Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase.
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ABSTRACT:
SUBMITTER: Yeoh GC
PROVIDER: S-EPMC1161030 | biostudies-other | 1979 Apr
REPOSITORIES: biostudies-other
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Project description:1. Adrenaline increased hepatic tyrosine aminotransferase activity when injected into foetal rats or 2-day-old rats. 2. The inhibition of the postnatal increase in tyrosine aminotransferase activity which occurred in adrenalectomized newborn rats rapidly overcome by injection of adrenaline or dibutyryl cyclic AMP. 3. The effects of adrenaline or dibutyryl cyclic AMP on the tyrosine aminotransferase activity in foetal, adrenalectomized newborn and 2-day-old rats could be partially or completely blocked by prior treatment with actinomycin D. 4. Dibutyryl cyclic AMP induced tyrosine aminotransferase activity in hepatocytes cultured from 15-day foetal rats in glucocorticoid-free medium. 5. Actinomycin D at 0.2 microgram/ml in the culture medium completely prevented the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP in cultured cells. 6. The results suggest that adrenaline and cyclic AMP stimulate a transcriptional event during induction of tyrosine aminotransferase in perinatal liver.
| S-EPMC1162147 | biostudies-other
Project description:The trypanosomatid parasite Leishmania infantum is the causative agent of visceral leishmaniasis (VL), which is usually fatal unless treated. VL has an incidence of 0.5 million cases every year and is an important opportunistic co-infection in HIV/AIDS. Tyrosine aminotransferase (TAT) has an important role in the metabolism of trypanosomatids, catalyzing the first step in the degradation pathway of aromatic amino acids, which are ultimately converted into their corresponding L-2-oxoacids. Unlike the enzyme in Trypanosoma cruzi and mammals, L. infantum TAT (LiTAT) is not able to transaminate ketoglutarate. Here, the structure of LiTAT at 2.35 Å resolution is reported, and it is confirmed that the presence of two Leishmania-specific residues (Gln55 and Asn58) explains, at least in part, this specific reactivity. The difference in substrate specificity between leishmanial and mammalian TAT and the importance of this enzyme in parasite metabolism suggest that it may be a useful target in the development of new drugs against leishmaniasis.
| S-EPMC4014323 | biostudies-literature
Project description:Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.
| S-EPMC3887452 | biostudies-literature
Project description:BackgroundHepatocyte differentiation inducer (HDI) lacks both glucose and arginine, but is supplemented with galactose and ornithine, and is added together with other reagents such as apoptosis inhibitor and oncostatin M. Although human induced pluripotent stem (iPS) cells initiate hepatocyte differentiation, most die within 7 days. In this study, we investigated both HDI and conventional media for their potential to improve cell survival.Materials and methods201B7 iPS cells were cultured in conventional media. This consisted of three cycles of 5-day culture in William's E (WE) medium, followed by a 2-day culture in HDI.ResultsExpression levels of α-feto protein (AFP) were higher in cells cultured in WE and in Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham (DF12). 201B7 cells expressed the highest AFP and albumin (ALB) when cultured in HDI for 2 days following 7-day culture in WE. After three cycles of 5-day culture in WE followed by 2 days in HDI, 201B7 cells expressed AFP and ALB 54 ± 2.3 (average ± standard deviation) and 73 ± 15.1 times higher, respectively, than those cultured in ReproFF (feeder-free condition).Conclusion201B7 cells survived culture in WE for 7 days followed HDI for 2 days. After three cycles of culture under these conditions, hepatocyte differentiation was enhanced, as evidenced by increased AFP and ALB expression.
| S-EPMC4830564 | biostudies-literature
Project description:Embryonic stem cell-derived hepatocyte precursor cells represent a promising model for clinical transplantations to diseased livers, as well as for establishment of in vitro systems for drug metabolism and toxicology investigations. This study aimed to establish an in vitro culture system for scalable generation of hepatic progenitor cells. We used stable transgenic clones of murine embryonic stem cells possessing a reporter/selection vector, in which the enhanced green fluorescent protein- and puromycin N-acetyltransferase-coding genes are driven by a common alpha-fetoprotein gene promoter. This allowed for "live" monitoring and puromycin selection of the desired differentiating cell type possessing the activated alpha-fetoprotein gene. A rotary culture system was established, sequentially yielding initially partially selected hepatocyte lineage-committed cells, and finally, a highly purified cell population maintained as a dynamic suspension spheroid culture, which progressively developed the hepatic gene expression phenotype. The latter was confirmed by quantitative RT-PCR analysis, which showed a progressive up-regulation of hepatic genes during spheroid culture, indicating development of a mixed hepatocyte precursor-/fetal hepatocyte-like cell population. Adherent spheroids gave rise to advanced differentiated hepatocyte-like cells expressing hepatic proteins such as albumin, alpha-1-antitrypsin, cytokeratin 18, E-cadherin, and liver-specific organic anion transporter 1, as demonstrated by fluorescent immunostaining. A fraction of adherent cells was capable of glycogen storage and of reversible up-take of indocyanine green, demonstrating their hepatocyte-like functionality. Moreover, after transplantation of spheroids into the mouse liver, the spheroid-derived cells integrated into recipient. These results demonstrate that large-scale hepatocyte precursor-/hepatocyte-like cultures can be established for use in clinical trials, as well as in in vitro screening assays.
