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RISS 인기검색어
- 검색키워드
- 저자 : Gyunggyu Lee (검색결과 6 건)
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Park, Ji Young,Han, Jiyou,Jung, Hyo Sung,Lee, Gyunggyu,Kim, Hyo Jin,Cho, Gun-Sik,Park, Han-Jin,Han, Choongseong,Kim, Jong Seung,Kim, Jong-Hoon Elsevier 2019 Biomaterials Vol.222 No.-
<P><B>Abstract</B></P> <P>Hepatocytes derived from human pluripotent stem cells (hPSCs) are promising candidates for cell therapy and drug discovery. However, it remains challenging to efficiently purify hepatocytes from undesired cell types after differentiation and to accurately monitor grafted cells after transplantation. Indocyanine Green (ICG), an FDA-approved, near-infrared (NIR) dye, has been used for various clinical purposes and is exclusively taken up by hepatocytes. However, ICG has a long emission wavelength (λ<SUB>em</SUB> > 800 nm) that is beyond the detection range of fluorescence-activated cell sorting (FACS) systems. Moreover, it is easily eliminated from hepatocytes, hindering its application for NIR imaging. Here, we designed and synthesized two different probes based on the properties of ICG; 1) hepatocyte purifying agent (<B>HPA,</B> λ<SUB>em</SUB> = 562 nm) for <I>in vitro</I> sorting and 2) hepatocyte imaging agent (<B>HIA,</B> λ<SUB>em</SUB> = 817 nm) for efficient <I>in vivo</I> NIR imaging. We obtained highly enriched populations of hPSC-derived hepatocytes (hPSC-Heps) from various hPSC lines using HPA probe-based FACS purification. In addition, HIA labelling and NIR imaging allowed the direct visualization and tracking of grafted hPSC-Heps in animals with liver injuries. These results demonstrated that these two probes could be used as powerful tools with hPSC-Heps in both cell replacement therapy and drug screening.</P>
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Kim, Jong Hyun,Jang, Yu Jin,An, Su Yeon,Son, Jeongsang,Lee, Jaehun,Lee, Gyunggyu,Park, Ji Young,Park, Han-Jin,Hwang, Dong-Youn,Kim, Jong-Hoon,Han, Jiyou ACADEMIC PRESS 2015 TOXICOLOGICAL SCIENCES Vol.147 No.1
<P>Highly homogeneous and functional stem cell-derived hepatocyte-like cells (HLCs) are considered a promising option in the cell-based therapy of liver disease and the development of effective <I>in vitro</I> toxicity screening tools. However, the purity of cells and expression and/or activity of drug metabolizing enzymes in stem cell-derived HLCs are usually too low to be useful for clinical or <I>in vitro</I> applications. Here, we describe a highly optimized hepatic differentiation protocol, which produces >90% (BGO1 and CHA15) albumin-positive HLCs with no purification process from human embryonic stem cell lines. In addition, we show that hepatic enzyme gene expressions and activities were significantly improved by generating 3D spheroidal aggregate of HLCs, compared with 2D HLCs. The 3D differentiation method increased expression of nuclear receptors (NRs) that regulate the proper expression of key hepatic enzymes. Furthermore, significantly increased hepatic functions such as albumin and urea secretion were observed in 3D hepatic spheroids, compared with 2D HLCs. HLCs in the spheroid exhibited morphological and ultrastructural features of normal hepatocytes. Importantly, we show that repeated exposures to xenobiotics facilitated further functional maturation of HLC, as confirmed by increased expression of genes for drug metabolizing enzymes and transcription factors. In conclusion, the 3D culture system with repeated exposures to xenobiotics may be a new strategy for enhancing hepatic metabolizing ability of stem cell-derived HLCs as a cell source for <I>in vitro</I> high-throughput hepatotoxicity models.</P>
