Publications by authors named "Atsushi Miyajima"

164 Publications

CD82 is a marker to isolate β cell precursors from human iPS cells and plays a role for the maturation of β cells.

Sci Rep 2021 May 5;11(1):9530. Epub 2021 May 5.

Institute for Quantitative Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Generation of pancreatic β cells from pluripotent stem cells is a key technology to develop cell therapy for insulin-dependent diabetes and considerable efforts have been made to produce β cells. However, due to multiple and lengthy differentiation steps, production of β cells is often unstable. It is also desirable to eliminate undifferentiated cells to avoid potential risks of tumorigenesis. To isolate β cell precursors from late stage pancreatic endocrine progenitor (EP) cells derived from iPS cells, we have identified CD82, a member of the tetraspanin family. CD82 cells at the EP stage differentiated into endocrine cells more efficiently than CD82 EP stage cells. We also show that CD82 cells in human islets secreted insulin more efficiently than CD82 cells. Furthermore, knockdown of CD82 expression by siRNA or inhibition of CD82 by monoclonal antibodies in NGN3 cells suppressed the function of β cells with glucose-stimulated insulin secretion, suggesting that CD82 plays a role in maturation of EP cells to β cells.
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http://dx.doi.org/10.1038/s41598-021-88978-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8100138PMC
May 2021

Small extracellular vesicles derived from interferon-γ pre-conditioned mesenchymal stromal cells effectively treat liver fibrosis.

NPJ Regen Med 2021 Mar 30;6(1):19. Epub 2021 Mar 30.

Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, 1-757 Asahimachi-dori, Chuo-ku, Niigata, 951-8510, Japan.

Mesenchymal stromal cells (MSCs) are used for ameliorating liver fibrosis and aiding liver regeneration after cirrhosis; Here, we analyzed the therapeutic potential of small extracellular vesicles (sEVs) derived from interferon-γ (IFN-γ) pre-conditioned MSCs (γ-sEVs). γ-sEVs effectively induced anti-inflammatory macrophages with high motility and phagocytic abilities in vitro, while not preventing hepatic stellate cell (HSC; the major source of collagen fiber) activation in vitro. The proteome analysis of MSC-derived sEVs revealed anti-inflammatory macrophage inducible proteins (e.g., annexin-A1, lactotransferrin, and aminopeptidase N) upon IFN-γ stimulation. Furthermore, by enabling CXCR1+ macrophage accumulation in the damaged area, γ-sEVs ameliorated inflammation and fibrosis in the cirrhosis mouse model more effectively than sEVs. Single cell RNA-Seq analysis revealed diverse effects, such as induction of anti-inflammatory macrophages and regulatory T cells, in the cirrhotic liver after γ-sEV administration. Overall, IFN-γ pre-conditioning altered sEVs resulted in efficient tissue repair indicating a new therapeutic strategy.
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http://dx.doi.org/10.1038/s41536-021-00132-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010072PMC
March 2021

Case series of elderly patients with scabies topically applied with ivermectin via whole-body bathing.

J Dermatol 2021 Apr 6;48(4):559-563. Epub 2021 Jan 6.

Tsubasa Home Care Clinic, Ohta, Japan.

As a novel method of ivermectin (IVM) administration for the treatment of scabies, we devised a whole-body bathing (WBB), in which patients are immersed in a fluid that contains IVM. A multi-institutional trial for elderly patients with scabies was conducted to investigate the efficacy and safety of IVM-WBB. Seven elderly patients with scabies were enrolled and received IVM-WBB up to four times at 1-week interval. The cure for scabies was defined as the absence of mites in two consecutive microscopic or dermoscopic examinations at weekly intervals and the absence of new skin lesions indicative of scabies. Consequently, the cure rate on day 22, the primary end-point, was 71.4%, and all patients had been cured until day 29. Additionally, neither significant adverse events nor clinically problematic abnormal blood test values were obtained. Furthermore, no IVM was detected in the optional plasma (five cases) collected for IVM measurement after bathing. These results suggest that IVM-WBB was effective to treat scabies, causing no serious adverse events and with a very low internal exposure of IVM.
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http://dx.doi.org/10.1111/1346-8138.15746DOI Listing
April 2021

Multi-omics analysis of hiPSCs-derived HLCs matured on-chip revealed patterns typical of liver regeneration.

Biotechnol Bioeng 2021 Jan 6. Epub 2021 Jan 6.

CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, Tokyo, Japan.

