Hepatocyte Differentiation Research Articles

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation

SummaryDuring mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4α is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4α′s Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4α during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation.

Modeling and identifying therapeutic targets in Hepatoblastoma.

e21518 Background: Hepatoblastoma, an aggressive pediatric liver tumor, most often affects young children under five years of age. Surgical resection and chemotherapy have saved many children’s lives; yet, the overall five-year survival rate is 70%, and worse for children who present with unresectable tumors. Addressing the clinical need for HB-targeted therapies requires a better understanding of how HB tumors maintain their tumorigenic potential. Recent studies suggest that YAP1, the transcriptional co-regulator, and the Wnt/β-catenin pathway cooperate for HB tumor initiation. Further YAP1 is hyperactivated in nearly 80% of children’s HB tumors; however, will YAP1 inhibition result in therapeutic benefit for pediatric patients? Methods: To assess the role of YAP1 signaling in HB tumor maintenance, we generated a conditional mouse model of HB driven by doxycycline-inducible YAP1S127A and constitutively active β-Catenin. Delivery of doxycycline induces YAP1S127A expression and mice develop HB tumors within six weeks. After tumor formation, doxycycline is withdrawn, turning off YAP1 expression, and tumor progression is monitored using in vivo bioluminescent imaging. Results: Here we show in our conditional mouse model of HB that YAP1 is essential for tumor maintenance. In vivo YAP1 withdrawal leads to > 90% tumor regression within 10 weeks. Transcriptional analyses reveal that YAP1 withdraw in regressing tumors potently induces hepatocyte differentiation and metabolism genes, including HNF4a and FoxA2 target genes. Conclusions: Establishment of a luminescent conditional model of HB provides a new platform to critically address and explore therapeutic vulnerabilities in HB. Specifically, our data suggest HB tumor maintenance requires YAP1. Finally, in vivo tumor regression following YAP1 withdraw serves as preclinical evidence for the therapeutic potential of YAP1 inhibitory therapies in children with HB.

Cytochrome P450 1A1/2, 2B6 and 3A4 HepaRG Cell-Based Biosensors to Monitor Hepatocyte Differentiation, Drug Metabolism and Toxicity.

Human hepatoma HepaRG cells express most drug metabolizing enzymes and constitute a pertinent in vitro alternative cell system to primary cultures of human hepatocytes in order to determine drug metabolism and evaluate the toxicity of xenobiotics. In this work, we established novel transgenic HepaRG cells transduced with lentiviruses encoding the reporter green fluorescent protein (GFP) transcriptionally regulated by promoter sequences of cytochromes P450 (CYP) 1A1/2, 2B6 and 3A4 genes. Here, we demonstrated that GFP-biosensor transgenes shared similar expression patterns with the corresponding endogenous CYP genes during proliferation and differentiation in HepaRG cells. Interestingly, differentiated hepatocyte-like HepaRG cells expressed GFP at higher levels than cholangiocyte-like cells. Despite weaker inductions of GFP expression compared to the strong increases in mRNA levels of endogenous genes, we also demonstrated that the biosensor transgenes were induced by prototypical drug inducers benzo(a)pyrene and phenobarbital. In addition, we used the differentiated biosensor HepaRG cells to evidence that pesticide mancozeb triggered selective cytotoxicity of hepatocyte-like cells. Our data demonstrate that these new biosensor HepaRG cells have potential applications in the field of chemicals safety evaluation and the assessment of drug hepatotoxicity.

Dual Delivery of HNF4α and Cisplatin by Mesoporous Silica Nanoparticles Inhibits Cancer Pluripotency and Tumorigenicity in Hepatoma-Derived CD133-Expressing Stem Cells.

