Miho Tamai

Polymeric Membranes for Chiral Separation of Pharmaceuticals and Chemicals

The optical resolution or chiral separation of one specific enantiomer from others is in demand for the production of pharmaceuticals because many pharmaceuticals exist as stereoisomers, with each enantiomer having different biological activity. There is considerable demand for separation techniques appropriate for the large-scale resolution of chiral molecules. Chiral separation of racemic mixtures of pharmaceuticals through chiral or achiral polymeric membranes with or without a chiral selector represents a promising system for future commercial application. This article reviews several polymeric materials for the chiral separation of pharmaceuticals. Several chiral separation membranes were prepared from chiral polymers where enantioselectivity was generated from chiral carbons in the main chain. However, it is rather difficult to generate excellent chiral separation membranes from chiral polymers alone, because racemic penetrants mainly encounter the flexible side chains of the membrane polymers. Therefore, chiral separation membranes were also prepared using polymers with a chiral branch. Furthermore, several molecules have been used for specific interactions between the molecules and specific pharmaceuticals or drugs in chiral separation membranes. Cyclodextrins, crown ether derivatives, albumin, and DNA are commonly used as stereoselective ligands in chiral separation membranes. Finally, this article discusses future trends in polymeric materials for chiral separation membranes.

Mitochondrial development of the in vitro hepatic organogenesis model with simultaneous cardiac mesoderm differentiation from murine induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells, resembling embryonic stem (ES) cells in many phenomena, including differentiation potential, colony morphology, and the expression of specific representative markers, were generated from differentiated somatic cells by exogenous expression of several transcriptional factors. In recent, the mitochondria of iPS cells were also reported to be rejuvenated to that of ES cells, however it is not known if the mitochondria have same potential for differentiation as ES cells. We have established the murine ES cell-derived in vitro hepatic organogenesis model, consisting of not only hepatocytes but also endothelial networks together with cardiac mesoderm differentiation, previously. By measuring oxygen concentration and pH in the culture medium, respectively corresponding to the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), we compared the metabolic patterns and bio-energetic profiles of both iPS and ES cells during the hepatic differentiation. The bio-energetic profiles of the in vitro hepatic organogenesis from iPS cells accorded with each differentiation steps, from proliferation stage as the initiation, spontaneously beating cardiac differentiation in the next, and finally liver tissue-formation, as well as that from ES cells. Both iPS and ES cells were differentiated into liver-like tissue with similar mitochondrial development.

Hybrid sponge comprised of galactosylated chitosan and hyaluronic acid mediates the co-culture of hepatocytes and endothelial cells

When constructing an in vitro model of liver tissue to mimic the in vivo liver microenvironment, the major challenge is to preserve and maintain the hepatocyte phenotype. The aim of this study was to develop a novel intelligent hybrid sponge for use in a dense co-culture system designed to simulate the liver microenvironment. We prepared a galactosylated chitosan (GCs)/hyaluronic acid (HA) hybrid sponge using a freeze-drying technique for the co-culture of primary hepatocytes and endothelial cells. Subsequently, we investigated the biocompatibility of the GCs/HA scaffold with primary hepatocytes and endothelial cells in terms of cell attachment, morphology, bioactivity, and maintenance of specific liver functions. The GCs/HA-hybrid sponge demonstrated good biocompatibility not only with primary hepatocytes, but also with endothelial cells. In our model, primary hepatocytes exhibited superior bioactivity and higher levels of liver-specific functions in terms of hepatocyte-specific gene expression, urea production, and testosterone metabolism as compared to a monoculture system. We succeeded in constructing a liver tissue-like model using the GCs/HA-hybrid sponge. Therefore, we anticipate that GCs/HA-hybrid sponges may be a promising matrix for the co-culture of hepatocytes and endothelial cells in liver tissue engineering, and might be employed as a novel co-culture model for applications in toxicology and drug metabolism.

Acetaminophen-induced Hepatotoxicity in a Liver Tissue Model Consisting of Primary Hepatocytes Assembling Around an Endothelial Cell Network

Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.

Isolation and characterization of portal branch ligation-stimulated Hmga2-positive bipotent hepatic progenitor cells.

Hepatic stem/progenitor cells are one of several cell sources that show promise for restoration of liver mass and function. Although hepatic progenitor cells (HPCs), including oval cells, are induced by administration of certain hepatotoxins in experimental animals, such a strategy would be inappropriate in a clinical setting. Here, we investigated the possibility of isolating HPCs in a portal branch-ligated liver model without administration of any chemical agents. A non-parenchymal cell fraction was prepared from the portal branch-ligated or non-ligated lobe, and seeded onto plates coated with laminin. Most of the cells died, but a small number were able to proliferate. These proliferating cells were cloned as portal branch ligation-stimulated hepatic cells (PBLHCs) by the limiting dilution method. The PBLHCs expressed cytokeratin19, albumin, and Hmga2. The PBLHCs exhibited metabolic functions such as detoxification of ammonium ions and synthesis of urea on Matrigel-coated plates in the presence of oncostatin M. In Matrigel mixed with type I collagen, the PBLHCs became rearranged into cystic and tubular structures. Immunohistochemical staining demonstrated the presence of Hmga2-positive cells around the interlobular bile ducts in the portal branch-ligated liver lobes. In conclusion, successful isolation of bipotent hepatic progenitor cell clones, PBLHCs, from the portal branch-ligated liver lobes of mice provides the possibility of future clinical application of portal vein ligation to induce hepatic progenitor cells.