Maturation of human-induced pluripotent stem cells (hiPSCs)-derived hepatocytes-like cells (HLCs) toward a complete hepatocyte phenotype remains a challenge as primitiveness patterns are still commonly observed. In this study, we propose a modified differentiation protocol for those cells which includes a prematuration in Petri dishes and a maturation in microfluidic biochip. For the first time, a large range of biomolecular families has been extracted from the same sample to combine transcriptomic, proteomic, and metabolomic analysis. After integration, these datasets revealed specific molecular patterns and highlighted the hepatic regeneration profile in biochips. Overall, biochips exhibited processes of cell proliferation and inflammation (via TGFB1) coupled with anti-fibrotic signaling (via angiotensin 1-7, ATR-2, and MASR). Moreover, cultures in this condition displayed physiological lipid-carbohydrate homeostasis (notably via PPAR, cholesterol metabolism, and bile synthesis) coupled with cell respiration through advanced oxidative phosphorylation (through the overexpression of proteins from the third and fourth complex). The results presented provide an original overview of the complex mechanisms involved in liver regeneration using an advanced in vitro organ-on-chip technology.
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http://dx.doi.org/10.1002/bit.27667DOI Listing
January 2021

Essential roles of oncostatin M receptor β signaling in renal crystal formation in mice.

Sci Rep 2020 10 13;10(1):17150. Epub 2020 Oct 13.

Department of Anatomy and Neurobiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama, 641-8509, Japan.

Oncostatin M (OSM), a member of the IL-6 family of cytokines, has important roles in renal diseases. The relationship between OSM and kidney stone disease, however, remains unclear. To investigate the roles of OSM in the development of kidney stone disease, we generated a mouse model of renal crystal formation using OSM receptor β (OSMRβ)-deficient mice (OSMRβ mice). There were fewer renal crystal deposits in OSMRβ mice than in wild-type (WT) mice. Crystal-binding molecules (osteopontin, annexin A1, and annexin A2), inflammatory cytokines (TNF-α and IL-1β), and fibrosis markers (TGF-β, collagen 1a2, and α-smooth muscle actin) were also decreased in the kidneys of OSMRβ mice compared with those in WT mice. Immunofluorescence staining showed that OSMRβ was expressed in renal tubular epithelial cells (RTECs) and renal fibroblasts in the model of renal crystal formation. In the cultured RTECs and renal fibroblasts, OSM directly induced the expression of crystal-binding molecules and fibrosis markers. Expressions of inflammatory cytokines were increased by stimulation with OSM in cultured renal fibroblasts. OSM may promote the formation of renal crystal deposits by directly acting on RTECs and renal fibroblasts to produce crystal-binding molecules and inflammatory cytokines.
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http://dx.doi.org/10.1038/s41598-020-74198-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553912PMC
October 2020

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Modulation of hepatitis B virus infection by epidermal growth factor secreted from liver sinusoidal endothelial cells.

Sci Rep 2020 09 1;10(1):14349. Epub 2020 Sep 1.

Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that liver sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC-derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.
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http://dx.doi.org/10.1038/s41598-020-71453-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7462976PMC
September 2020

Analysis of hiPSCs differentiation toward hepatocyte-like cells upon extended exposition to oncostatin.

Differentiation 2020 Jul - Aug;114:36-48. Epub 2020 May 19.

CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan. Electronic address:

The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.
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http://dx.doi.org/10.1016/j.diff.2020.05.006DOI Listing
May 2020

Multidimensional imaging of liver injury repair in mice reveals fundamental role of the ductular reaction.

Commun Biol 2020 06 5;3(1):289. Epub 2020 Jun 5.

Laboratory of Stem Cell Therapy, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Upon severe and/or chronic liver injury, ectopic emergence and expansion of atypical biliary epithelial-like cells in the liver parenchyma, known as the ductular reaction, is typically induced and implicated in organ regeneration. Although this phenomenon has long been postulated to represent activation of facultative liver stem/progenitor cells that give rise to new hepatocytes, recent lineage-tracing analyses have challenged this notion, thereby leaving the pro-regenerative role of the ductular reaction enigmatic. Here, we show that the expanded and remodelled intrahepatic biliary epithelia in the ductular reaction constituted functional and complementary bile-excreting conduit systems in injured parenchyma where hepatocyte bile canalicular networks were lost. The canalicular collapse was an incipient defect commonly associated with hepatocyte injury irrespective of cholestatic statuses, and could sufficiently provoke the ductular reaction when artificially induced. We propose a unifying model for the induction of the ductular reaction, where compensatory biliary epithelial tissue remodeling ensures bile-excreting network homeostasis.
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http://dx.doi.org/10.1038/s42003-020-1006-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275065PMC
June 2020

Efficacy and safety of a combination regimen of phenothrin and ivermectin lotion in patients with head lice in Okinawa, Japan.

J Dermatol 2020 Jul 7;47(7):720-727. Epub 2020 May 7.

National Sanatorium Tamazenshoen, Higashimurayama, Japan.