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An, Su Yeon,Jang, Yu Jin,Lim, Hee-Joung,Han, Jiyou,Lee, Jaehun,Lee, Gyunggyu,Park, Ji Young,Park, Seo-Young,Kim, Ji Hyang,Do, Byung-Rok,Han, Choongseong,Park, Hee-Kyung,Kim, Ok-Hee,Song, Myeong Jun,Ki Elsevier 2017 Gastroenterology Vol.152 No.5
<P><B>Background & Aims</B></P> <P>Mesenchymal stem cells (MSCs) mediate tissue repair and might be used to prevent or reduce liver fibrosis. However, little is known about the anti-fibrotic factors secreted from MSCs or their mechanisms.</P> <P><B>Methods</B></P> <P>Umbilical cord-derived MSCs (UCMSCs) were differentiated into hepatocyte-like cells (hpUCMSCs), medium was collected, and secretome proteins were identified and quantified using nanochip-liquid chromatography/quadrupole time-of-flight mass spectrometry. Liver fibrosis was induced in mice by intraperitoneal injection of thioacetamide or CCl<SUB>4</SUB>; some mice were then given injections of secretomes or proteins. Liver tissues were collected and analyzed by histology or polymerase chain reaction array to analyze changes in gene expression patterns. We analyzed the effects of MSC secretomes and potential anti-fibrotic proteins on transforming growth factor β 1 (TGFβ1)-mediated activation of human hepatic stellate cell (HSC) lines (hTert-HSC and LX2) and human primary HSCs. Liver tissues were collected from 16 patients with liver cirrhosis and 16 individuals without cirrhosis (controls) in Korea and analyzed by immunohistochemistry and immunoblots.</P> <P><B>Results</B></P> <P>In mice with fibrosis, accumulation of extracellular matrix proteins was significantly reduced 3 days after injecting secretomes from UCMSCs, and to a greater extent from hpUCMSCs; numbers of activated HSCs that expressed the myogenic marker α-smooth muscle actin (α-SMA, encoded by <I>ACTA2</I> [actin, alpha 2, smooth muscle]) were also reduced. Secretomes from UCMSCs, and to a greater extent from hpUCMSCs, reduced liver expression of multiple fibrotic factors, collagens, metalloproteinases, TGFβ, and Smad proteins in the TGFβ signaling pathways. In HSC cell lines and primary HSCs, TGFβ1-stimulated upregulation of α-SMA was significantly inhibited (and SMAD2 phosphorylation reduced) by secretomes from UCMSCs, and to a greater extent from hpUCMSCs. We identified 32 proteins in secretomes of UCMSCs that were more highly concentrated in secretomes from hpUCMSCs and inhibited TGFβ-mediated activation of HSCs. One of these, milk fat globule-EGF factor 8 (MFGE8), was a strong inhibitor of activation of human primary HSCs. We found MFGE8 to down-regulate expression of TGFβ type I receptor by binding to α<SUB>v</SUB>β<SUB>3</SUB> integrin on HSCs and to be secreted by MSCs from umbilical cord, teeth, and bone marrow. In mice, injection of recombinant human MFGE8 had anti-fibrotic effects comparable to those of the hpUCMSC secretome, reducing extracellular matrix deposition and HSC activation. Co-injection of an antibody against MFGE8 reduced the anti-fibrotic effects of the hpUCMSC secretome in mice. Levels of MFGE8 were reduced in cirrhotic liver tissue from patients compared with controls.</P> <P><B>Conclusions</B></P> <P>MFGE8 is an anti-fibrotic protein in MSC secretomes that strongly inhibits TGFβ signaling and reduces extracellular matrix deposition and liver fibrosis in mice.</P>
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Jiyou Han,Su Yoen An,Jong Hyun Kim,Yu Jin Jang,Jeong Sang Son,Jae Hoon Lee,Gyunggyu Lee,Ji Young Park,Jong-Hoon Kim 한국발생생물학회 2013 한국발생생물학회 학술발표대회 Vol.2013 No.8
