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Hepatocyte Isolation

Takeshi Katsuda, Takahiro Ochiya
Bipotent liver progenitor cells (LPCs) are promising cell sources for cell transplantation therapy in hepatic disorders as well as biliary dysfunctions. Using a cocktail of small molecules, we recently reported a novel approach to generate bipotent LPCs, named chemically induced liver progenitors (CLiPs), from adult rat hepatocytes. In this chapter, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat hepatocytes and then describe how to induce CLiPs from the hepatocytes...
2019: Methods in Molecular Biology
Junichi Kino, Norihisa Ichinohe, Masayuki Ishii, Toshihiro Mitaka
This protocol showed procedures to isolate and expand small hepatocytes (SHs), as hepatocytic progenitor cells, from a rat liver. SHs are identified as a subpopulation of mature hepatocytes in a healthy liver. SHs can proliferate to form colonies in serum-free medium on hyaluronic acid-coated dishes, of which cells show CD44 positivity (CD44+ SHs). CD44+ SHs can be separated and purified from colonies by using anti-CD44 antibodies after enzymatic dissociation. CD44+ SHs can proliferate to form colonies on Engelbreth-Holm-Swarm gel (EHS-gel)-coated dishes in the serum-free medium for a long period and subculture for several times...
2019: Methods in Molecular Biology
Naoki Tanimizu
Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. The Notch, TGFβ, and Hippo pathways have been implicated in lineage determination of LPCs during development and regeneration. However, the molecular mechanisms governing the lineage specification have not been fully elucidated, yet. Epithelial adhesion molecule (EpCAM) is a marker of cholangiocytes and of LPCs. We found that EpCAM+ cells isolated from neonatal liver contain LPCs that clonally proliferate and are bipotential in vitro and in vivo...
2019: Methods in Molecular Biology
Wen-Chung Huang, Ya-Ling Chen, Hui-Chia Liu, Shu-Ju Wu, Chian-Jiun Liou
Ginkgolide C, isolated from Ginkgo biloba , is a diterpene lactone that has multiple biological functions and can improve Alzheimer disease and platelet aggregation. Ginkgolide C also inhibits adipogenesis in 3T3-L1 adipocytes. The present study evaluated whether ginkgolide C reduced lipid accumulation and regulated the molecular mechanism of lipogenesis in oleic acid-induced HepG2 hepatocytes. HepG2 cells were treated with 0.5 mM oleic acid for 48 h to induce a fatty liver cell model. Then, the cells were exposed to various concentrations of ginkgolide C for 24 h...
December 2018: Saudi Pharmaceutical Journal: SPJ: the Official Publication of the Saudi Pharmaceutical Society
Yue Wang, Jing-Lin Wang, Hu-Cheng Ma, Zhen-Ting Tang, Hao-Ran Ding, Xiao-Lei Shi
In recent years, transplantation of mesenchymal stem cells (MSCs) has attracted much attention as a potential cell-based therapy for acute liver failure (ALF). As an inducible enzyme, heme oxygenase 1 (HO-1) has been reported to have cytoprotective, anti-apoptotic and immunoregulatory effects. Autophagy, a conserved catabolic process in cells, may be an important pathway for MSCs to treat ALF. In this study, we aimed to explore whether MSCs treat ALF by regulating autophagy and whether HO-1 was involved in the same pathway...
December 5, 2018: Biochemical and Biophysical Research Communications
Guanliu Yu, Yawen Wang, Minmin Zhang, Yun Lin, Yi Tang, Youxiang Diao
Hydropericardium hepatitis syndrome (HHS) caused by group I fowl adenovirus serotype 4 (FAdV-4) is an acute and infectious disease in fowl, particularly in broilers aged 3-5 weeks. In June 2015, a highly pathogenic disease outbroke in 25-40 day-old ducklings in Shandong province, characterized similar symptom to HHS. In order to determine the pathogenic mechanism of FAdV-4 (SDSX strain) in meat ducks. We divided 90 25-day-old Cherry Valley meat ducks into three groups (oral, subcutaneous, and control; 30 ducks in each group) and infected them with the virus...