In Japan, pyrethroid-resistant head lice have been increasing; however, only 0.4% phenothrin, a pyrethroid drug, is available as an over the counter formulation. In recent years, Sumithrin Lotion containing 5% phenothrin (PHT) was approved for scabies. In the USA, Sklice Lotion containing 0.5% ivermectin (IVM) is used for the treatment of pyrethroid-resistant head lice. Therefore, to enhance the treatment of head lice in Japan, we conducted a clinical study to confirm the efficacy and safety of a combination regimen of PHT and IVM (PI regimen). Twelve cases were enrolled and PHT was applied to all patients on day 1. On day 8, five patients (41.7%) were lice free, and PHT was applied again. Notably, seven patients were not lice free and were switched to IVM. The rate of patients who were lice free on the PI regimen, which was the primary end-point, was 75.0% on day 15 and 91.7% on day 22. No adverse events were reported. A genetic analysis of the head lice collected at each visit revealed a kdr mutation in all patients. These results suggest that the PI regimen is safe and effective for the treatment of pyrethroid-resistant head lice in Japan.
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http://dx.doi.org/10.1111/1346-8138.15348DOI Listing
July 2020

Characterization of liver zonation-like transcriptomic patterns in HLCs derived from hiPSCs in a microfluidic biochip environment.

Biotechnol Prog 2020 09 22;36(5):e3013. Epub 2020 May 22.

CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, Tokyo, Japan.

The liver zonation is an important phenomenon characterized by a gradient of several functions along the liver acinus. However, this gradient remains difficult to reproduce in in-vitro conditions, making the obtention of an in-vitro method to recapitulate the liver zonation a challenging issue. In this study, we evaluated the spatial evolution of the transcriptome profile of human induced pluripotent stem cells (hiPSCs) differentiated toward hepatocytes-like cells (HLCs) phenotype in a microfluidic biochip environment. Cells collected at the inlet of the biochip, where the oxygen concentration is higher, were identified by the expression of genes involved in metabolic pathways related to cellular reorganization and cell proliferation. Cells collected in the middle and at the outlet of the biochips, where oxygen concentrations are lower, were characterized by the upregulation of genes involved in cellular detoxification processes (CYP450), PPAR signaling or arginine biosynthesis. A subset of 16 transcription factors (TFs) was extracted and identified as upstream regulators to HNF1A and PPARA. These TFs are also known as regulators to target genes engaged in the Wnt/βcatenin pathway, in the TGFβ/BMP/SMAD signaling, in the transition between epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET), in the homeostasis of lipids, bile acids and carbohydrates homeostasis, in drug metabolism, in the estrogen processing and in the oxidative stress response. Overall, the analysis allowed to confirm a partial zonation-like pattern in hiPSCs-derived HLCs in the microfluidic biochip environment. These results provide important insights into the reproduction of liver zonation in-vitro for a better understanding of the phenomenon.
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http://dx.doi.org/10.1002/btpr.3013DOI Listing
September 2020

Reversible expansion of pancreatic islet progenitors derived from human induced pluripotent stem cells.

Genes Cells 2020 May 6;25(5):302-311. Epub 2020 Mar 6.

Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan.

Transplantation of pancreatic islets is an effective therapy for severe type 1 diabetes. As donor shortage is a major problem for this therapy, attempts have been made to produce a large number of pancreatic islets from human pluripotent stem cells (hPSCs). However, as the differentiation of hPSCs to pancreatic islets requires multiple and lengthy processes using various expensive cytokines, the process is variable, low efficiency and costly. Therefore, it would be beneficial if islet progenitors could be expanded. Neurogenin3 (NGN3)-expressing pancreatic endocrine progenitor (EP) cells derived from hPSCs exhibited the ability to differentiate into pancreatic islets while their cell cycle was arrested. By using a lentivirus vector, we introduced several growth-promoting genes into NGN3-expressing EP cells. We found that SV40LT expression induced proliferation of the EP cells but reduced the expression of endocrine lineage-commitment factors, NGN3, NEUROD1 and NKX2.2, resulting in the suppression of islet differentiation. By using the Cre-loxP system, we removed SV40LT after the expansion, leading to re-expression of endocrine-lineage commitment genes and differentiation into functional pancreatic islets. Thus, our findings will pave a way to generate a large quantity of functional pancreatic islets through the expansion of EP cells from hPSCs.
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http://dx.doi.org/10.1111/gtc.12759DOI Listing
May 2020

Tissue substructure-specific deposition of the β3-containing laminin-332 in the biliary epithelium of human and mouse livers.

Biochem Biophys Res Commun 2020 04 31;524(2):465-471. Epub 2020 Jan 31.

Laboratory of Stem Cell Therapy, Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0032, Japan. Electronic address:

Laminin is a family of basement membrane proteins, whose selective and spatiotemporal expression profiles are linked to their various functions in development, maintenance, and functional regulation of different tissues. In the liver, α1-and α5-containing laminin isoforms have been documented to be critically involved in the developmental process of the epithelial tissue of the bile duct. However, possible roles of other laminin isoforms in bile duct formation and function remain elusive. Here, we evaluated public single-cell RNA sequencing databases on human liver cells to reveal expression landscape of laminin genes, and found that genes for laminin-332 subunits were conjointly expressed in the EPCAM biliary epithelial cell population. Expression of the β3 and γ2 subunit genes was restricted to biliary epithelial cells in the liver and, remarkably, showed apparent heterogeneity among them. We confirmed the heterogeneous nature of the laminin-β3 expression in murine livers, which was firmly related to morphological substructures in the biliary epithelium. Finally, we generated the liver epithelial tissue-specific laminin- β3 knockout mice and found that this laminin subunit was dispensable under physiological conditions. Together, our present findings have identified the β3 subunit and the related laminin-332 isoform as useful markers and potentially important regulatory molecules for future understanding of pathophysiology in the hepatobiliary system.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.104DOI Listing
April 2020

Transcriptome profiling of hiPSC-derived LSECs with nanoCAGE.

