymetrix human focus arrays. Sets of differentially expressed genes were functionally categorized in order to understand which pathways drive the hepatic conversion and interesting targets were validated by Q-PCR. Results ATSC-derived hepatocyte-like cells activate several genes associated with specific liver functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted downregulation of transcripts associated with the mesenchymal lineage, while epithelial-related genes were overexpressed. Conclusion Our data suggest that the in vitro system used in this study drove ATSCs toward a hepatic conversion through a subtle regulation of molecular pathways controlling lineage commitment that promote mesenchymal-epithelial transition.

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte-like phenotypes. When transplanted intrasplentically into carbon tetrachloride-injured livers of SCID mice, EGFP-tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three-dimensional architecture, and differentiate into hepatocyte-like cells expressing human albumin and alpha-1-anti-trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury.

Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection

BackgroundIn order to elucidate how Hepatitis C Virus (HCV) interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D) rotating wall vessel (RWV) bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells.ResultsWhen cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D) monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4α, Albumin, TTR and α1AT), were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, β-Catenin and E-Cadherin) significantly increased and exhibiting apical, lateral and/or basolateral localization.ConclusionThese findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system.

Exocrine pancreas trans-differentiation to hepatocytes—A physiological response to elevated glucocorticoid in vivo

Damage or ectopic expression of some growth factors can lead to the appearance of hepatocyte-like cells within the pancreas. Since glucocorticoids promote liver hepatocyte phenotype in vitro, the effect of glucocorticoid on pancreatic differentiation in vivo was examined. Treatment of rats with glucocorticoid for 25 days at levels that significantly inhibited weight gain resulted in the appearance of acinar cells expressing cytokeratin 7 and hepatocyte markers glutamine synthetase, carbamoyl phosphate synthetase and cytochrome P450 2E (the nomenclature employed is that given at http://drnelson.utmem.edu/CytochromeP450.html). Using a plastic pancreatic acinar cell line, this response was shown to be associated with changes in the regulation of WNT signalling-related gene expression and a repression of WNT signalling activity. These data suggest that a pathological response of the pancreas in vivo to elevated glucocorticoid is a differentiation of exocrine pancreatic cells or pancreatic progenitor cells to an hepatocyte-like phenotype.

The use of stem cells in liver disease

Cell transplantation to restore liver function as an alternative to whole liver transplantation has thus far not been successful in humans. Adult mature hepatocytes and various populations of liver progenitors and stem cells are being studied for their regenerative capabilities. Hepatocyte transplantation to treat metabolic deficiencies has shown promising early improvement in liver function; however, long-term success has not been achieved. Liver progenitor cells can now be identified and were shown to be capable to differentiate into a hepatocyte-like phenotype. Despite evidence of mesenchymal stem cell fusion in animal models of liver regeneration, encouraging results were seen in a small group of patients receiving autologous transplantation of CD133 mesenchymal stem cells to repopulate the liver after extensive hepatectomy for liver masses. Ethical issues, availability, potential rejection and limited understanding of the totipotent capabilities of embryonic stem cells are the limitations that prevent their use for restoration of liver function. The effectiveness of embryonic stem cells to support liver function has been proven with their application in the bioartificial liver model in rodents. There is ongoing research to restore liver function in cell biology, animal models and clinical trials using mature hepatocytes, liver progenitor cells, mesenchymal stem cells and embryonic stem cells.

Passage of bone-marrow-derived liver stem cells in a proliferating culture system

To explore the feasibility of passage of bone-marrow-derived liver stem cells (BDLSCs) in culture systems that contain cholestatic serum. Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay. The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units (H-CFUs) were formed. H-CFUs expressed markers of embryonic hepatocytes (alpha-fetoprotein, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and cytochrome P450-2b1), and hepatocyte nuclear factors 1alpha and -3beta). They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype. In vitro, we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo, we investigated co-expression of hepatocyte (albumin, alpha-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and alpha-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro. Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype. Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.

Native Umbilical Cord Matrix Stem Cells Express Hepatic Markers and Differentiate Into Hepatocyte-like Cells

Umbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage. We isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins. In vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated. We conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies.

Human Cord Blood-Derived Endothelial Progenitor Cells Engraft Following In Utero Transplantation, Integrate into the Developing Cytoarchitecture and Contribute to Ongoing Vasculogenesis.

