Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes.

Interindividual differences in hepatic metabolism, which are mainly due to genetic polymorphism in its gene, have a large influence on individual drug efficacy and adverse reaction. Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells have the potential to predict interindividual differences in drug metabolism capacity and drug response. However, it remains uncertain whether human iPSC-derived HLCs can reproduce the interindividual difference in hepatic metabolism and drug response. We found that cytochrome P450 (CYP) metabolism capacity and drug responsiveness of the primary human hepatocytes (PHH)-iPS-HLCs were highly correlated with those of PHHs, suggesting that the PHH-iPS-HLCs retained donor-specific CYP metabolism capacity and drug responsiveness. We also demonstrated that the interindividual differences, which are due to the diversity of individual SNPs in the CYP gene, could also be reproduced in PHH-iPS-HLCs. We succeeded in establishing, to our knowledge, the first PHH-iPS-HLC panel that reflects the interindividual differences of hepatic drug-metabolizing capacity and drug responsiveness.

A diphtheria toxin receptor deficient in epidermal growth factor-like biological activity.

Targeted cell ablation in animals is a powerful method for analyzing the physiological function of cell populations and generating various animal models of organ dysfunction. To achieve more specific and conditional ablation of target cells, we have developed a method termed Toxin Receptor mediated Cell Knockout (TRECK). A potential shortcoming of this method, however, is that overexpression of human heparin-binding epidermal growth factor-like growth factor (hHB-EGF) as a diphtheria toxin (DT) receptor in target cells or tissues may cause abnormalities in transgenic mice, since hHB-EGF is a member of the EGF growth factor family. To create novel DT receptors that are defective in growth factor activity and resistant to metalloprotease-cleavage, we mutated five amino acids in the extracellular EGF-like domain of hHB-EGF, which contains both DT-binding and protease-cleavage sites. Two of the resultant hHB-EGF mutants, I117A/L148V and I117V/L148V, possessed little growth factor activity but retained DT receptor activity. Furthermore, these mutants were resistant to metalloprotease-cleavage by 12-O-tetradecanoylphorbol-13-acetate stimulation, which is expected to enhance DT receptor activity. These novel DT receptors should be useful for the generation of transgenic mice by TRECK.

Suppression of hepatitis C virus replicon by adenovirus vector-mediated expression of tough decoy RNA against miR-122a.

Recent studies have demonstrated that the liver-specific microRNA (miRNA) miR-122a plays an important role in the replication of hepatitis C virus (HCV). Antisense nucleotides against miR-122a, including locked nucleic acid (LNA), have shown promising results for suppression of HCV replication; however, a liver-specific delivery system of antisense nucleotides has not been fully developed. In this study, an adenovirus (Ad) vector that expresses tough decoy (TuD)-RNA against miR-122a (TuD-122a) was developed to suppress the HCV replication in the liver hepatocytes. Ad vectors have been well established to exhibit a marked hepatotropism following systemic administration. An in vitro reporter gene expression assay demonstrated that Ad vector-mediated expression of TuD-122a efficiently blocked the miR-122a in Huh-7 cells. Furthermore, transduction with the Ad vector expressing TuD-122a in HCV replicon-expressing cells resulted in significant reduction in the HCV replicon levels. These results indicate that Ad vector-mediated expression of TuD-122a would be a promising tool for treatment of HCV infection.

Optimization of a microRNA expression vector for function analysis of microRNA.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs endogenously expressed in a tissue-type specific pattern. Recent studies have demonstrated that miRNAs are involved in almost all cellular biological processes, including cellular development, differentiation, apoptosis, and proliferation. To elucidate the function of miRNAs in biological processes, it is crucial that we develop miRNA-expressing vectors for the efficient expression of miRNAs in cultured cells and animals. At the present time, however, no fully optimized miRNA-expressing vectors have been developed, since such vectors require consideration of the choice of promoters and several other complex factors. In this study, we constructed various types of plasmid vectors expressing human miR-199a. There are two genes encoding miR-199a in the different chromosomes, resulting in expression of two different precursors which produce the two mature miRNAs from the 5'- and 3'-strands (miR-199a-5p and -3p). When the miR-199a precursors containing the genomic sequences flanking the hairpin were expressed, the cytomegalovirus (CMV) promoter, CMV promoter/enhancer containing the intron A (CMVi), and CMV enhancer/ß-actin (CA) promoter were more effective than the human phosphoglycerate kinase (PGK) promoter. The reduction levels by the human U6 promoter-transcribed miR-199a were different between the cell lines. The suppressive effects of miR-199a on the reporter gene expression were different between miR-199a-5p and -3p, especially when miR-199a was expressed as a stem-loop structure under the control of the U6 promoter. Expression of miR-199a as a short-hairpin RNA (shRNA) with an artificial hairpin sequence and independent expression of the mature miR-199a and its complementary strand were effective for distinguishing the function of the 5'- and 3'-strand of the miRNA. In addition, expression of miRNAs as an shRNA and separate expression of mature miRNAs and their complementary strand would be promising methods under conditions in which the processing steps of miRNAs are impaired. The results of this study provide important information on the construction of miRNA-expressing vectors for miRNA function analysis as well as for gene therapy using miRNA-expressing vectors.

Adenovirus vector-mediated efficient transduction into human embryonic and induced pluripotent stem cells.

We examined the transduction efficiency in human embryonic stem (ES) and induced pluripotent stem (iPS) cells using an adenovirus (Ad) vector. RT-PCR analysis revealed the expression of the coxsackievirus and adenovirus receptor, a receptor for Ad, in these cells. However, gene expression after the transduction with an Ad vector was observed only in the periphery of ES and iPS cell colonies, when human ES and iPS cells were passaged as small colonies. This suggests that the Ad vector could not enter inside the ES and iPS cell colonies by their tight connection. We thus attempted to transduce foreign genes into the dissociated form of human ES and iPS cells, which were passaged using Rho-associated kinase inhibitor. In this condition, transduction efficiency in human ES and iPS cells was markedly increased and transgene expression was observed even inside the colonies by using Ad vectors. Furthermore, Ad vector-mediated transduction did not alter the expression of undifferentiated markers such as Oct-3/4, Nanog, and SSEA-4. Our results indicate that Ad vectors are effective tools for transduction into human ES and iPS cells.

Mechanism of acid adaptation of a fish living in a pH 3.5 lake.

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.