Publications by authors named "Taketomo Kido"

27 Publications

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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

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

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

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

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

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

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

Spatiotemporal Expression of Three Secretoglobin Proteins, SCGB1A1, SCGB3A1, and SCGB3A2, in Mouse Airway Epithelia.

J Histochem Cytochem 2019 06 15;67(6):453-463. Epub 2019 Feb 15.

Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Secretoglobins (SCGBs) are cytokine-like small molecular weight secreted proteins with largely unknown biological functions. Three SCGB proteins, SCGB1A1, SCGB3A1, and SCGB3A2, are predominantly expressed in lung airways. To gain insight into the possible functional relationships among the SCGBs, their protein and mRNA expression patterns were examined in lungs during gestation and in adult mice, using Scgb3a1-null and Scgb3a2-null mice as negative controls, by immunohistochemistry and by qRT-PCR analysis, respectively. The three SCGBs exhibited unique spatiotemporal expression patterns during embryogenesis. The lack of Scgb3a1 or Scgb3a2 did not affect expression of the other Scgb genes as determined by mRNA measurements. Moreover, the lack of Scgb3a1 or Scgb3a2 did not affect development of the pulmonary neuroepithelial bodies during embryogenesis, while the lack of Scgb3a2 may have resulted in slightly fewer ciliated cells than in the wild-type. These results suggest that SCGB1A1, SCGB3A1, and SCGB3A2 each may possess its own unique biological function.
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http://dx.doi.org/10.1369/0022155419829050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542149PMC
June 2019

Induction of Functional Hepatocytes from Human iPSCs.

Methods Mol Biol 2019 ;1905:131-142

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

Human-induced pluripotent stem cells (iPSCs) could be a useful source for production of hepatocytes. Here, we develop protocols to generate iPSC-derived liver progenitor cells, liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs). We also establish long-term two-dimensional co-culture system to induce functional hepatocytes from iPSC-derived liver cells.
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http://dx.doi.org/10.1007/978-1-4939-8961-4_12DOI Listing
June 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

An In Vitro Human Liver Model by iPSC-Derived Parenchymal and Non-parenchymal Cells.

Stem Cell Reports 2017 08 27;9(2):490-498. Epub 2017 Jul 27.

Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan. Electronic address:

During liver development, hepatoblasts and liver non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) constitute the liver bud where they proliferate and differentiate. Accordingly, we reasoned that liver NPCs would support the maturation of hepatocytes derived from human induced pluripotent stem cells (hiPSCs), which usually exhibit limited functions. We found that the transforming growth factor β and Rho signaling pathways, respectively, regulated the proliferation and maturation of LSEC and HSC progenitors isolated from mouse fetal livers. Based on these results, we have established culture systems to generate LSECs and HSCs from hiPSCs. These hiPSC-derived NPCs exhibited distinctive phenotypes and promoted self-renewal of hiPSC-derived liver progenitor cells (LPCs) over the long term in the two-dimensional culture system without exogenous cytokines and hepatic maturation of hiPSC-derived LPCs. Thus, a functional human liver model can be constructed in vitro from the LPCs, LSECs, and HSCs derived from hiPSCs.
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http://dx.doi.org/10.1016/j.stemcr.2017.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549957PMC
August 2017

Comparison of the transcriptomic profile of hepatic human induced pluripotent stem like cells cultured in plates and in a 3D microscale dynamic environment.

Genomics 2017 01 29;109(1):16-26. Epub 2016 Nov 29.

CIBIS, Institute of Industrial Science, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8505, Japan; 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 have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore, the results of the transcriptomic profile, coupled with immunostaining, and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells, hepatocytes like cells, and endothelial like cells. However, the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless, the biochip culture provided a specific micro-environment in which a complex multicellular differentiation toward liver could be oriented.
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http://dx.doi.org/10.1016/j.ygeno.2016.11.008DOI Listing
January 2017

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

Stem Cell Reports 2015 Oct 10;5(4):508-15. Epub 2015 Sep 10.

