LT, Others : O-066 ; Three-dimensionally co-cultured human adipose-derived stem cell with human hepatocyte can be used as an alternative source of liver transplantation.

( Da Yoon No ),( Yoon Young Choi ),( Seung A Lee ),( Dong Sik Kim ),( Sang Hoon Lee ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.1

Background: The number of patients with chronic liver disease or cirrhosis is increasing. For these patients, liver transplantation has been the only solution, but, because of the shortage of donors and immune rejection, human-liver-cell-based therapies using autologous liver resection might be an alternative approach. However, continuous supply of human primary hepatocytes (hHeps) is challenging because of the poor viability of isolated hHeps from partial hepatectomy. Here, we overcame these obstacles by co-culturing hHeps with human adipose- derived stem cells (hADSCs), which can be obtained in large quantities, repeatedly, under local anesthesia. Methods: hHeps were isolated from resection margins after partial hepatectomy using a two-step collagenase technique. hADSCs were from subcutaneous adipose tissue. Spheroids were generated using polydimethylsiloxane-based concavemicromolds. For spheroid formation, a 1:1 mixture of hHeps and hADSCs was inoculated and cultured on microwells. Results: Co-cultured spheroids aggregated more rapidly, and the viability was significantly higher than mono-cultured spheroids. Co-cultured spheroids exhibited higher liver-specific functions compared to mono-cultured, though they had only half the number of hHeps. The albumin and urea level of co-cultured spheroids was 15% and 20% higher on day 7, respectively. A quantitative cytochrome P450 assay showed that enzymatic activity of co-cultured spheroids was 20% higher than mono-cultured spheroids. Conclusions: hADSCs co-cultured with hHeps assist the compact formation of hepatic spheroids and behave like hHeps. Co-cultured spheroids might enable autologous hHeps as a safer method to whole organ transplantation to cure patients, overcoming donor shortages. Additionally, they could be used in the bioartificial liver maintaining cell viability and function.

SV 40 Large T 유전자를 이용해 불멸화시킨 사람 태아 간세포주의 확립

박중원(Joong Won Park),이주영(Joo Young Lee),이효석(Hyo Suk Lee),박주배(Joo Bae Park),김정룡(Chung Yong Kim) 대한내과학회 1994 대한내과학회지 Vol.46 No.4

N/A Background: Hepatocyte culture represents a cell model for analyzing the mechanism involved in car- cinogenesis of carcinogens, a tool for measuring hepatotoxicity of drugs and a simple model for studying hepatitis virus life cycle. However, problems raised by both the short-term survival and the poor functional stability of hepatocytes in culture hindered scientist in using of this in vitro system. Thus we planed to obtain the immortalized and differentiated human hepatocyte cell line by modifying the genome by transfecting the cells with viral specific DNA, always available in studying the liver disease. Method: After primary culture of hepatocytes obtained by therapeutic abortion at 18 weeks of gestation, SV40 large T gene was transfected into the cells by using Polybren-DMSO method, And then transfected hepatocytes were selected in G418 containing medium. Selected, transformed hepatocytes were subcultured in 10% fetal bovine serum containg F-12 medium. Morphological characteristics of subcultured cells was followed by phase-contrast microscope and electoron microscope. The immunocytochemisty using anti-human albumin and anti-human alpha fetoprotein and the immunofluorescence using snti-SV40 T antigen were performed for proving the differentiation of sub-cultured hepatocytes. Results: Electron microscope revealed subcultured cell to be epithelial cell. After more than 20 passages over a period of 7 months, the cells retained an epitheloid morphology. All the SV40 transformed hepatocyte cell lines were 100% positive for T antigen. Significant anti-alpha fetoprotein staining and week anti-albumin staining were observed in cytoplasm around nucleus and so we confirmed the synthesis of liver specific protein of transformed hepatocytes, Conclusion: Human fetal immortalized hepatocytes cell line secreting the liver specific proteins was established.

LT, Other : O-066 ; Three-dimensionally co-cultured human adipose-derived stem cell with human hepatocyte can be used as an alternative source of liver transplantation

( Da Yoon No ),( Yoon Young Choi ),( Seung A Lee ),( Dong Sik Kim ),( Sang Hoon Lee ) 대한간학회 2012 춘·추계 학술대회 (KASL) Vol.2012 No.-