2018: Frontiers in Microbiology
Beom Jin Lim, Woon-Kyu Lee, Hyun Woong Lee, Kwan Sik Lee, Ja Kyung Kim, Hye Young Chang, Jung Il Lee
BACKGROUND: Platelet-derived growth factor receptor α (PDGFRα) expression is increased in activated hepatic stellate cells (HSCs) in cirrhotic liver, while normal hepatocytes express PDGFRα at a negligible level. However, cancerous hepatocytes may show upregulation of PDGFRα, and hepatocellular carcinoma is preceded by chronic liver injury. The role of PDGFRα in non-cancerous hepatocytes and liver fibrosis is unclear. We hypothesized that upon liver injury, PDGFRα in insulted hepatocytes contributes to liver fibrosis by facilitating intercellular crosstalk between hepatocytes and HSCs...
December 3, 2018: Cell Communication and Signaling: CCS
Rachana Haliyur, Xin Tong, May Sanyoura, Shristi Shrestha, Jill Lindner, Diane C Saunders, Radhika Aramandla, Greg Poffenberger, Sambra D Redick, Rita Bottino, Nripesh Prasad, Shawn E Levy, Raymond D Blind, David M Harlan, Louis H Philipson, Roland W Stein, Marcela Brissova, Alvin C Powers
Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment...
December 3, 2018: Journal of Clinical Investigation
Eman Al-Sayed, Mohamed M Abdel-Daim, Mohamed Abdelrazik Khattab
The hepatoprotective activity of praecoxin A, an ellagitannin from Melaleuca ericifolia, was determined against CCl4 -induced toxicity in mice. Praecoxin A was administered (25, 50, and 100 mg/kg) for 5 days followed by CCl4 . Praecoxin A markedly ameliorated the CCl4 -induced increase in AST (by 19, 52, and 56%), ALP (22, 45, and 48%), ALT (11, 47, and 54%), total bilirubin (14, 27, and 28%), and MDA (26, 44, and 51%) at the tested doses, respectively, as compared with CCl4 group. It was evident that praecoxin A significantly (p < 0...
December 3, 2018: Phytotherapy Research: PTR
Soo-Jin Kim, Kyeong-Ryoon Lee, Seiji Miyauchi, Yuichi Sugiyama
We investigated whether human serum albumin (HSA) would affect the uptake clearance of anionic drugs with high binding to HSA by suspended human hepatocytes and improve the extrapolation of in vivo hepatic clearance from in vitro uptake clearance by the hepatocytes via the "albumin-mediated" hepatic uptake mechanism. The uptake clearances for total forms ( PS inf ) and for unbound forms ( PS u,inf ) of 11 anionic drugs (all of which were organic anion-transporting polypeptide (OATP) substrates) were determined with suspended human hepatocytes in varying concentrations of HSA...
November 30, 2018: Drug Metabolism and Disposition: the Biological Fate of Chemicals
Lei Zhang, Yan Chen, Li-Jun Zhang, Ming Wang, Dong-Lei Chang, Wei-Wei Wan, Bi-Xiang Zhang, Wan-Guang Zhang, Xiao-Ping Chen
Although hepatitis B virus (HBV)-related cirrhosis and hepatocellular carcinoma (HCC) cause a sever health problem worldwide, the underlying mechanisms are still elusive. This study aimed to investigate the responses of different cell types isolated from HBV transgenic mice. A cross-sectional set of hepatocytes and oval cells were obtained from HBV transgenic and control mice. Flow cytometry, immunohistochemistry and microarray were applied to investigate the cell biology of the hepatocytes and oval cells. Our results showed that HBV induced the proliferation of both cell oval cells and hepatocytes, and induced cell death of HBV hepatocytes while had minimal effects on oval cells...