Laboratory of Cell Growth and Differentiation, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan. Electronic address:

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.
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http://dx.doi.org/10.1016/j.stemcr.2015.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624956PMC
October 2015

Transgenically-expressed secretoglobin 3A2 accelerates resolution of bleomycin-induced pulmonary fibrosis in mice.

BMC Pulm Med 2015 Jul 16;15:72. Epub 2015 Jul 16.

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Background: Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated in vivo. The aim of this study was to address these questions in mice.

Methods: A transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis.

Results: Adult Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs.

Conclusions: These results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.
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http://dx.doi.org/10.1186/s12890-015-0065-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4504078PMC
July 2015

Secretoglobin superfamily protein SCGB3A2 deficiency potentiates ovalbumin-induced allergic pulmonary inflammation.

Mediators Inflamm 2014 27;2014:216465. Epub 2014 Aug 27.

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Building 37, Room 3106, NIH, 9000 Rockville Pike, Bethesda, MD 20892, USA.

Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, which may play a role in lung inflammation, is predominantly expressed in airway epithelial cells. In order to understand the physiological role of SCGB3A2, Scgb3a2(-/-) mice were generated and characterized. Scgb3a2(-/-) mice did not exhibit any overt phenotypes. In ovalbumin- (OVA-) induced airway allergy inflammation model, Scgb3a2(-/-) mice in mixed background showed a decreased OVA-induced airway inflammation, while six times C57BL/6NCr backcrossed congenic Scgb3a2(-/-) mice showed a slight exacerbation of OVA-induced airway inflammation as compared to wild-type littermates. These results indicate that the loss of SCGB3A2 function was influenced by a modifier gene(s) in mixed genetic background and suggest that SCGB3A2 has anti-inflammatory property. The results further suggest the possible use of recombinant human SCGB3A2 as an anti-inflammatory agent.
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http://dx.doi.org/10.1155/2014/216465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163287PMC
June 2015

Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse.

Mol Cancer 2012 Dec 12;11:90. Epub 2012 Dec 12.

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

Background: The CCAAT/enhancer binding proteins (C/EBPs) play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known.

Methods: A transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP) gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis bioassay in the presence and absence of doxycycline, and the effect of abolition of DNA binding activities of C/EBPs on lung carcinogenesis was examined.

Results: A-C/EBP expression was found not to interfere with tumor development; however, it suppressed the malignant conversion of adenoma to carcinoma during NNK-induced lung carcinogenesis. The results suggested that Ki67 may be used as a marker for lung carcinomas in mouse.

Conclusions: The DNA binding of C/EBP family members can be used as a potential molecular target for lung cancer therapy.
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http://dx.doi.org/10.1186/1476-4598-11-90DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548712PMC
December 2012

Inhibition of Stabilin-2 elevates circulating hyaluronic acid levels and prevents tumor metastasis.

Proc Natl Acad Sci U S A 2012 Mar 27;109(11):4263-8. Epub 2012 Feb 27.

Laboratory of Cell Growth and Differentiation and Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.

Hyaluronic acid (HA) has been implicated in the proliferation and metastasis of tumor cells. However, most previous studies were conducted on extracellular matrix or pericellular HA, and the role of circulating HA in vivo has not been studied. HA is rapidly cleared from the bloodstream. The scavenger receptor Stabilin-2 (Stab2) is considered a major clearance receptor for HA. Here we report a dramatic elevation in circulating HA levels in Stab2-deficient mice without any overt phenotype. Surprisingly, the metastasis of B16F10 melanoma cells to the lungs was markedly suppressed in the Stab2-deficient mice, whereas cell proliferation was not affected. Furthermore, administration of an anti-Stab2 antibody in Stab2(+) mice elevated serum HA levels and prevented the metastasis of melanoma to the lung, and also suppressed spontaneous metastasis of mammary tumor and human breast tumor cells inoculated in the mammary gland. Administration of the antibody or high-dose HA in mice blocked the lodging of melanoma cells to the lungs. Furthermore, HA at high concentrations inhibited the rolling/tethering of B16 cells to lung endothelial cells. These results suggest that blocking Stab2 function prevents tumor metastasis by elevating circulating HA levels. Stab2 may be a potential target in antitumor therapy.
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http://dx.doi.org/10.1073/pnas.1117560109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306694PMC
March 2012