Endothelial progenitor cells (EPC), whether isolated from the bone marrow (BM), peripheral (PB), or cord blood (CB), represent a promising tool for the development of novel cell therapies. EPC have been shown to contribute to re-endothelialization and neovascularization of damaged tissue, and have been proposed to be some of the primary regulators of tissue regeneration in organs such as the liver. Many studies have looked at the role of EPC in vasculogenic processes, but very few, if any, have focused their efforts on determining the complete differentiative potential of EPC upon transplantation in an experimental model that permits the robust formation of donor-derived tissue-specific cells in the absence of selective pressure to drive differentiation towards a specific phenotype. To this end, CB-derived EPC were obtained as previously described (Ingram et al. Blood:104,2004), transduced with a retroviral vector expressing dsRed, and transplanted (Tx) into 55–60 days old fetal sheep recipients (n=8) at concentrations ranging from 0.5–1.5 × 106cells/fetus. Recipients were then evaluated at 85 days post-transplant for the presence of donor (human)-specific cell types using flow cytometry and confocal microscopy. Using these methods, we found that levels of EPC engraftment in liver, as detected by dsRed expression, correlated directly with the Tx cell dose. Furthermore co-localization of CD31 or vWF was found within the dsRed+ cells. In animals receiving lower cell doses, EPC engrafted throughout the liver at the overall level of 0.12±0.03%; this number doubled in animals that received 2.6 × 106cells. Importantly, there was a preferential distribution of EPC around the vessels, with the EPC comprising 10 to 25% of the cells located around the perivascular areas, with some contributing directly to the endothelial layer of these vessels. Furthermore, expression of Connexin-43 and 45 in engrafted EPC demonstrated that the EPC had not only engrafted, but had also functionally integrated into the developing blood vessels. In addition, co-expression of albumin and alpha-fetoprotein in some of the engrafted EPC suggests that some of these cells may also have contributed to cells with a hepatocyte-like phenotype. Flow cytometric analysis of BM and PB of the transplanted sheep demonstrated that EPC engrafted and proliferated in the BM, with cells expressing CD105 (6.2±2.2) and CD146 (0.6±0.1), and continued to circulate in the PB with cells positive for CD105 (1.4±0.4) and CD146 (0.9±0.2). Of interest is that a CD45 negative aminopeptidase N+ (APN/CD13) population was found in both BM (18±7) and PB (5.6±2). This is particularly interesting, since CD13/APN is a potent regulator of vascular endothelial morphogenesis during angiogenesis. In conclusion, CB derived EPC are able to engraft and proliferate in vivo, integrate into the developing cytoarchitecture, and establish a circulating EPC pool ensuring long-term contribution to ongoing vasculogenesis.

Human Mesenchymal Stem Cells Differentiate Promptly into Tissue-Specific Cell Types without Cell Fusion, Mitochondrial or Membrane Vesicular Transfer in Fetal Sheep.

Despite the exciting reports regarding the ability of human mesenchymal stem cells (MSC) to differentiate into different cells of different organs, the mechanism by which this process occurs remains controversial. Several possible explanations have been put forth as an alternative to the existence of a true differentiation mechanism. We previously showed that MSC, at a single cell level, are able to differentiate into cells of different germ cell layers. In the present study, we investigated whether transfer of mitochondria or membrane-derived vesicles between cells and/or cell fusion participate in the events that lead to the change of phenotype of MSC upon transplantation (Tx). To this end, 54 sheep fetuses (55–60 gestational days) were Tx intra-peritoneally with Stro-1+,CD45−, Gly-A- MSC labeled prior to Tx with either CFSE, that irreversibly couples to both intracellular and cell-surface proteins, or DiD that efficiently labels all cell membranes and intracellular organelles, such as mitochondria. Evaluation of the recipients' different organs started at 20h post-Tx and continued at 25,30,40,60 and 120h. MSC reached the liver at 25h post-Tx (0.033%±0.0) with maximal engraftment at 40h (0.13%±0.02). MSC were first detected in the lung (0.028%±0.0) and brain (0.034%±0.0) at 30h and 40h respectively. In the brain, engraftment peaked at 60 hours post-Tx (0.08%±0.0) and in the lung at 120h (0.09%±0.01). Normalization of the number of engrafted cells per tissue mass and number of Tx cells revealed that 26% of the Tx MSC reached the lung; 2% the liver; and 3% the brain. Since the decreasing number of CFSE+ and DiD+ cells detected after 120h could be due to cell division, Ki67 staining was performed and revealed that 85–95% of the engrafted cells proliferated upon lodging in the organs, and divided throughout the evaluation period. To determine MSC differentiative timeline, confocal microscopy was performed to assess whether CFSE+ or DiD+ cells expressed tissue-specific markers (MSC were negative for these markers prior to transplant) within the engrafted organs. In the liver at 25h post-Tx, all CFSE+ or DiD+ cells co-expressed alpha-fetoprotein, demonstrating the rapid switch from an MSC to a fetal hepatocyte-like phenotype. In the lung, co-localization of pro-surfactant protein and CFSE/DiD was first detected at 30h post-Tx, but cells remained negative for Caveolin1; a phenotype that is consistent with differentiation to a type II epithelial cell, but not to a more mature type I. In the brain, MSC expressed Tau promptly, but synaptophysin expression was not detected until 120h. In situ hybridization on serial sections using either a human- or sheep-specific probe, with simultaneous visualization of CFSE+ or DiD+ cells allowed us to show that no membrane or mitochondrial transfer had occurred, since none of the sheep cells contained CFSE or DiD, and all of the dye+ cells hybridized only to the human probe. Furthermore, this combined methodology enabled us to determine that differentiation to all of the different cell types had occurred in the absence of cell fusion. In conclusion, MSC engraft multiple tissues rapidly, undergo proliferation, and give rise to tissue-specific cell types in the absence of cellular fusion or the transfer of mitochondria or membrane vesicles.

Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation

The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte-like morphology and acquired liver-specific markers on protein and gene expression levels. Furthermore, these fibroblast-derived hepatocyte-like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal-epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte-like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha-fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver-specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal-derived hepatocyte-like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels.

Murine gallbladder epithelial cells can differentiate into hepatocyte-like cells in vitro

We determined whether extrahepatic biliary epithelial cells can differentiate into cells with phenotypic features of hepatocytes. Gallbladders were removed from transgenic mice expressing hepatocyte-specific beta-galactosidase (beta-Gal) and cultured under standard conditions and under experimental conditions designed to induce differentiation into a hepatocyte-like phenotype. Gallbladder epithelial cells (GBEC) cultured under standard conditions exhibited no beta-Gal activity. beta-Gal expression was prominent in 50% of cells cultured under experimental conditions. Similar morphological changes were observed in GBEC from green fluorescent protein transgenic mice cultured under experimental conditions. These cells showed higher levels of mRNA for genes expressed in hepatocytes, but not in GBEC, including aldolase B, albumin, hepatocyte nuclear factor-4alpha, aldehyde dehydrogenase 1, and glutamine synthetase, and they synthesized bile acids. Additional functional evidence of a hepatocyte-like phenotype included LDL uptake and enhanced benzodiazepine metabolism. Connexin-32 expression was evident in murine hepatocytes and in cells cultured under experimental conditions, but not in cells cultured under standard conditions. Notch 1, 2, and 3 and Notch ligand Jagged 1 mRNAs were downregulated in these cells compared with cells cultured under standard conditions. CD34, alpha-fetoprotein, and Sca-1 mRNA were not expressed in cells cultured under standard conditions, suggesting that the hepatocyte-like cells did not arise from hematopoietic stem cells or oval cells. These results point to future avenues for investigation into the potential use of GBEC in the treatment of liver disease.

Mesenchymal Stem Cells Over-expressing Hepatocyte Growth Factor Improve Small-for-size Liver Grafts Regeneration

Ischemia-reperfusion (I/R) associated with small-for-size liver transplantation (SFSLT) impairs liver graft regeneration. Mesenchymal stem cells (MSCs) have the capability, under specific conditions, of differentiating into hepatocytes. Hepatocyte growth factor (HGF) has potent anti-apoptotic and mitogenic effects on hepatocytes during liver injury, and has been utilized in many experimental and clinical applications. In this study, we implanted HGF-expressing MSCs into liver grafts via the portal vein, using a 30% small-for-size rat liver transplantation model. HGF, c-met expression, hepatic injury and liver regeneration were assessed after liver transplantation. Our study demonstrated that MSCs over-expressing HGF prevented liver failure and reduced mortality in rats after SFSLT. These animals also exhibited improved liver function and liver weight recovery during the early post-transplantation period. Using green fluorescent protein (GFP) gene as a marker, we demonstrated that the engrafted cells and their progeny incorporated into remnant livers and produced albumin. These findings suggest that MSCs genetically modified to over-express HGF and implanted in the liver graft, may offer a novel approach to promoting liver regeneration after small-for-size transplantations.