November 29, 2018: Cancer Letters
Tiantian Guo, Qingchao Liu, Pengbo Hou, Fahui Li, Shoudong Guo, Weiguo Song, Hai Zhang, Xueying Liu, Shengyong Zhang, Jianchun Zhang, Chi-Tang Ho, Naisheng Bai
Three new stilbenoids (1-3) and 16 known stilbenoids (4-6) and cannabinoids (7-19) were isolated from the leaves of hemp (Cannabis sativa L.). The structures of the three new compounds were identified as α,α'-dihydro-3',4,5'-trihydroxy-4'-methoxy-3-isopentenylstilbene (HM1), α,α'-dihydro-3,4',5-trihydroxy-4-methoxy-2,6-diisopentenylstilbene (HM2), and α,α'-dihydro-3',4,5'-trihydroxy-4'-methoxy-2',3-diisopentenylstilbene (HM3) by 1D and 2D NMR spectroscopy, LC-MS, and HRESIMS. The known α,α'-dihydro-3,4',5-trihydroxy-4,5'-diisopentenylstilbene (5) and combretastatin B-2 (6) were isolated for the first time from C...
November 30, 2018: Food & Function
Atsushi Kawase, Ayaka Kaneto, Mao Ishibashi, Akihiro Kobayashi, Hiroaki Shimada, Masahiro Iwaki
Direct hepatotoxic effects of drugs can occur when a parent drug and/or its reactive metabolites induces the formation of reactive oxygen species. Reactive metabolites of diclofenac (DIC) such as DIC acyl-β-D-glucuronide (DIC-AG) bind covalently to proteins, potentially decreasing protein function or inducing an immune response. However, it is unclear whether the macrophages and GSH depletion participate in DIC-induced cytotoxicity. Mouse hepatocytes (Hep) co-cultured with peritoneal macrophages (PM) were used to clarify the effects of presence of PM with GSH depletion on DIC-induced cytotoxicity in Hep...
November 29, 2018: Toxicology Mechanisms and Methods
Fernanda Marconi Roversi, Nathalia Moreno Cury, Matheus Rodrigues Lopes, Karla Priscila Ferro, João Agostinho Machado-Neto, Marisa Claudia Alvarez, Gabriela Pereira Dos Santos, Renata Giardini Rosa, Ana Leda Longhini, Adriana da Silva Santos Duarte, Fernando Vieira Pericole, Patricia Favaro, José Andres Yunes, Sara Teresinha Olalla Saad
The role of tumour microenvironment in neoplasm initiation and malignant evolution has been increasingly recognized. However, the bone marrow mesenchymal stromal cell (BMMSC) contribution to disease progression remains poorly explored. We previously reported that the expression of serine protease inhibitor kunitz-type2 (SPINT2/HAI-2), an inhibitor of hepatocyte growth factor (HGF) activation, is significantly lower in BMMSC from myelodysplastic syndromes (MDS) patients compared to healthy donors (HD). Thus, to investigate whether this loss of expression was due to SPINT2/HAI-2 methylation, BMMSC from MDS and de novo acute myeloid leukaemia (de novo AML) patients were treated with 5-Azacitidine (Aza), a DNA methyltransferase inhibitor...
November 28, 2018: Journal of Cellular and Molecular Medicine
Aleksija S Neimanis, Harri Ahola, Ulrika Larsson Pettersson, Ana M Lopes, Joana Abrantes, Siamak Zohari, Pedro J Esteves, Dolores Gavier-Widén
BACKGROUND: Prior to 2010, the lagoviruses that cause rabbit hemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus) and European brown hare syndrome (EBHS) in hares (Lepus spp.) were generally genus-specific. However, in 2010, rabbit hemorrhagic disease virus 2 (RHDV2), also known as Lagovirus europaeus GI.2, emerged and had the distinguishing ability to cause disease in both rabbits and certain hare species. The mountain hare (Lepus timidus) is native to Sweden and is susceptible to European brown hare syndrome virus (EBHSV), also called Lagovirus europaeus GII...