Hepatocellular carcinoma (HCC) is one of the most prevalent and deadly malignancies characterized by high rate of recurrence. Tumor recurrence is often attributed to the presence of a subpopulation of cells with stem cell properties, referred to as cancer stem cells (CSCs). Traditionally, cancer therapies target the entire bulk of tumor cells; however, they are poorly effective against CSCs, characterized by higher drug resistance. Therefore, approaches targeting CSCs may be required in addition to conventional chemotherapy to prevent tumor recurrence. In this study, we investigated an approach to target HCC by combining the conventional chemotherapeutic drug, cisplatin, to target the bulk of tumor cells, and differentiation therapy by delivering the gene encoding HNF4α, an important regulator of hepatocyte differentiation, to target CSCs. We used the Huh7 cell line as an in vitro model of HCC, which is characterized by a high proportion of CD133-expressing CSCs. By using flow cytometry, we separated CD133+ and CD133- Huh7 cell subpopulations and have shown that the former has highly pronounced in vivo tumorigenic capacity in contrast to the latter, which could not generate tumors in vivo. For the dual delivery of HNF4α-encoding plasmid and cisplatin, we used polyethyleneimine-modified mesoporous silica nanoparticles (PMSNs) as the nanocarriers. Here, we show that the treatment of CD133-expressing Huh7 cells with HNF4α-loaded PMSNs can suppress their proliferation rate, decrease the proportion of CSCs, downregulate stemness-associated genes, and increase the expression of mature hepatocyte-associated genes. At the same time, the treatment of Huh7 with PMSNs loaded with both HNF4α-encoding plasmid and cisplatin could block them in the S-phase of the cell cycle and cause apoptosis. In addition, dually loaded PMSNs were the most efficient formulation in suppressing tumor growth in vivo. To summarize, in this study, we tested the nanoparticle-based delivery system as both chemotherapy and gene-based therapy agents, which has great potential for development of effective treatment of HCC.

Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response

Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response. We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response. The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect. LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

Opposing Roles of the Forkhead Box Factors FoxM1 and FoxA2 in Liver Cancer

The forkhead box transcription factor FoxM1 is essential for hepatocellular carcinoma (HCC) development, and its overexpression coincides with poor prognosis. Here, we show that the mechanisms by which FoxM1 drives HCC progression involve overcoming the inhibitory effects of the liver differentiation gene FoxA2. First, the expression patterns of FoxM1 and FoxA2 in human HCC are opposite. We show that FoxM1 represses expression of FoxA2 in G1 phase. Repression of FoxA2 in G1 phase is important, as it is capable of inhibiting expression of the pluripotency genes that are expressed mainly in S-G2 phases. Using a transgenic mouse model for oncogenic Ras-driven HCC, we provide genetic evidence for a repression of FoxA2 by FoxM1. Conversely, FoxA2 inhibits expression of FoxM1 and inhibits FoxM1-induced tumorigenicity. Also, FoxA2 inhibits Ras-induced HCC progression that involves FoxM1. IMPLICATIONS: The observations provide strong genetic evidence for an opposing role of FoxM1 and FoxA2 in HCC progression. Moreover, FoxM1 drives high-grade HCC progression partly by inhibiting the hepatocyte differentiation gene FoxA2.

SUN-023 HNF4a Isoforms Regulate the Circadian Balance between Carbohydrate and Fatty Acid Metabolism in the Liver

Nuclear receptor genes often contain alternative promoters but the physiological relevance of the different promoters or the proteins they generate is largely unknown. Hepatocyte Nuclear Factor 4α (HNF4α), master regulator of hepatocyte differentiation and the most abundant nuclear receptor in the liver, is regulated by two promoters (P1 and P2). P1-HNF4α, a tumor suppressor, is the major isoform in the adult liver while P2-HNF4α is expressed in fetal liver and liver cancer: a role for P2-HNF4α in normal adult liver has not been identified. Here we show for the first time that P2-HNF4α is expressed at ZT9 and ZT21 in the normal adult liver and use P2-HNF4α-expressing exon swap mice and ‘omics approaches to demonstrate that P2-HNF4α orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions. Cytochrome P450 and sex-specific gene expression is impacted as well as carbohydrate and fatty acid metabolism. The exon swap mice exhibit subtle differences in circadian gene regulation and disruption of the clock increases expression of P2-HNF4α. They also exhibit notable differences in carbohydrate versus fatty acid metabolism with P1-HNF4α driving gluconeogenesis and P2-HNF4α driving ketogenesis. Our results also show that P2-HNF4α is required for the elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure in the Hnfa gene is designed, at least in part, to respond to the availability of food resources and maintain the balance between gluconeogenesis and ketogenesis in a circadian and sex-specific fashion. (Funding: NIH NIDDK, West Cost Metabolomics Center, NIEHS T32, USDA NIFA, CCFA, Superfund Research Program)

Modeling of Hepatic Drug Metabolism and Responses in CYP2C19 Poor Metabolizer Using Genetically Manipulated Human iPS cells.

Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human induced pluripotent stem cells (human iPS cells) and our highly efficient genome-editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between wild-type iPS cell-derived hepatocyte-like cells (WT HLCs) and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S-mephenytoin metabolites using ultra-performance liquid chromatography-tandem mass spectrometry. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogrel- and ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogrel- and DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome-editing technologies for pharmaceutical research. SIGNIFICANCE STATEMENT: Although unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 poor metabolizers, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 poor metabolizers because there is currently no model for this phenotype. Using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells as a novel CYP2C19 poor metabolizer model for drug development and research.

Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells

Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota’s Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs.

Dennd1a, a susceptibility gene for polycystic ovary syndrome, is essential for mouse embryogenesis.

The DENND1A has been identified as a guanine nucleotide exchange factor for small GTPase Rab35, which functions in endocytic trafficking to mediate the recycling of selective cargos. Genetic alterations within the DENND1A gene have been implicated in human disease such as polycystic ovary syndrome (PCOS). However, the role of DENND1A in developmental and reproductive processes is largely unknown. Using Dennd1a gene knockout mice, we uncovered that homogeneous Dennd1a-/- mutants died around embryonic day (E) 14.5. The brain of Dennd1a-/- embryos exhibited defects, partially attributed to the dysregulation of cell division and survival in the telencephalon. The transcription of Fgf8 mRNA was ectopically elevated in the dorsal midline of telencephalon, concomitant with a decrease of active β-catenin and Axin2 in the brain of Dennd1a-/- embryos. During liver morphogenesis, the ablation of Dennd1a impaired hepatic cell proliferation, the differentiation of hepatocyte, and hepatic hematopoiesis. In addition, loss of Dennd1a also affected the development of primordial germ cells. We demonstrate that Dennd1a, a susceptibility gene for PCOS, is essential for embryogenesis, probably through the mediation of endocytic recycling of selective cargos that are involved in cell signaling crucial for the development of multiple embryonic organ systems.

THU-375-Transcription factor TRIM33 controls liver progenitor cell towards hepatocyte differentiation through synergizing with SMAD2/3 following massive parenchymal loss

THU-375-Transcription factor TRIM33 controls liver progenitor cell towards hepatocyte differentiation through synergizing with SMAD2/3 following massive parenchymal loss

FGF signal is not required for hepatoblast differentiation of human iPS cells

Human induced pluripotent stem cell-derived hepatocyte-like cells are expected to be utilized in pharmaceutical research and regenerative medicine. In general, human induced pluripotent stem (iPS) cells are differentiated into hepatocyte-like cells through definitive endoderm cells and hepatoblast-like cells using various growth factors that are essential for liver development. Although recombinant bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are widely used in the hepatoblast differentiation, hepatoblast differentiation process has not been fully modified. In this study, we examined the roles of BMPs and FGFs in the hepatoblast differentiation from human iPS cells. Surprisingly, the gene expression levels of hepatoblast markers were upregulated by the removal of FGFs. In addition, the percentages of hepatoblast markers-positive cells were increased by the removal of FGFs. Furthermore, the hepatocyte differentiation potency was also significantly increased by the removal of FGFs. To examine whether FGF signals are completely unnecessary for the hepatoblast differentiation, the expression levels of endogenous FGF ligands and receptors were examined. The definitive endoderm cells highly expressed the FGF ligand, FGF2, and the FGF receptor, FGFR1. To examine the role of endogenous FGF signals, an FGFR inhibitor was treated during the hepatoblast differentiation. The hepatoblast differentiation was promoted by using FGFR inhibitor, suggesting that endogenous FGF signals are also unnecessary for the hepatoblast differentiation. In conclusion, we found that FGF signals are not essential for hepatoblast differentiation. We believe that our finding will be useful for generating functional hepatocyte-like cells for medical applications.