Mol Omics 2020 04 28;16(2):138-146. Epub 2020 Jan 28.

CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

Liver Sinusoidal Endothelial Cells (LSECs) are an important component of the liver as they compose the microvasculature which allows the supply of oxygen, blood, and nutrients. However, maintenance of these cells in vitro remains challenging as they tend to rapidly lose some of their characteristics such as fenestration or as their immortalized counterparts present poor characteristics. In this work, human induced pluripotent stem cells (hiPSCs) have been differentiated toward an LSEC phenotype. After differentiation, the RNA quantification allowed demonstration of high expression of specific vascular markers (CD31, CD144, and STAB2). Immunostaining performed on the cells was found to be positive for both Stabilin-1 and Stabilin-2. Whole transcriptome analysis performed with the nanoCAGE method further confirmed the overall vascular commitment of the cells. The gene expression profile revealed the upregulation of the APLN, LYVE1, VWF, ESAM and ANGPT2 genes while VEGFA appeared to be downregulated. Analysis of promoter motif activities highlighted several transcription factors (TFs) of interest in LSECs (IRF2, ERG, MEIS2, SPI1, IRF7, WRNIP1, HIC2, NFIX_NFIB, BATF, and PATZ1). Based on this investigation, we compiled the regulatory network involving the relevant TFs, their target genes as well as their related signaling pathways. The proposed hiPSC-derived LSEC model and its regulatory network were then confirmed by comparing the experimental data to primary human LSEC reference datasets. Thus, the presented model appears as a promising tool to generate more complex in vitro liver multi-cellular tissues.
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http://dx.doi.org/10.1039/c9mo00135bDOI Listing
April 2020

Endodermal differentiation of human induced pluripotent stem cells using simple dialysis culture system in suspension culture.

Regen Ther 2019 Dec 30;12:14-19. Epub 2019 May 30.

Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

A differentiation of human induced pluripotent stem cells (hiPSCs) into definitive endoderm linage is required for a preparation of metabolic organ derived cells. The differentiation consumed high-priced cytokines and small molecules, which have hampered the manufacturability of differentiated cells. Although the cytokines and small molecules are remained or cells produce the autocrine factors, daily culture medium change should be proceeded to remove toxic metabolites generated from cells. In this study, we developed a simple dialysis culture system to refine the medium during definitive endodermal differentiation. We demonstrated that dialysis culture prevented cell damage to remove lactate. The hiPSCs cultured with dialysis also differentiated similarly as usual differentiation without dialysis even if they were not supplied Activin A for latter culture days in the differentiation. With this dialysis culture system, hiPSCs were differentiated into endodermal lineage with medium refinement and recycling and autocrine factors as well as cytokines, which may lead to reduce differentiation cost.
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http://dx.doi.org/10.1016/j.reth.2019.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6933453PMC
December 2019

Metabolomic profiling during the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

Differentiation 2020 Mar - Apr;112:17-26. Epub 2019 Dec 10.

CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan. Electronic address:

Human induced pluripotent stem cells (hiPSCs) are potentially an invaluable source of cells for regenerative medicine, disease modeling and drug discovery. However, the differentiation of hiPSCs into fully functional hepatocytes remains a major challenge. Despite the importance of the information carried by metabolomes, the exploitation of metabolomics for characterizing and understanding hiPSC differentiation remains largely unexplored. Here, to increase knowledge of hiPSC maturation into mature hepatocytes, we investigated their metabolomics profiles during sequential step-by-step differentiation: definitive endoderm (DE), specification into hepatocytes (HB-pro (hepatoblast progenitors)), progenitor hepatocytes (Pro-HEP) and mature hepatocyte-like cells (HLCs). Metabolomics analysis illustrated a switch from glycolysis-based respiration in DE step to oxidative phosphorylation in HLCs step. DE was characterized by fatty acid beta oxidation, sorbitol metabolism and pentose phosphate pathway, and glutamine and glucose metabolisms as various potential energy sources. The complex lipid metabolism switch was monitored via the reduction of lipid production from DE to HLCs step, whereas high glycerol production occurred mainly in HLCs. The nitrogen cycle, via urea production, was also a typical mechanism revealed in HLCs step. Our analysis may contribute to better understanding of differentiation and suggest new targets for improving iPSC maturation into functional hepatocytes.
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http://dx.doi.org/10.1016/j.diff.2019.10.006DOI Listing
December 2019

Analysis of the transcription factors and their regulatory roles during a step-by-step differentiation of induced pluripotent stem cells into hepatocyte-like cells.