MFG-E8 (Milk fat globule-epidermal growth factor VIII), also called lactadherin or BA46, SED1 is a glycoprotein found in milk and mammary epithelial cells, it is a major protein component associated with milk fat globule membrane. Previously, our study showed that expression of MFG-E8 is gradually increased with hepatic differentiation of human embryonic stem cells (hESCs). Therefore, we hypothesized that MFG-E8 would be an early cancer stem cell marker, which may predict cancer progression. Our results showed that MFG-E8 was expressed in various human cancer cell lines such as HepG2, Hep3B, and Huh7. Production and secretion of the MFG-E8 were also confirmed in the conditioned media of those three cell lines using enzyme-linked immunosorbent assay. Next, we analyzed the MFG-E8 expression in 11 clinical cases of cholangiocellular carcinoma (CC) and 33 cases of hepatocellular carcinoma (HCC) by immunohistochemistry and examined the potential correlation with β-catenin and AFP, which are known cancer markers. According to hitological criteria, the progression of HCC and CC was evaluated and classified into high, low, metastatic, and well-, moderate-, poor-differentiated, respectively. Statistical analysis indicated that incidence of both HCC and CC is significantly associated with male compared to female (P<0.05). Tumor size also has positive correlation with age (r2=08948). Our immunohistochemistry data showed that MFG-E8 was expressed both HCC and CC tissue. Interestingly, the MFG-E8 expression was significantly increased with cancer progression (P<0.05) in both cases. Additionally, b-cateninexpression was increased and its localization was changed from membrane to cytoplasm and nucleus with the degree of HCC. Likely b-catenin, AFP was also increased with the degree of HCC but it was not correlated with severalty of CC. Importantly, both AFP and b-catenin were highly co-localized with MFG-E8 in HCC. These results suggest that MFG-E8 may have important physiological roles and its expression in HCC and CC would be considered as an important prognostic factor.
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Ji Young Park,Jiyou Han,Soo Jin Uhm,Jong Hyun Kim,Jeong Sang Son,Yu Jin Jang,Jae Hun Lee,Gyunggyu Lee,Jong Hoon Kim,Jong Seung Kim 한국발생생물학회 2015 한국발생생물학회 학술발표대회 Vol.2015 No.9
Hepatocytes and hepatic progenitors derived from human ES cells may be a useful source for clinical application. Therefore, identification and purification of these cell types would be following important issues. There are very few candidate surface markers that can be used to identify and purify hepatic progenitor cells. In addition, indocyanine-green can be uptaken by mature hepatocytes, but cannot be applied for fluorescence activated cell sorting (FACS) due to its long emission wavelength. In the present study, we tested EpCAM as a potential marker for magnetic-activated cell sorting (MACS) of hepatic progenitors and also modified indocyanine-green into fluorescent indomonocarbocyanine for FACS-mediated sorting of mature hepatocytes after differentiation of human ES cells. Hepatic progenitor cells were sorted by MACS after incubation with anti-human EpCAM antibodies. After the final differentiation, the differentiated cells and mouse primary hepatocytes (control group) were incubated with indomonocarbocyanine and were sorted by FACS. MACS and immunocytochemistry data showed that approximately 45% of differentiated cells were EpCAM-positive cells. EpCAM-positive cells expressed α-fetoprotein, FOXa2, HnF4a, and CK18. Differentiation efficiency into albumin-positive cells was significantly higher in EpCAM-positive cells, compared to EpCAM-negative cells. Importantly, indomonocarbocyanine successfully stained cells that expressed ALB. Furthermore, FACS analysis data showed that the purity of hepatocytes that expressed albumin was significantly increased after purification of indomonocarbocyanine-positive cells. Our data demonstrated that human ES cell-derived hepatic progenitors can be efficiently isolated by MACS using EpCAM antibody. In addition, we also showed that indomonocarbocyanine can be successfully used to identify and purify mature hepatocytes using FACS.
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