FOXA1 plays a role in regulating secretoglobin 1a1 expression in the absence of CCAAT/enhancer binding protein activities in lung in vivo.

Am J Physiol Lung Cell Mol Physiol 2011 Mar 17;300(3):L441-52. Epub 2010 Dec 17.

Laboratory or Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

Secretoglobin (SCGB) 1A1, also called Clara cell secretor protein (CCSP) or Clara cell-specific 10-kDa protein (CC10), is a small molecular weight secreted protein mainly expressed in lung, with anti-inflammatory/immunomodulatory properties. Previous in vitro studies demonstrated that CCAAT/enhancer-binding proteins (C/EBPs) are the major transcription factors for the regulation of Scbg1a1 gene expression, whereas FOXA1 had a minimum effect on the transcription. To determine the in vivo role of C/EBPs in the regulation of SCGB1A1 expression, experiments were performed in which A-C/EBP, a dominant-negative form of C/EBP that interferes with DNA binding activities of all C/EBPs, was specifically expressed in lung. Surprisingly, despite the in vitro findings, expression of SCGB1A1 mRNA was not decreased in vivo in the absence of C/EBPs. This may be due to a compensatory role assumed by FOXA1 in the regulation of Scgb1a1 gene expression in lung in the absence of active C/EBPs. This disconnect between in vitro and in vivo results underscores the importance of studies using animal models to determine the role of specific transcription factors in the regulation of gene expression in intact multicellular complex organs such as lung.
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http://dx.doi.org/10.1152/ajplung.00435.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064285PMC
March 2011

Oncostatin M regulates secretoglobin 3A1 and 3A2 expression in a bidirectional manner.

Am J Respir Cell Mol Biol 2009 May 31;40(5):620-30. Epub 2008 Oct 31.

Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Secretoglobin (SCGB) 3A1 and 3A2 are members of the small molecular weight secretoglobin gene superfamily. SCGB3A1 is a tumor suppressor gene, whereas SCGB3A2 has anti-inflammatory properties. Both genes are mainly expressed in the lung and trachea in mice. Whether the expression and/or function of these two genes are related is not known. Here we show that the expression of SCGB3A1 and SCGB3A2 are bidirectionally regulated by oncostatin M (OSM) when examined in a mouse transformed Clara cell line (mtCC); SCGB3A1 is up-regulated by OSM, while SCGB3A2 is down-regulated in a time- and dose-dependent manner. OSM-activated STAT3/5, through binding to the STAT-binding element located at -201 to -209 bp in the mouse Scgb3a1 gene promoter, and the extracellular signal-regulated kinase (ERK)- and p38-mitogen-activated protein kinase (MAPK) pathways are responsible for the OSM-induced up-regulation of SCGB3A1 expression. On the other hand, the -113 to -273 bp region in the Scgb3a2 promoter appears to be responsible for the OSM induced down-regulation of the gene. No significant differences in the levels or patterns of specific DNA-binding proteins were found in the -113 to -273 bp region as determined by electrophoretic mobility shift assays. Neither the ERK- nor p38-MAPK pathways were involved in the OSM-induced reduction of Scgb3a2 promoter activity. These results suggest that OSM-induced suppression of SCGB3A2 expression is an indirect effect of OSM. Expression of the Clara cell marker, CYP2F2, was markedly decreased upon OSM treatment in parallel with the decrease of SCGB3A2 expression in mtCC cells. The differential regulation of Scgb3a1 and Scgb3a2 gene expression by OSM may explain the unique functions of these genes in the lung.
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http://dx.doi.org/10.1165/rcmb.2008-0062OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677441PMC
May 2009

CAATT/enhancer-binding proteins alpha and delta interact with NKX2-1 to synergistically activate mouse secretoglobin 3A2 gene expression.