Characterization of WIF-B9/R cells as an in vitro model with hepatocyte-like polarity and enhanced expression of canalicular ABC transporters involved in phase III of hepatic detoxification

The rat hepatoma/human fibroblast hybrid cell line WIF-B9 was developed to be used in studies requiring maintained hepatocyte-like polarity. To enhance their usefulness in order to investigate hepatic phase III detoxification process, we have characterized a subline of WIF-B9 cells (WIF-B9/R) obtained by exposure to progressively increasing cisplatin concentrations (up to 10 μM) and double sub-clonal selection. As compared to WIF-B9 cells, the cytostatic effect of cisplatin and doxorubicin on WIF-B9/R cells was lower (>10-fold), whereas the ability to reduce cell loading of cisplatin, doxorubicin, rhodamine 123 and calcein was higher. As their parent cells, WIF-B9/R cells express hepatocyte-like polarity. However, they have enhanced stable expression of Mdr1, Mrp1, Mrp2, Mrp3 and BCRP, but not Bsep/BSEP, as determined by real-time quantitative RT-PCR and western blot. Differentiation to hepatocyte-like phenotype was characterized by the formation of canalicular-like structures, containing in their membranes immunocytochemically detectable Mdr1, Mrp2 and BCRP. Functionality of these ABC transporters was evaluated by using specific substrates and inhibitors. Thus, canalicular-like structures were able to concentrate calcein, rhodamine 123 and doxorubicin. Moreover, verapamil, probenecid and Hoechst-33342 inhibited doxorubicin efflux and enhanced its content in WIF-B9/R cells. Probenecid inhibited calcein efflux and increased calcein cell load, but had no effect on cell loading of rhodamine 123, which was increased by verapamil and Hoechst-33342. In conclusion, WIF-B9/R cells are a useful model of polarized cells to study, in the absence of Bsep/BSEP, hepatic phase III of the detoxification process of several compounds whose canalicular transport is mediated by ABC proteins.

Human Cord Blood Cells Transplanted Into Chronically Damaged Liver Exhibit Similar Characteristics to Functional Hepatocytes

Human umbilical cord blood (CB) cells have many advantages as a source for stem cell transplantation because of immaturity and availability. It has been reported that CB cells transplanted into an injured liver displayed hepatocyte-like phenotypes. However, there have been few studies to characterize CB-derived hepatocyte-like cells (HLCs). In this study, CB cells were transplanted into mice with 2 types of liver damage: transient and chronic damage. We analyzed the expression of hepatic differentiation markers in CB-derived HLCs. In the liver of NOD/SCID mice with transient damage, CB-derived HLCs were detected infrequently at 3 weeks after transplantation. In contrast, in the liver of SCID mice damaged chronically by a urokinase-type plasminogen activator transgene under the control of albumin promotor/enhancer (ALB-uPA/SCID mice), more human HLCs colonized the host liver compared with hosts with transiently damaged livers. The CB-derived HLCs in both the transiently and the chronically damaged liver expressed a few markers of human hepatocytes, whereas the transcripts related to mature hepatic functions, including cytochrome P450s, were detected only in the ALB-uPA/SCID mice. These data indicated that CB cells were able to display a similar phenotype to functional hepatocytes in the recipient liver with chronic damage. CB cells may represent a transplantable source for chronic decompensated liver disease.

Hepatic potential of bone marrow stromal cells: Development of in vitro co-culture and intra-portal transplantation models

Bone marrow comprises heterogeneous cell populations and is thought to contain certain progenitors with the ability to differentiate into multiple mesenchymal cell lineages. To identify any differentiation plasticity of adult bone marrow stromal cells (BMSCs) into hepatocyte-like phenotypes, we developed a co-culture model with damaged liver tissue and an animal model of engraftment in cirrhotic liver via intra-portal transplantation. After 10 days of co-culture with injured liver tissues, BMSC expressed specific markers for hepatocytes by RT-PCR and Western blot. The two animal models for liver injury employed used carbon tetrachloride (CCl 4) induction or bile duct ligation. For tracing BMSC resident in the liver after intra-portal transplantation, green fluorescent protein (GFP)-positive BMSCs or in situ hybdization of Y-chromosome Sry gene in male BMSC were used in a cross-sex transplantation model. Expression of hepatocyte-specific markers in recipients' liver tissues was determined by fluorescence immunohistochemistry. Our findings demonstrated that about 16% parenchyma cells were GFP-positive cells derived from infused BMSCs, and expression of albumin was detected in these cells in engrafted liver tissues. In conclusion, BMSCs exhibited hepatocyte-like phenotypes after co-cultivation with liver tissue and transplanted into the injured liver. The presented evidence indicated the trans differentiation potential of BMSC developing to the hepatocytes.