Mol Omics 2019 12;15(6):383-398

CNRS UMI 2820, Laboratory for Integrated Micro Mechatronic Systems, Institute of Industrial Science, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan.

We investigated the human induced pluripotent stem cells (hiPSCs) during a sequential in vitro step-by-step differentiation into hepatocyte-like cells (HLCs) using nanoCAGE, a method for promoters, transcription factors, and transcriptome analysis. Specific gene clusters reflected the different steps of the hepatic differentiation. The proliferation step was characterized by a typical cell cycle and DNA replication. The hepatic endoderm and the HLC steps were marked by a common signature including cell interactions with extracellular matrix (ECM), lipoproteins and hepatic biomarkers (such as albumin and alpha-fetoprotein). The specific HLC profile was characterized by important transcription factors such as HIF1A, JUN, MAF, KLF6, BMP4 and with a larger expression of genes related to Wnt signaling, extracellular matrix, lipid metabolism, urea cycle, drugs, and solute transporters. HLC profile was also characterized by the activation of upstream regulators such as HNF1A, MEIS2, NFIX, WRNIP1, SP4, TAL1. Their regulatory networks highlighted HNF4a as a bridge and linked them to important processes such as EMT-MET transitions, ECM remodeling and liver development pathways (HNF3, PPARA signaling, iron metabolism) along the different steps of differentiation.
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http://dx.doi.org/10.1039/c9mo00122kDOI Listing
December 2019

Filling a gYap in Hepato-Biliary Tissue Integration in Liver Homeostasis and Regeneration.

Cell Stem Cell 2019 07;25(1):5-6

Laboratory of Stem Cell Therapy, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan. Electronic address:

Liver bile ducts serve primarily as drainage for bile and undergo extensive remodeling in response to hepatocyte injuries. In this issue of Cell Stem Cell, Pepe-Mooney et al. (2019) and Planas-Paz et al. (2019) show that Yap signaling can be activated by bile acids and is critical for biliary tissue homeostasis and dynamics.
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http://dx.doi.org/10.1016/j.stem.2019.06.006DOI Listing
July 2019

Hepatic ferroptosis plays an important role as the trigger for initiating inflammation in nonalcoholic steatohepatitis.

Cell Death Dis 2019 06 18;10(6):449. Epub 2019 Jun 18.

Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.

Nonalcoholic steatohepatitis (NASH) is a metabolic liver disease that progresses from simple steatosis to the disease state of inflammation and fibrosis. Previous studies suggest that apoptosis and necroptosis may contribute to the pathogenesis of NASH, based on several murine models. However, the mechanisms underlying the transition of simple steatosis to steatohepatitis remain unclear, because it is difficult to identify when and where such cell deaths begin to occur in the pathophysiological process of NASH. In the present study, our aim is to investigate which type of cell death plays a role as the trigger for initiating inflammation in fatty liver. By establishing a simple method of discriminating between apoptosis and necrosis in the liver, we found that necrosis occurred prior to apoptosis at the onset of steatohepatitis in the choline-deficient, ethionine-supplemented (CDE) diet model. To further investigate what type of necrosis is involved in the initial necrotic cell death, we examined the effect of necroptosis and ferroptosis inhibition by administering inhibitors to wild-type mice in the CDE diet model. In addition, necroptosis was evaluated using mixed lineage kinase domain-like protein (MLKL) knockout mice, which is lacking in a terminal executor of necroptosis. Consequently, necroptosis inhibition failed to block the onset of necrotic cell death, while ferroptosis inhibition protected hepatocytes from necrotic death almost completely, and suppressed the subsequent infiltration of immune cells and inflammatory reaction. Furthermore, the amount of oxidized phosphatidylethanolamine, which is involved in ferroptosis pathway, was increased in the liver sample of the CDE diet-fed mice. These findings suggest that hepatic ferroptosis plays an important role as the trigger for initiating inflammation in steatohepatitis and may be a therapeutic target for preventing the onset of steatohepatitis.
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http://dx.doi.org/10.1038/s41419-019-1678-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579767PMC
June 2019

Optimized protocol for the hepatic differentiation of induced pluripotent stem cells in a fluidic microenvironment.

Biotechnol Bioeng 2019 07 29;116(7):1762-1776. Epub 2019 Mar 29.

Laboratory for Integrated Micro Mechatronic Systems, CNRS UMI 2820, Institute of Industrial Science, The University of Tokyo, Tokyo, Japan.

In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for complete and partial on-chip differentiation were tested. Unlike full on-chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin-1 or albumin, while no stabilin-1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 10 cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha ( ESRRA), hepatic nuclear factor 1 ( HNF1A), hepatic nuclear factor 4 ( HNF4A), transcription factor 4 ( TCF4), and CCAAT enhancer binding protein alpha ( CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the ɑ-fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.
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http://dx.doi.org/10.1002/bit.26970DOI Listing
July 2019

Generation of mesothelial progenitor-like cells from mouse-induced pluripotent stem cells.