Energy metabolism couples hepatocyte integrin-linked kinase to liver glucoregulation and postabsorptive responses of mice in an age-dependent manner.

Integrin-linked kinase (ILK) is a critical intracellular signaling node for integrin receptors. Its role in liver development is complex, as ILK deletion at E10.5 (before hepatocyte differentiation) results in biochemical and morphological differences that resolve as mice age. Nevertheless, mice with ILK depleted specifically in hepatocytes are protected from the hepatic insulin resistance during obesity. Despite the potential importance of hepatocyte ILK to metabolic health, it is unknown how ILK controls hepatic metabolism or glucoregulation. The present study tested the role of ILK in hepatic metabolism and glucoregulation by deleting it specifically in hepatocytes, using a cre-lox system that begins expression at E15.5 (after initiation of hepatocyte differentiation). These mice develop the most severe morphological and glucoregulatory abnormalities at 6 wk, but these gradually resolve with age. After identifying when the deletion of ILK caused a severe metabolic phenotype, in depth studies were performed at this time point to define the metabolic programs that coordinate control of glucoregulation that are regulated by ILK. We show that 6-wk-old ILK-deficient mice have higher glucose tolerance and decreased net glycogen synthesis. Additionally, ILK was shown to be necessary for transcription of mitochondrial-related genes, oxidative metabolism, and maintenance of cellular energy status. Thus, ILK is required for maintaining hepatic transcriptional and metabolic programs that sustain oxidative metabolism, which are required for hepatic maintenance of glucose homeostasis.

Extensive reprogramming of the nascent transcriptome during iPSC to hepatocyte differentiation

Hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs) provide a renewable source of cells for drug discovery, disease modelling and cell-based therapies. Here, by using GRO-Seq we provide the first genome-wide analysis of the nascent RNAs in iPSCs, HLCs and primary hepatocytes to extend our understanding of the transcriptional changes occurring during hepatic differentiation process. We demonstrate that a large fraction of hepatocyte-specific genes are regulated at transcriptional level and identify hundreds of differentially expressed non-coding RNAs (ncRNAs), including primary miRNAs (pri-miRNAs) and long non-coding RNAs (lncRNAs). Differentiation induced alternative transcription start site (TSS) usage between the cell types as evidenced for miR-221/222 and miR-3613/15a/16-1 clusters. We demonstrate that lncRNAs and coding genes are tightly co-expressed and could thus be co-regulated. Finally, we identified sets of transcriptional regulators that might drive transcriptional changes during hepatocyte differentiation. These included RARG, E2F1, SP1 and FOXH1, which were associated with the down-regulated transcripts, and hepatocyte-specific TFs such as FOXA1, FOXA2, HNF1B, HNF4A and CEBPA, as well as RXR, PPAR, AP-1, JUNB, JUND and BATF, which were associated with up-regulated transcripts. In summary, this study clarifies the role of regulatory ncRNAs and TFs in differentiation of HLCs from iPSCs.

MiR-15/16 mediate crosstalk between the MAPK and Wnt/β-catenin pathways during hepatocyte differentiation from amniotic epithelial cells

MiR-15/16 play an important role in liver development and hepatocyte differentiation, but the mechanisms by which these miRNAs regulate their targets and downstream genes to influence cell fate are poorly understood. In this study, we showed up-regulation of miR-15/16 during HGF- and FGF4-induced hepatocyte differentiation from amniotic epithelial cells (AECs). To elucidate the role of miR-15/16 and their targets in hepatocyte differentiation, we investigated the roles of miR-15/16 in both the MAPK and Wnt/β-catenin pathways, which were predicted to be involved in miR-15/16 signaling. Our results demonstrated that the transcription of miR-15/16 was enhanced by c-Fos, c-Jun, and CREB, important elements of the MAPK pathway, and miR-15/16 in turn directly targeted adenomatous polyposis coli (APC) protein, a major member of the β-catenin degradation complex. MiR-15/16 destroyed these degradation complexes to activate β-catenin, and the activated β-catenin combined with LEF/TCF7L1 to form a transcriptional complex that enhanced transcription of hepatocyte nuclear factor 4 alpha (HNF4α). HNF4α also bound the promoter region of miR-15/16 and promoted its transcription, thereby forming a regulatory circuit to promote the differentiation of AECs into hepatocytes. Endogenous miRNAs are, therefore, involved in hepatocyte differentiation from AECs and should be considered during the development of an effective hepatocyte transplant therapy for liver damage.