Background: The number of patients with chronic liver disease or cirrhosis is increasing. For these patients, liver transplantation has been the only solution, but, because of the shortage of donors and immune rejection, human-liver-cell-based therapies using autologous liver resection might be an alternative approach. However, continuous supply of human primary hepatocytes (hHeps) is challenging because of the poor viability of isolated hHeps from partial hepatectomy. Here, we overcame these obstacles by co-culturing hHeps with human adipose- derived stem cells (hADSCs), which can be obtained in large quantities, repeatedly, under local anesthesia. Methods: hHeps were isolated from resection margins after partial hepatectomy using a two-step collagenase technique. hADSCs were from subcutaneous adipose tissue. Spheroids were generated using polydimethylsiloxane-based concavemicromolds. For spheroid formation, a 1:1 mixture of hHeps and hADSCs was inoculated and cultured on microwells. Results: Co-cultured spheroids aggregated more rapidly, and the viability was significantly higher than mono-cultured spheroids. Co-cultured spheroids exhibited higher liver-specific functions compared to mono-cultured, though they had only half the number of hHeps. The albumin and urea level of co-cultured spheroids was 15% and 20% higher on day 7, respectively. A quantitative cytochrome P450 assay showed that enzymatic activity of co-cultured spheroids was 20% higher than mono-cultured spheroids. Conclusions: hADSCs co-cultured with hHeps assist the compact formation of hepatic spheroids and behave like hHeps. Co-cultured spheroids might enable autologous hHeps as a safer method to whole organ transplantation to cure patients, overcoming donor shortages. Additionally, they could be used in the bioartificial liver maintaining cell viability and function.

Functional Comparison of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells as Sources of Hepatocyte-Like Cells

정재민,김규남,정민성,김한준 한국조직공학과 재생의학회 2016 조직공학과 재생의학 Vol.13 No.6

Pluripotent stem cells can differentiate into many cell types including mature hepatocytes, and can be used in the development of new drugs, treatment of diseases, and in basic research. In this study, we established a protocol leading to efficient hepatic differentiation, and compared the capacity to differentiate into the hepatocyte lineage of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Optimal combinations of cytokines and growth factors were added to embryoid bodies produced by both types of cell. Differentiation of the cells was assessed with optical and electron microscopes, and hepatic-specific transcripts and proteins were detected by quantitative reverse transcription polymerase chain reaction and immunocytochemistry, respectively. Both types of embryoid body produced polygonal hepatocyte-like cells accompanied by time-dependent up regulation of genes for α-fetoprotein, albumin (ALB), asialoglycoprotein1, CK8, CK18, CK19, CYP1A2, and CYP3A4, which are expressed in fetal and adult hepatocytes. Both types of cell displayed functions characteristic of mature hepatocytes such as accumulation of glycogen, secretion of ALB, and uptake of indocyanine green. And these cells are transplanted into mouse model. Our findings indicate that hESCs and hiPSCs have similar abilities to differentiate into hepatocyte in vitro using the protocol developed here, and these cells are transplantable into damaged liver.

In Vitro Differentiation of Human Amniotic Membrane-derived Stem Cells into Hepatocyte-like Cells

국민지,박수연,강현미,김해권 한국발생생물학회 2006 발생과 생식 Vol.10 No.1

간질환 환자의 대부분은 간 조직 손상으로 인해 간세포의 재생 능력이 감소한다. 간세포 이식은 이러한 간질환을 치료하는데 있어 혁신적인 방법으로 대두되고 있으나, 여전히 많은 의문과 문제점이 제기되고 있다. 사람의 양막으로부터 얻은 줄기 세포를 이용하여 간세포 분화를 위한 최적의 조건을 알아 보고자 하였다. 세포내 알부민에 대한 면역 화학적 방법, 세포내 글리코겐의 특이 염색법, 세포의 형태적 변화 연구 방법 등을 이용하여 여러가지 배양 조건을 조사한 결 This study aimed to find out suitable culture conditions for the differentiation of human amniotic membrane-derived stem cells(HAM) into hepatocyte-like cells. Almost homogenous population of fibroblast-like cells was successfully isolated from the amniotic membrane. In comparison to the non-coated plates and in the absence of insulin/transferrin/selenium(ITS), HAM cultured on the fibronectin-coated plates and in the presence of ITS showed the more intense immunocytochemical staining against the albumin. Addition of both fibroblast growth factor(FGF)-1 and -2 to the differentiation medium gave stronger staining compared to the treatment with FGF-1 or -2 alone. Periodic acid Schiff's base staining of glycogen and morphological turnover of fibroblast-like appearance of HAM into round shape matched the results of immunocytochemical studies. When the efficiency of two-step culture method was examined on the differentiation of HAM into hepatocyte-like cells, all of the results of immunocytochemical staining, periodic acid Schiff's staining and morphological change exhibited effective hepatic differentiation of HAM compared to the continuous culture method. Immunoblot analyses of HAM- conditioned media against the albumin showed that the culture of HAM in the presence of both ITS and fibronectin always gave a stronger staining intensity than those in the absence of them, and that the addition of ether mixture of FGF-4 and either FGF-2 or transforming growth to the culture medium significantly enhanced the albumin secretion by HAM. Based on these observations, it is suggested that HAM could differentiate into hepatocyte-like cells under a culture condition consisting of fibronectin and ITS, and addition of FGF-4 with either one of FGF-2 or could enhance the hepatic differentiation of HAM.

The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02

Ning Mu,Hong-Bao Liu,Qiu-Hong Meng,De-Wei Du,Yi Jiang,Huan-Zhang Hu 대한외과학회 2014 Annals of Surgical Treatment and Research(ASRT) Vol.88 No.1

Purpose: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02. Methods: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry. Results: With the MACS method, (5?10) × 10⁴/mL hMAPCs could be separated from 1 × 10?/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5. Conclusion: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.