FEBS Lett 2019 02 18;593(4):386-394. Epub 2019 Jan 18.

Institute for Quantitative Biosciences, The University of Tokyo, Japan.

Mesothelial cells, which cover the surface of visceral organs and serous cavities in mammals, play a crucial role in preventing adhesion. We previously reported that primary mesothelial progenitor cells (MPCs) can not only prevent postoperative adhesion but also promote liver regeneration after hepatectomy. Induced pluripotent stem cells (iPSCs) have the potential to be used for regenerative medicine. Here, we have established a differentiation protocol for mouse iPSC-derived MPCs (miMPCs) via the exposure to defined factors, as well as purification using MPC-specific cell surface antigens. Furthermore, the miMPCs had the ability to suppress postoperative adhesion and facilitate liver regeneration. This is the first report highlighting the generation of functional miMPCs, which may offer potential for de novo cell therapy.
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http://dx.doi.org/10.1002/1873-3468.13325DOI Listing
February 2019

Nrf2 Activation Ameliorates Hepatotoxicity Induced by a Heme Synthesis Inhibitor.

Toxicol Sci 2019 01;167(1):227-238

Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba, Sendai 980-8575, Japan.

Transcription factor Nrf2 protects hepatocytes against various toxicants by upregulating cytoprotective genes. The heme synthesis inhibitor 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) leads to liver injury around the portal vein, unlike other groups of toxicants that cause hemorrhage and necrosis in the centrilobular area. To examine whether and how Nrf2 protects livers from the injury, we fed DDC to Nrf2 knockout (Nrf2KO), wild-type (WT), Keap1flox/flox (Keap1-knockdown; Keap1KD), and liver-specific Keap1 knockout (Keap1-Alb) mice, as these lines of mice exhibit stepwise increases in Nrf2 protein expression levels. Liver-specific Keap1::Nrf2 double-knockout (Keap1::Nrf2-Alb) mice were also exploited to examine the contribution of Nrf2. Two weeks after DDC feeding, Keap1-Alb mice were fully recovered from body weight loss, but the WT and Nrf2KO mice were not. The liver-to-body-weight ratio of Keap1-Alb mice was significantly larger than that of WT and Nrf2KO mice. Two indicators of hepatotoxicity, alanine aminotransferase and bilirubin in plasma, were both elevated in WT mice, but downregulated in Keap1-Alb mice after the DDC-feeding. DDC-induced porphyrin accumulation was reduced in the livers of Keap1-Alb and Keap1KD mice compared with that of WT mice. When assessed by the Nqo1 level, Nrf2 expression was further enhanced by DDC in Keap1-Alb mice, suggesting that DDC may have a Keap1 independent potential to activate Nrf2. Genetic activation of Nrf2 in Keap1-Alb mice increased the extracellular excretion of porphyrins, but contrary to our expectation, hepatic damages in Nrf2KO mice appeared to be similar to that of WT mice. Based on these observations, we conclude that Nrf2 activation protects livers against DDC-elicited hepatotoxicity.
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http://dx.doi.org/10.1093/toxsci/kfy233DOI Listing
January 2019

Enhanced Hepatic Differentiation of Human Induced Pluripotent Stem Cells Using Gas-Permeable Membrane.

Tissue Eng Part A 2019 03 12;25(5-6):457-467. Epub 2018 Dec 12.

1 Department of Bioengineering and School of Engineering, University of Tokyo, Tokyo, Japan.

Impact Statement: Although oxygen is a vital nutrient for the hepatocytes , few reports have focused on its effect during hepatic differentiation of induced pluripotent stem cells (iPSCs). In this report, we performed the hepatic differentiation of human iPSCs (hiPSCs) under different atmospheric oxygen concentrations and oxygen supply fluxes to investigate the effects of oxygen in terms of both the concentration and the supply flux. Results demonstrate that direct oxygenation through a polydimethylsiloxane (PDMS) membrane enhances the maturation and efficient production of hiPSC-derived hepatocyte-like cells (iHeps). Thus, direct oxygenation through a PDMS membrane is a better alternative culture method over conventional tissue culture-treated polystyrene (TCPS) plates for the maturation of hiPSC-derived hepatocytes .
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http://dx.doi.org/10.1089/ten.TEA.2018.0084DOI Listing
March 2019

Characterization of Peribiliary Gland-Constituting Cells Based on Differential Expression of Trophoblast Cell Surface Protein 2 in Biliary Tract.

Am J Pathol 2018 09 3;188(9):2059-2073. Epub 2018 Jul 3.

Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan; Laboratory of Stem Cell Regulation, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan. Electronic address:

Peribiliary glands (PBGs) are accessory glands with mucinous and serous acini in the biliary tree. The PBG is composed of a heterogeneous cell population, such as mucus- and pancreatic enzyme-producing epithelial cells, whereas it constitutes niches for multipotential stem/progenitor cells in the human extrahepatic bile duct (EHBD). By contrast, the nature of PBGs in the mouse EHBD remains unclear. Our aim was to establish a method for isolating and characterizing PBG-constituting cells in the mouse EHBD. We found that trophoblast cell surface protein 2 (Trop2) was expressed in the luminal epithelium of mouse EHBD exclusively, but not in the PBG. On the basis of the differential expression profile of Trop2, lumen-forming biliary epithelial cells (LBECs) and PBG-constituting epithelial cells (PBECs) were separately isolated for further characterization. Gene expression analysis revealed that the isolated mouse PBECs expressed several marker genes related to human PBGs. In the colony formation assay, PBECs showed significantly higher colony formation capacity than LBECs. In the organoid formation assay, PBECs formed cystic organoid with LBEC-like phenotype. Interestingly, PBECs proliferated, accompanied by reexpression of Trop2 in vivo after bile duct ligation. Furthermore, the unique expression profile of Trop2 was conserved in human EHBD. Our findings indicate that Trop2 is a useful marker in investigating the pathophysiological roles and characteristics of mouse and human PBGs in biliary diseases.
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http://dx.doi.org/10.1016/j.ajpath.2018.05.016DOI Listing
September 2018

Differential expression of Lutheran/BCAM regulates biliary tissue remodeling in ductular reaction during liver regeneration.

Elife 2018 07 30;7. Epub 2018 Jul 30.

Department of Regenerative Medicine, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan.

Under chronic or severe liver injury, liver progenitor cells (LPCs) of biliary origin are known to expand and contribute to the regeneration of hepatocytes and cholangiocytes. This regeneration process is called ductular reaction (DR), which is accompanied by dynamic remodeling of biliary tissue. Although the DR shows apparently distinct mode of biliary extension depending on the type of liver injury, the key regulatory mechanism remains poorly understood. Here, we show that Lutheran (Lu)/Basal cell adhesion molecule (BCAM) regulates the morphogenesis of DR depending on liver disease models. Lu and Lu biliary cells isolated from injured liver exhibit opposite phenotypes in cell motility and duct formation capacities in vitro. By overexpression of Lu, Lu biliary cells acquire the phenotype of Lu biliary cells. Lu-deficient mice showed severe defects in DR. Our findings reveal a critical role of Lu in the control of phenotypic heterogeneity of DR in distinct liver disease models.
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http://dx.doi.org/10.7554/eLife.36572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107333PMC
July 2018

Efficient functional cyst formation of biliary epithelial cells using microwells for potential bile duct organisation in vitro.

Sci Rep 2018 07 23;8(1):11086. Epub 2018 Jul 23.

Center for International Research on Integrative Biomedical Systems (CIBiS), Institute of Industrial Science (IIS), The University of Tokyo, Tokyo, Japan.

Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 µm-size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-based assays.
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http://dx.doi.org/10.1038/s41598-018-29464-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056467PMC
July 2018

Neutrophils alleviate fibrosis in the CCl-induced mouse chronic liver injury model.

Hepatol Commun 2018 Jun 12;2(6):703-717. Epub 2018 Apr 12.

Institute of Molecular and Cellular Biosciences University of Tokyo Tokyo Japan.

Tribbles pseudokinase 1 () is a negative regulator of CCAAT/enhancer binding protein α (C/EBPα) and is known to induce granulopoiesis while suppressing monocyte differentiation. Loss of was previously shown to increase the neutrophil population in the spleen but lead to M2-like macrophage reduction. Because M2 macrophages are anti-inflammatory and promote tissue repair by producing fibrogenic factors, we investigated liver fibrosis in -deficient mice. Interestingly, loss of suppressed fibrosis in the CCl-induced chronic liver injury model. knockout increased neutrophils but had a minimal effect on the macrophage population in the liver. Hepatic expressions of neutrophil matrix metalloproteinases () and were increased, but the production of fibrogenic factors, including transforming growth factor β1, was not affected by loss of . These results suggest that neutrophils are responsible for the suppression of fibrosis in -deficient liver. Consistently, transplantation of -deficient bone marrow cells into wild-type mice alleviated CCl-induced fibrosis. Furthermore, expression of chemokine (C-X-C motif) ligand 1 () by adeno-associated viral vector in the normal liver recruited neutrophils and suppressed CCl-induced fibrosis; infusion of wild-type neutrophils in CCl-treated mice also ameliorated fibrosis. Using recombinant adeno-associated virus-mediated expression of and alleviated liver fibrosis. Finally, neutrophil depletion by infusion of Ly6G antibody significantly enhanced CCl-induced fibrosis. : While neutrophils are well known to exacerbate acute liver injury, our results demonstrate a beneficial role of neutrophils in chronic liver injury by promoting fibrolysis. ( 2018;2:703-717).
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http://dx.doi.org/10.1002/hep4.1178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983199PMC
June 2018

The transcription factor Klf5 is essential for intrahepatic biliary epithelial tissue remodeling after cholestatic liver injury.

J Biol Chem 2018 04 9;293(17):6214-6229. Epub 2018 Mar 9.