Intrasplenic Transplantation of Cytotoxic T-Lymphocyte Associated Protein 4-Fas Ligand--Modified Hepatic Oval Cells for Acute Liver Injury in Rats

BackgroundIntrasplenic transplantation of xenogeneic hepatic oval cells (HOCs) may provide metabolic support for acute liver injury. However, xenoreactive lymphocyte-mediated immune response hinders HOCs' survival in the xeno-spleen parenchyma. Cytotoxic T-lymphocyte associated protein 4-Fas ligand (CTLA4.FasL), a fusion product integrating 2 inhibitory elements against lymphocytes into 1 molecule, effectively inhibited the proliferation of allogeneic and autoimmune lymphocytes. The purpose of this study was to explore the effect of CTLA4.FasL on the proliferation of xenoreactive lymphocytes and evaluate the therapeutic efficacy of CTLA4.FasL-modified HOC transplantation on acute liver injury in rats. MethodsThe effect of CTLA4.FasL-modified mouse liver epithelial progenitor cells (CTLA4.FasL-LEPCs) on the proliferation of rat lymphocytes in xeno-mixed lymphocyte reaction was investigated. Furthermore, CTLA4.FasL-LEPCs were intrasplenically transplanted in carbon tetrachloride- and partial hepatectomy-treated rats, and the therapeutic effect was evaluated using hematoxylin and eosin staining and alanine aminotransferase and aspartate aminotransferase assays. The hepatocytic differentiation of CTLA4.FasL-LEPCs in xenogeneic spleen was monitored by immunohistochemical staining for albumin. ResultsIn xeno-mixed lymphocyte reaction, CTLA4.FasL-LEPCs substantially inhibited the rat lymphocytes proliferation. CTLA4.FasL-LEPC transplantation significantly ameliorated liver injury compared with mCherry-modified LEPC and LEPC transplantation, as assessed by hematoxylin and eosin staining, alanine aminotransferase, and aspartate aminotransferase assays. Albumin positive cells appeared only in CTLA4.FasL-LEPCs group, but not in the mCherry-modified LEPCs group and LEPCs group. ConclusionsOur results indicate CTLA4.FasL-LEPCs substantially improved liver function and structure in carbon tetrachloride- and partial hepatectomy-induced acute liver injury rats through long-term hepatocytic differentiation.

Fine Tuning of Hepatocyte Differentiation from Human Embryonic Stem Cells: Growth Factor vs. Small Molecule-Based Approaches.

Human embryonic stem cells (hESCs) are being utilized in diverse areas of studies such as development and disease modeling, cell replacement therapy, or drug toxicity testing because of their potential to be differentiated into any cell type in the body. The directed differentiation of hESCs into hepatocytes could provide an invaluable source of liver cells for various liver-based applications. Therefore, several protocols have been established in the past for hESC-hepatocyte differentiation based on the knowledge of signaling pathways and growth factors involved in different stages of embryonic hepatogenesis. Although successful derivation of hepatocytes has been achieved through these protocols, the efficiency is not always ideal. Herein, we have tested several combinations of published protocols, for example, growth factor vs. small molecule and different time durations of treatment for definitive endoderm (DE) induction and further hepatocyte differentiation to develop an efficient DE induction and hepatocyte differentiation in a highly reproducible manner based on the stage-specific marker expression and functional analysis.

Collagen vitrigel promotes hepatocytic differentiation of induced pluripotent stem cells into functional hepatocyte-like cells.