| S-EPMC3454367 | biostudies-literature
Project description:1. The acquisition of dexamethasone-inducibility of tyrosine aminotransferase activity by hepatocytes cultured from 15-day-foetal rat liver is blocked in the presence of cytosine arabinoside. 2. Similar results are obtained in the presence of bormodeoxyuridine. 3. No effects on steroid-inducibility of tyrosine aminotransferase are obtained with either of the above compounds in hepatocytes cultured from 19-day-foetal liver. 4. the inhibitory effects of the agents are substantially reversed after their removal from the culture medium. 5. The effects of bromodeoxyuridine suggest that cell differentiation, with respect to tyrosine aminotransferase-inducibility, occurs in cultures of 15-day-doetal hepatocytes. 6. The effects of cytosine arabinoside suggest that such an event is dependent on mitosis.
| S-EPMC1161980 | biostudies-other
Project description:In the first group of samples, we are looking into the different metabolites and their concentrations within our human embryonic stem cells over the course of differentiation. Our goal is to study how the metabolism of stem cells are changing as they undergo this conversion to become hepatocyte-like cells. In the second group of samples labeled HyCell, we would like to understand the concentrations of metabolites within our CHO cells in order to update the parameters for our metabolic model. Included are two samples from different days of a suspension batch culture which may have different lactate production qualities.
2017-08-02 | ST000837 | MetabolomicsWorkbench
Project description:Leishmania infantum is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The disease is fatal without treatment, which has been based on antimonial pentavalents for more than 60 years. Due to resistances, relapses and toxicity to current treatment, the development of new drugs is required. The structure of the L. infantum tyrosine aminotransferase (LiTAT) has been recently solved showing important differences with the mammalian orthologue. The characterization of LiTAT is reported herein. This enzyme is cytoplasmic and is over-expressed in the more infective stages and nitric oxide resistant parasites. Unlike the mammalian TAT, LiTAT is able to use ketomethiobutyrate as co-substrate. The pharmacophore model of LiTAT with this specific co-substrate is described herein. This may allow the identification of new inhibitors present in the databases. All the data obtained support that LiTAT is a good target candidate for the development of new anti-leishmanial drugs.
| S-EPMC4266777 | biostudies-literature
Project description:Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine and other aromatic amino acids. The enzyme is thought to play a role in tyrosinemia type II, hepatitis and hepatic carcinoma recovery. The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases. Mouse TAT (mTAT) was cloned from a mouse cDNA library, and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques. The recombinant mTAT is able to catalyze the transamination of tyrosine using α-ketoglutaric acid as an amino group acceptor at neutral pH. The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxy-phenylpyruvate, phenylpyruvate and alpha-ketocaproic acid as amino group acceptors. Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9 Å resolution. The crystal structure revealed the interaction between the pyridoxal-5'-phosphate cofactor and the enzyme, as well as the formation of a disulphide bond. The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent. Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity. The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.
| S-EPMC3023147 | biostudies-literature
Project description:AimTo explore whether a co-culture of cynomolgus monkey embryonic stem (cES) cells with embryonic liver cells could promote their differentiation into hepatocytes.MethodsMouse fetal liver-derived cells (MFLCs) were prepared as adherent cells from mouse embryos on embryonic d (ED) 14, after which undifferentiated cES cells were co-cultured with MFLCs. The induction of cES cells along a hepatic lineage was examined in MFLC-assisted differentiation, spontaneous differentiation, and growth factors (GF) and chemicals-induced differentiations (GF-induced differentiation) using retinoic acid, leukemia inhibitory factor (LIF), FGF2, FGF4, hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone.ResultsThe mRNA expression of alpha-fetoprotein, albumin (ALB), alpha-1-antitrypsin, and hepatocyte nuclear factor 4alpha was observed earlier in the differentiating cES cells co-cultured with MFLCs, as compared to cES cells undergoing spontaneous differentiation and those subjected to GF-induced differentiation. The expression of cytochrome P450 7a1, a possible marker for embryonic endoderm-derived mature hepatocytes, was only observed in cES cells that had differentiated in a co-culture with MFLCs. Further, the disappearance of Oct3/4, a representative marker of an undifferentiated state, was noted in cells co-cultured with MFLCs, but not in those undergoing spontaneous or GF-induced differentiation. Immunocytochemical analysis revealed an increased ratio of ALB-immunopositive cells among cES cells co-cultured with MFLCs, while glycogen storage and urea synthesis were also demonstrated.ConclusionMFLCs showed an ability to induce cES cells to differentiate toward hepatocytes. The co-culture system with MFLCs is a useful method for induction of hepatocyte-like cells from undifferentiated cES cells.
| S-EPMC4087437 | biostudies-literature