November 26, 2018: BMC Veterinary Research
Juan Chen, Qiu-Yun Li, Yong-Heng Luo, En-Hua Xiao
AIMS: To investigate the effect of zebularine, a stable inhibitor of DNA methylation, on hepatic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) under liver-specific factors induction in vitro. MAIN METHODS: BM-MSCs were isolated from the mononuclear cell fraction of rabbit bone marrow samples. The identification of these cells was carried out by immunophenotype analysis. The three hepatic differentiation protocols of BM-MSCs were as follows: liver-specific factors (hepatocyte growth factor and epidermal growth factor) without zebularine, liver-specific factors combined with a 24 h zebularine pre-treatment, and liver-specific factors combined with continuous zebularine treatment...
December 15, 2018: Life Sciences
Halina Falfushynska, Oksana Horyn, Agnieszka Brygider, Olga Fedoruk, Bogdan Buyak, Dmytro Poznansky, Barbara Poniedziałek, Mikołaj Kokociński, Piotr Rzymski
As yet European strains of Raphidiopsis raciborskii (previously Cylindrospermopsis raciborskii) have not been found to produce known cyanotoxins although their extracts have caused adverse effects in mammals, as shown using in vitro and in vivo experimental models. The present study investigated whether R. raciborskii isolated from Western Poland and Ukraine can affect fish cells using in vitro exposures of hepatocytes and red blood cells (RBC), and brain homogenates obtained from common carp (Cyprinus carpio) to 1...
November 17, 2018: Aquatic Toxicology
Yuko Yamauchi, Tetsuya Okuyama, Toshinari Ishii, Tadayoshi Okumura, Yukinobu Ikeya, Mikio Nishizawa
Pruni Cortex is a herbal drug from the bark of the Japanese flowering cherries, Prunus jamasakura or Prunus verecunda, and is included in the traditional Japanese herbal (Kampo) formula Jumihaidokuto, which is administered orally to patients suffering from inflammatory skin diseases. The flavanones contained in Pruni Cortex (e.g., sakuranetin and naringenin) have potent anti-inflammatory, anti-allergic, and anti-microbial activities. Although the effects of Pruni Cortex on skin disease have been well studied, reports regarding its pharmacological effects on the liver are limited...
November 22, 2018: Journal of Natural Medicines
Donna W Lee, Shelly Zhong, Rama Pai, Julie Rae, Siddharth Sukumaran, Eric G Stefanich, Jeff Lutman, Estelle Doudement, Xiaoting Wang, Brandon Harder, Annemarie Lekkerkerker, Ann Herman, Wenjun Ouyang, Dimitry M Danilenko
Although Interleukin-22 (IL-22) is produced by various leukocytes, it preferentially targets cells with epithelial origins. IL-22 exerts essential roles in modulating various tissue epithelial functions, such as innate host defense against extracellular pathogens, barrier integrity, regeneration, and wound healing. Therefore, IL-22 is thought to have therapeutic potential in treating diseases associated with infection, tissue injury or chronic tissue damage. A number of in vitro and in vivo nonclinical studies were conducted to characterize the pharmacological activity and safety parameters of UTTR1147A, an IL-22 recombinant fusion protein that links the human cytokine IL-22 with the Fc portion of a human immunoglobulin...
December 2018: Pharmacology Research & Perspectives
Nicolas Capelli, Olivier Marion, Martine Dubois, Sophie Allart, Justine Bertrand-Michel, Sébastien Lhomme, Florence Abravanel, Jacques Izopet, Sabine Chapuy-Regaud
Hepatitis E virus (HEV) is a common cause of acute viral hepatitis worldwide. Most HEV infections are asymptomatic but immunocompromised patients infected with HEV3, HEV4 or HEV7 may develop chronic infections. The HEV particles in stools are naked (nHEV) while those in the serum and culture supernatants (eHEV) are associated with lipids. Hepatocytes are polarized epithelial cells that have basolateral (to blood) and apical (to bile) exosomal pathways. We isolated a subclone, F2, from the human hepatocarcinoma cell line HepG2/C3A that grew as a polarized monolayer culture and had better HEV production than HepG2/C3A...
November 21, 2018: Journal of Virology
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