J Biol Chem 2008 Sep 16;283(37):25617-25627. Epub 2008 Jul 16.

Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892. Electronic address:

Secretoglobin (SCGB) 3A2 is a small molecular weight secreted protein predominantly expressed in lung airways. We previously demonstrated that the expression of SCGB3A2 is regulated by homeodomain transcription factor NKX2-1. Here we show that CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPdelta, regulate mouse Scgb3a2 gene transcription in vivo and in vitro by binding to specific sites located in the Scgb3a2 promoter and the activity is synergistically enhanced through cooperative interaction with NKX2-1. Six C/EBP binding sites lie within 500 bp of the Scgb3a2 gene promoter, of which two sites, located at -44 to -54 bp and -192 to -201 bp, appear to be critical for the synergistic activation of Scgb3a2 gene transcription with NKX2-1. All three transcription factors, C/EBPalpha, C/EBPdelta, and NKX2-1, are expressed in the epithelial cells of airways, particularly the bronchus, where high expression of SCGB3A2 is found. The expression of these transcription factors markedly increases toward the end of gestation, which coincides with the marked increase of SCGB3A2, suggesting the importance of C/EBPalpha and C/EBPdelta, and their synergistic interaction with NKX2-1 in mouse Scgb3a2 gene transcription and lung development.
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http://dx.doi.org/10.1074/jbc.M805140200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2533088PMC
September 2008

Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally established porcine intestinal epitheliocyte cell line.

Biochim Biophys Acta 2008 Feb 22;1780(2):134-44. Epub 2007 Nov 22.

Food Immunology Group, Laboratory of Animal Products Chemistry, Graduate School of Agricultural Science, Tohoku University, Aobaku, Sendai 981-8555, Japan.

To study the immune responses of porcine intestinal epithelial cells to gram-negative bacteria via toll-like receptors (TLRs), originally established porcine intestinal epitheliocyte (PIE) cells were treated with lipopolysaccharide (LPS) or swine-specific enterotoxigenic Escherichia coli (ETEC). Real-time quantitative PCR revealed that PIE cells expressed TLR1-9 and MD-2 mRNAs, preferentially expressed TLR4/MD-2. Immunostaining of PIE cells revealed that TLR4 was precisely expressed in PIE cells at the protein level. PIE cells treated with LPS had up-regulated expression of several TLRs (TLR2, 3, 4, 5 and 8), type 1 helper T (Th1) cytokines (interleukin (IL)-1alpha, IL-1beta, IL-6, IL-15, 18, leukemia inhibitory factor (LIF), and interferon (IFN)-beta), and chemokines (monocyte chemoattractant protein (MCP)-1 and IL-8). ETEC enhanced the expression of TLR2, Th1 type cytokines (IL-1alpha, IL-12p35 and IL-6) and chemokines (MCP-1 and IL-8). These results indicate that PIE induces inflammatory responses by up-regulating Th1 cytokines and chemokines in response to LPS or ETEC, suggesting that PIE is a useful cell line for studying inflammatory responses via TLR4/MD-2 in intestinal epithelial cells.
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http://dx.doi.org/10.1016/j.bbagen.2007.11.006DOI Listing
February 2008

Staining patterns for actin and villin distinguish M cells in bovine follicle-associated epithelium.

Res Vet Sci 2007 Apr 1;82(2):141-9. Epub 2006 Sep 1.

Laboratory of Functional Morphology, Tohoku University, 1-1 Tsutsumidori, Amamiyamachi, Aoba-ku, 981-8555 Sendai, Japan.