From the Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032 and

Under various conditions of liver injury, the intrahepatic biliary epithelium undergoes dynamic tissue expansion and remodeling, a process known as ductular reaction. Mouse models defective in inducing such a tissue-remodeling process are more susceptible to liver injury, suggesting a crucial role of this process in liver regeneration. However, the molecular mechanisms regulating the biliary epithelial cell (BEC) dynamics in the ductular reaction remain largely unclear. Here, we demonstrate that the transcription factor Krüppel-like factor 5 (Klf5) is highly enriched in mouse liver BECs and plays a key role in regulating the ductular reaction, specifically under cholestatic injury conditions. Although mice lacking Klf5 in the entire liver epithelium, including both hepatocytes and BECs (Klf5-LKO (liver epithelial-specific knockout) mice), did not exhibit any apparent phenotype in the hepatobiliary system under normal conditions, they exhibited significant defects in biliary epithelial tissue remodeling upon 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholangitis, concomitantly with exacerbated cholestasis and reduced survival rate. In contrast, mice lacking Klf5 solely in hepatocytes did not exhibit any such phenotypes, confirming Klf5's specific role in BECs. RNA-sequencing analyses of BECs isolated from the Klf5-LKO mouse livers revealed that the Klf5 deficiency primarily affected expression of cell cycle-related genes. Moreover, immunostaining analysis with the proliferation marker Ki67 disclosed that the Klf5-LKO mice had significantly reduced BEC proliferation levels upon injury. These results indicate that Klf5 plays a critical role in the ductular reaction and biliary epithelial tissue expansion and remodeling by inducing BEC proliferation and thereby contributing to liver regeneration.
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http://dx.doi.org/10.1074/jbc.RA118.002372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5925809PMC
April 2018

Portal fibroblasts marked by the surface antigen Thy1 contribute to fibrosis in mouse models of cholestatic liver injury.

Hepatol Commun 2017 05 22;1(3):198-214. Epub 2017 Mar 22.

Laboratory of Cell Growth and Differentiation Institute of Molecular and Cellular Biosciences, University of Tokyo Tokyo Japan.

Liver fibrosis, a condition that is characterized by excessive production and accumulation of extracellular matrix, including collagen, is the most common outcome of chronic liver injuries of different etiologies. Vitamin A-storing hepatic stellate cells (HSCs) are considered to be the main source of this collagen production, with activation in response to liver injury. In contrast, the contribution of other cell types to this fibrogenic response remains largely elusive due to the lack of specific surface markers to identify and isolate these cells for detailed analysis. Here, we identify a mesenchymal population of thymus cell antigen 1 (Thy1) CD45 cells (Thy1 MCs) in the mouse liver; these cells reside near the portal vein and indicate profibrogenic characteristics , shown by their expression of collagen and α-smooth muscle actin. Flow cytometric analysis of mouse liver nonparenchymal cells revealed that vitamin A storage and Thy1 expression were mutually exclusive, indicating that Thy1 MCs are distinct from HSCs. Importantly, Thy1 MCs reacted and contributed to the development of liver fibrosis specifically in mouse models of cholestatic liver injury. With the occurrence of cholestatic liver injury, collagen-producing Thy1 MCs expanded in cell number and inhibited collagen degradation through up-regulation of matrix metalloproteinase inhibitor expression, thereby promoting the accumulation of extracellular matrix in the periportal area. : This study establishes Thy1 as a useful cell surface marker to prospectively identify and isolate periportal fibroblasts and further highlights a significant contribution of these cells to the pathogenesis of liver fibrosis caused by cholestatic liver injuries. We suggest that Thy1 MCs may be an interesting therapeutic target for treating liver fibrosis in addition to the well-characterized HSCs. ( 2017;1:198-214).
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http://dx.doi.org/10.1002/hep4.1023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721447PMC
May 2017

Identification of the novel 3' UTR sequences of human IL-21 mRNA as potential targets of miRNAs.

Sci Rep 2017 08 10;7(1):7780. Epub 2017 Aug 10.

Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma worldwide. However, the strategy of HBV to escape from the host immune system remains largely unknown. In this study, we examined extracellular vesicles (EVs) secreted from human hepatocytes infected with HBV. EVs includeing exosomes are nano-size vesicles with proteins, mRNAs, and microRNAs (miRNAs), which can be transmitted to different cells. We found that 104 EV associated miRNAs were increased in hepatocytes more than 2-fold by HBV infection. We then selected those that were potentially implicated in immune regulation. Among them, five HBV-induced miRNAs were found to potentially target multiple sequences in the 3'UTR of IL-21, a cytokine that induces anti-viral immunity. Moreover, expression of a reporter gene with the 3' UTR of human IL-21 mRNA was suppressed by the five miRNAs individually. Finally, IL-21 expression in cloned human T cells was down-regulated by the five miRNAs. Collectively, this study identified the novel 3' UTR sequences of human IL-21 mRNA and potential binding sites of HBV-induced EV-miRNAs.
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http://dx.doi.org/10.1038/s41598-017-07853-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5552845PMC
August 2017