Expression of the rodent-specific alternative splice variant of tryptophanyl-tRNA synthetase in murine tissues and cells

Tryptophanyl-tRNA synthetase (TrpRS) catalyzes the aminoacylation of tRNATrp. mRNA of a rodent-specific alternative splice variant of TrpRS (SV-TrpRS), which results in the inclusion of an additional hexapeptide at the C-terminus of full-length TrpRS (FL-TrpRS), has been identified in murine embryonic stem (ES) cells. In the present study, we evaluated the expression of mouse TrpRS mRNA by real-time reverse transcription PCR. We show that SV-TrpRS and FL-TrpRS mRNAs are highly expressed in murine ES cells, embryo, spleen, lung, liver and uterus, and that the relative expression of SV-TrpRS compared to FL-TrpRS is significantly less in the brain. Moreover, we found that interferon-γ increases the expression of TrpRS in a mouse cell line. These results provide the first evidence for tissue-specific expression and alternative splicing of mouse TrpRS.

Nitric oxide is critical for avoiding hepatic lipid overloading via IL-6 induction during liver regeneration after partial hepatectomy in mice

Nitric oxide (NO), generated from L-arginine by three different isoforms of nitric oxide synthase (NOS), is a pleiotropic factor to regulate physiological functions in almost every organ and tissue. Each knockout mouse of iNOS or eNOS has been used to suggest that NO has a crucial role in liver regeneration after partial hepatectomy (PH), for NO may inhibit caspase 3 activity and is required for EGFR signaling. In previous reports, defective mitochondrial β-oxidation was observed in eNOS KO mice, and hepatic steatosis was often correlated to deficient liver regeneration, so we focused on metabolic perspective and hypothesized that NO depletion in PH mice would affect hepatocytic lipolysis and impair hepatocytes proliferation. We inhibited all NOS isoforms by administrating L-NG-nitroarginine methyl ester (L-NAME) to PH mice, and hepatocyte DNA synthesis was severely inhibited at 40–44 h post PH in L-NAME (+) group. IL-6 was robustly secreted into circulating blood in L-NAME (−) group, but not in L-NAME (+) group. Down-regulation of carnitine palmytoyltransferase 1A, massive lipid accumulation and elevated endoplasmic reticulum (ER) stress relative genes expression level were observed in L-NAME (+) group mouse liver. The expression level of C/EBP homologous protein, a mediator of ER stress induced apoptosis, significantly increased in L-NAME (+) group. Our findings suggest the lack of NO affected IL-6 induction and hepatocyte lipolysis after PH, consequently leading to excessive hepatic lipid accumulation, elevated ER stress and impaired hepatocyte proliferation.

An in vitro liver model consisting of endothelial vascular networks surrounded by human hepatoma cell lines allows for improved hepatitis B virus replication

The life cycle of viruses, from infection to budding, is dependent upon the physiological activity of the host cells, such as expression of cell surface proteins, activities of organelles and transcription factors and so on. Human hepatitis viruses exploit multiple hepatocyte pathways during their life cycle; however, primary hepatocytes dramatically lose function and die when cultured as a monolayer in vitro. We previously reported the development of an in vitro liver model, IVL, consisting of endothelial networks and mouse primary hepatocytes. Hepatocytes cultured using the IVL achieved higher hepatic gene expression and drug sensitivity. In this study, human IVLs were constructed by using the human hepatoma cell lines, Hep G2 and HuH-7, and human umbilical vein endothelial cell networks on Engelbreth-Holm-Swarm gels. In order that these human IVLs could serve as in vitro models of human viral hepatitis, these human hepatoma cell lines were stably transfected with the hepatitis B virus (HBV) genome. The levels of HBV markers observed in the supernatant of the IVL cultures were significantly increased as compared to those obtained in transfected monocultures. Furthermore, the hepatocytes in the human IVL cultures became polarized, leading to efficient HBV replication and release in vitro. This finding suggests that the IVL culture system could be an effective model for HBV replication.

In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVLmES, which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVLmES culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. l-Ornithine is an intermediate of the urea cycle, but supplemental l-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVLmES, supplemental l-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVLmES displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVLmES will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

Development of hepatoma-derived, bidirectional oval-like cells as a model to study host interactions with hepatitis C virus during differentiation

Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.