ABSTRACTDifferentiation of stem cells to hepatocytes provides an unlimited supply of human hepatocytes and therefore has been vigorously studied. However, to date, the stem cell-derived hepatocytes were suggested to be of immature features. To obtain matured hepatocytes from stem cells, we tested the effect of culturing human-induced pluripotent stem (hiPS) cell-derived endoderm cells on collagen vitrigel membrane and compared with our previous reported nanofiber matrix. We cultured hiPS cell-derived endoderm cells on a collagen vitrigel membrane and examined the expression profiles, and tested the activity of metabolic enzymes. Gene expression profile analysis of hepatocytic differentiation markers revealed that upon culture on collagen vitrigel membrane, immature markers of AFP decreased, with a concomitant increase in the expression of mature hepatocyte transcription factors and mature hepatocyte markers such as ALB, ASGR1. Mature markers involved in liver functions, such as transporters, cytochrome P450 enzymes and phase II metabolic enzymes were also upregulated. We observed the upregulation of the liver markers for at least 2 weeks. Gene array profiling analysis revealed that hiPS cell-derived hepatocyte-like cells (hiPS-hep) resemble those of the primary hepatocytes. Functions of the CYP enzyme activities were tested in multi-institution and all revealed high CYP1A, CYP2C19, CYP2D6, CYP3A activity, which could be maintained for at least 2 weeks in culture. Taken together, the present approach identified that collagen vitrigel membrane provides a suitable environment for the generation of hepatocytes from hiPS cells that resemble many characteristics of primary human hepatocytes.

Spatial patterning of liver progenitor cell differentiation mediated by cellular contractility and Notch signaling.

The progenitor cells of the developing liver can differentiate toward both hepatocyte and biliary cell fates. In addition to the established roles of TGFβ and Notch signaling in this fate specification process, there is increasing evidence that liver progenitors are sensitive to mechanical cues. Here, we utilized microarrayed patterns to provide a controlled biochemical and biomechanical microenvironment for mouse liver progenitor cell differentiation. In these defined circular geometries, we observed biliary differentiation at the periphery and hepatocytic differentiation in the center. Parallel measurements obtained by traction force microscopy showed substantial stresses at the periphery, coincident with maximal biliary differentiation. We investigated the impact of downstream signaling, showing that peripheral biliary differentiation is dependent not only on Notch and TGFβ but also E-cadherin, myosin-mediated cell contractility, and ERK. We have therefore identified distinct combinations of microenvironmental cues which guide fate specification of mouse liver progenitors toward both hepatocyte and biliary fates.

Activation of Polyamine Catabolism by N¹,N11-Diethylnorspermine in Hepatic HepaRG Cells Induces Dedifferentiation and Mesenchymal-Like Phenotype.

Tumorigenesis is accompanied by the metabolic adaptation of cells to support enhanced proliferation rates and to optimize tumor persistence and amplification within the local microenvironment. In particular, cancer cells exhibit elevated levels of biogenic polyamines. Inhibitors of polyamine biosynthesis and inducers of their catabolism have been evaluated as antitumor drugs, however, their efficacy and safety remain controversial. Our goal was to investigate if drug-induced modulation of polyamine metabolism plays a role in dedifferentiation using differentiated human hepatocyte-like HepaRG cell cultures. N1,N11-diethylnorspermine (DENSpm), a potent inducer of polyamine catabolism, triggered an epithelial-mesenchymal transition (EMT)-like dedifferentiation in HepaRG cultures, as shown by down-regulation of mature hepatocytes markers and upregulation of classical EMT markers. Albeit the fact that polyamine catabolism produces H2O2, DENSpm-induced de-differentiation was not affected by antioxidants. Use of a metabolically stable spermidine analogue showed furthermore, that spermidine is a key regulator of hepatocyte differentiation. Comparative transcriptome analyses revealed, that the DENSpm-triggered dedifferentiation of HepaRG cells was accompanied by dramatic metabolic adaptations, exemplified by down-regulation of the genes of various metabolic pathways and up-regulation of the genes involved in signal transduction pathways. These results demonstrate that polyamine metabolism is tightly linked to EMT and differentiation of liver epithelial cells.