Efficient Derivation of Hepatogenic Endoderm from Human Embryonic Stem Cells Using Serum-Free Conditioned Medium from L-Wnt3a Cells

( Hyun Ju You ),( Jiyou Han ),( Dong Hun Woo ),( Su Yeon An ),( Jong Hyun Kim ),( Yu Jin Jang ),( Jeong Sang Son ),( Suel Kee Kim ),( Jong Hoon Kim ) 한국조직공학·재생의학회 2011 조직공학과 재생의학 Vol.8 No.6

The directed differentiation of human embryonic stem cells (hESCs) into hepatocytes is considered a promising new approach to generate a transplantable and limitless cell source for the treatment of acute and chronic liver diseases. Current protocols for generating hepatocytes from hESCs need to be improved because of the inefficient differentiation procedures which lead to low yields and large cellular heterogeneity. In this study, we describe a simple and efficient three-stage process for differentiating hESCs into functional hepatocyte-like cells. The first step of this optimized protocol was to induce hESCs towards mesendoderm (co-precursor stage of the mesoderm and endoderm) using 80% Wnt3a conditioned medium (Wnt3a-CM) and 20 ng/ml activin A for 5 days. In the second step, the mesoendodermal cells were treated with 40 ng/ml bone morphogenetic protein 4 (BMP4) for 4 days. In the third stage of hepatocyte maturation, cells were cultured in hepatocyte growth factor (HGF), oncostatin M (OSM), and dexamethasone (DEX). During the first stage, the definitive endodermal markers were significantly increased, and under the further differentiation, the expression of hepatocyte precursor markers, especially, albumin and CYP genes, induced sequentially. Furthermore, these cells showed functions associated with hepatocytes, which include γ-glutamyltranspeptidase (GGT), glycogen storage, indocyanine green (ICG) uptake, and albumin secretion. These results may form the basis for further investigation into understanding the fundamental mechanisms of liver development, applying cell therapy and screening cytotoxicity in a new drug to treat liver diseases.

Hepatic Differentiation of Human Eyelid Adipose-Derived Stem Cells

박수연,박세아,강현미,김해권 한국발생생물학회 2008 발생과 생식 Vol.12 No.2

A variety of stem cells has been emerging as therapeutic cells that can replace organ transplantation in human liver diseases. The present study focused on whether human eyelid adipose-derived stem cells (HAD) might differentiate into functional hepatocyte-like cells in vitro. HAD were isolated from human eyelid adipose tissue. Effect of dimethyl sulfoxide (DMSO), fibroblast growth factor (FGF)-2 and FGF-4 on the hepatic differentiation of HAD have been examined in vitro. Immunocytochemical analysis and PAS staining showed that HAD cultured in both DMSO and FGF-4 exhibited the most intense staining than HAD of the other experimental groups. These HAD expressed numerous hepatocyte-related genes. Immunoblotting analyses showed that HAD cultured in the presence of DMSO and FGF-4 secreted higher amount of human albumin than HAD cultured in other conditions. Urea analysis also demonstrated that these HAD produced higher amount of urea than any other groups of HAD. In conclusion, combined treatment of DMSO and FGF-4 could effectively induce the functional differentiation of HAD into hepatocyte-like cells.

사람 태아 간세포 및 섬유아세포의 장기 일차 배양 및 계대 배양

이효석(Hyo Suk Lee),김용태(Yong Tae Kim),김정룡(Chung Yong Kim) 대한내과학회 1991 대한내과학회지 Vol.41 No.1

N/A Cultured normal hepatocytes might be a useful system in probing the effect of hepatotoxic, carcinogenic and viral agents as well as in studying the regulation of specific hepatocellular functions, and cultured human fibroblasts might provide a helpful system in researching developement of fibrosis in the pathogenesis of liver cirrhosis. This study was designed to establish the long-term primary culture and subculture of hepatocyte and fibroblasts using 2 different culture media, namely, a serum-free completely defined medium and a medium containing 10% fetal bovine serum, respectively. Cells obtained by collagenase dissociation from the livers of human fetus taken by abortion at 18 to 22 weeks of gestation were maintained on a substratum of positively charged plastic. 1) In a serum-free medium, epitheloid polygonal cells which were morphologically considered hepatocytes, proliferated to form a confluent culture and retained their initial shape for more than 3 weeks and could be subcultured. 2) In a medium supplemented with 10% fetal bovine serum, spindle-shaped cells, which had the morphological characteristics of fibroblasts, proliferated and could be subcultured for more than 36 passages over a period of 6 months. Thus, we established a long-term culture and passage of human fetal hepatocyte.

Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

Cho, Yong-Yeon,Jeong, Hyeon-Uk,Kim, Jeong-Han,Lee, Hye Suk Dove Medical Press 2014 Drug design, development and therapy Vol.8 No.-

<P>Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of <I>Magnolia officinalis</I> and <I>Magnolia grandiflora</I>, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin <I>O</I>-deethylase and CYP3A4-catalyzed midazolam 1′-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.</P>