M cells play a central role in the initiation of mucosal immune responses. However, a primary source of difficulty for investigations of this is the lack of an available specific marker for bovine M cells. As M cells possess irregular and short microvilli, we investigated the distribution and localization of the microvillar proteins actin and villin by immunohistochemistry of the gut of calves. In ileum of the calf, actin and villin were clearly and continuously immunostained in the brush border of the villous epithelia, however, discontinuous immunostaining with patches of no staining were observed in follicle-associated epithelium (FAE). Electron microscopy revealed that M cells had irregular microvilli and lacked the typical brush border, and it was inferred that these patches of no staining might be the intercellular crevices of M cells. As the microvilli of M cells were very sparse, there were several areas of weak immunostaining in calf jejunal FAE. These results suggest that M cells in calf FAE are detectable by the absence of staining for actin and villin.
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http://dx.doi.org/10.1016/j.rvsc.2006.05.009DOI Listing
April 2007

Apoptotic process of porcine intestinal M cells.

Cell Tissue Res 2006 Mar 10;323(3):425-32. Epub 2005 Nov 10.

Laboratory of Functional Morphology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, 981-8555, Sendai, Japan.

Membranous (M) cells of the follicle-associated epithelium (FAE) are believed to sample antigens from the gut lumen. However, the origin, differentiation mechanism, and cell death of M cells are still a matter of controversy. Therefore, we investigated the process of M cell differentiation and determined their fate in the intestine of three-way crossbred female pigs. We used anti-cytokeratin 18 and anti-PCNA antibodies to distinguish M cells and proliferative cells and performed immunohistochemistry, enzyme histochemistry, and scanning electron microscopy on fresh ileal Peyer's patches. Cell migration and apoptotic cells were detected by BrdU labeling and the TUNEL method, respectively. The turnover of the FAE was similar to that of the villi. M cells were mostly observed from the FAE crypt to the FAE periphery, but not in the FAE apex. As proliferative M cells (cytokeratin 18(+)/PCNA(+) cells) have previously been detected in the FAE crypt, porcine M cells may be directly derived from intestinal epithelial stem cells and committed as a distinct cell lineage in the crypts. M cells from the FAE periphery were unstained or only weakly stained for alkaline phosphatase, whereas cytokeratin 18(+)/alkaline phosphatase(+) cells lying near to the FAE apex showed a columnar shape similar to that of adjacent enterocytes. These data suggest that the committed M cells differentiate to mature M cells by contact with lymphocytes at the FAE periphery, and that they trans-differentiate to enterocytes and are finally excluded near the FAE apex.
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http://dx.doi.org/10.1007/s00441-005-0086-zDOI Listing
March 2006

Identification of bovine dendritic cell phenotype from bovine peripheral blood.

Res Vet Sci 2006 Aug 25;81(1):40-5. Epub 2005 Oct 25.

Laboratory of Functional Morphology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, 981-8555 Sendai, Japan.

Dendritic cells (DCs) are professional antigen presenting cells, which initiate primary immune responses and also play an important role in the generation of peripheral tolerance. There is no reliable method established for the isolation of bovine peripheral blood DCs, and furthermore, the phenotypes and the functions of bovine DCs are still not fully clear. In the present study, we have attempted to identify bovine peripheral blood DCs by negative-selection. In bovine peripheral blood mononuclear cells (PBMC), we have newly characterized the phenotype of DCs, which is CD11c+/CD172a+. These cells display features of myeloid type DCs. In the thymic medulla, CD11c+/CD172a+ cells were also present and CD1+/CD172a+ cells were additionally detected as a population of DCs. The data suggest that one of the bovine DCs phenotypes from PBMC is derived from myeloid lineages lacking a CD1 molecule, which then drift to several tissues, and that they then may express a CD1 molecule upon their functional differentiation.
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http://dx.doi.org/10.1016/j.rvsc.2005.09.003DOI Listing
August 2006