Expression of hepatocyte-like phenotypes in bone marrow stromal cells after HGF induction

Bone marrow comprises heterogeneous cell populations, of which certain progenitors have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. This study demonstrates the bone marrow stromal cells (BMSCs) with intrinsic plasticity to differentiate into hepatocyte-like phenotypes under in vitro induction of hepatocyte growth factor (HGF). BMSCs isolated from rat femurs and tibias were cultured and passaged 3–4 times in the presence of HGF. Cells were harvested on days 0, 10, and 20 and subjected to examination of any hepatocyte characteristics by flow cytometry, RT-PCR, Western blot, and immunocytochemistry. Expression of albumin and α-fetoprotein at both mRNA and protein levels was detectable on day 10. By contrast, c-Met mRNA was significantly decreased in BMSC in the course of HGF induction. Here BMSC was shown to differentiate into hepatocyte-like phenotypes given the HGF induction, as an alternative source for adult stem cell transplantation in liver repair.

Origin and characterization of a human bipotent liver progenitor cell line

Background & Aims: Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells. Methods: Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages. Results: HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules. Conclusions: HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.

Differentiative potential of human metanephric mesenchymal cells.

To evaluate the ability of mesenchymal cells derived from nonhematopoietic organs to form blood and other tissues in vitro and in vivo. Because of its mesodermic derivation, human fetal kidney was used as a source of mesenchymal cells. Two populations of kidney cells were studied at a nonclonal level: a crude preparation, and an adherent fraction that was derived from the first by propagation in vitro (MNMC). Both populations were transplanted into sheep fetuses and analyzed at intervals for the presence of human cells in different organs by flow cytometry, PCR, immunohistochemistry, and in situ hybridization. Secondary transplantation studies were performed using human hematopoietic cells obtained from the bone marrow (BM) of primary recipients. MNMC were Thy-1(+), CD51(+), CD44(+), CD45(-), and vimentin(+), a phenotype consistent with that of metanephric mesenchyme. The crude population displayed the same phenotype but was contaminated with 0.4% CD34(+)CD45(+) cells. Cells with hepatocyte-like morphology and phenotype were obtained from the MNMC after culture in specific inducing media. After transplantation, both populations of cells produced multilineage hematopoietic engraftment and gave rise to CD34(+) cells. Successful hematopoietic engraftment in secondary recipients demonstrated the generation of long-term engrafting hematopoietic stem cells from MNMC. PCR analysis confirmed human hematopoietic engraftment and revealed that human cells were also present within other organs. Liver sections of transplanted animals contained human albumin-producing hepatocyte-like cells. A human metanephric mesenchymal cell population simultaneously gave rise to human blood and liver-like cells, suggesting that mesenchymal cells may represent a broad population of putative stem cells in multiple adult organs.

Phenotypic characterization of rat hepatoma cell lines and lineage-specific regulation of gene expression by differentiation agents

Hepatoma cell lines can be characterized by their expression of hepatocyte- and biliary-specific genes and by their response to differentiating agents in a lineage-dependent manner. These characteristics can be used to map the maturational lineage position of the cell lines. Tissue-specific gene expression and regulation by heparin, dimethylsulfoxide (DMSO), and sodium butyrate (SB) were examined in three rat hepatoma cell lines and two rat liver epithelial cell lines. Based on antigenic profiles and gene expression in serum-supplemented medium, the hepatoma cell lines could be organized in distinct categories of hepatic differentiation. All three hepatomas expressed the following five genes: γ-glutamyl transpeptidase (GGT), glutathione-S-transferase pi (Yp), glutamine synthetase, and α5 and β1 integrin. Cell line H 4AzC 2 also expressed α-fetoprotein (AFP), albumin, IGF II receptor, and the biliary/oval cell antigens OC.2 and OC.3, a phenotype characteristic of fetal hepatocytes. FTO-2B cells lacked AFP, OC.2, and OC.3 but expressed albumin and IGF II receptor in addition to the five commonly expressed genes, consistent with a more hepatocyte-like phenotype. Cell line H5D.7 expressed neither albumin nor the IGF II receptor, but did express OC.2, OC.3, and α3 integrin in addition to the five commonly expressed genes, characteristic of biliary epithelial cells. Regulation of gene expression by heparin, DMSO, and SB was examined in cells cultured in hormonally defined medium. The patterns of regulation of AFP, albumin, GGT, and Yp were dependent upon the state of differentiation of the cell. FTO-2B cells regulated genes in a manner similar to that of E16 fetal hepatocytes, H 4AzC 2 regulated genes characteristic of both hepatocytic and biliary lineages, and H5D.7 regulated only biliary genes. Suppression of GGT by DMSO was uniformly observed. The three cell lines expressed equal amounts of HNF-4, but FTO-2B cells expressed more HNF-3β and less HNF-3α, while the reverse was true of H 4AzC 2 and H5D.7 cells.