AIM To establish a model to enrich and characterize stemlike cells from murine normal liver and hepatocellular carcinoma(HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesench...AIM To establish a model to enrich and characterize stemlike cells from murine normal liver and hepatocellular carcinoma(HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition(EMT).METHODS In this study,we utilized a stem cell conditioned serumfree medium to enrich stem-like cells from mouse HCC and normal liver cell lines,Hepa 1-6 and AML12,respectively.We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating theRNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR(q RTPCR).Next,we examined the relationship between stem cells and EMT using q RT-PCR.RESULTS Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor,basic fibroblast growth factor and heparin sulfate.The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell(CSC) marker Cd44 compared to parental cells grown as adherent cultures.We report that epithelial markers E-cadherin and ZO-1 were downregulated,while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres.The 3-dimensional spheres also exhibited changes in expression of Snai,Zeb and Twist family of EMT transcription factors.CONCLUSION Our novel method successfully enriched stem-like cells which possessed an EMT phenotype.The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.展开更多
AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from ...AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes： 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester（CFSE）, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein（Di I-Ac LDL）, and green fluorescent protein（GFP）. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo.RESULTS： EPCs labeled with CFSE and Di I-Ac LDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28 d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and Di I-Ac LDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina.CONCLUSION： The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and Di I-Ac LDL are suitable for short-term EPClabeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.展开更多
基金Supported by The Gallipoli Medical Research Foundation,Australia,No.016092the Cyril Gilbert Foundation,Australia,No.017348
文摘AIM To establish a model to enrich and characterize stemlike cells from murine normal liver and hepatocellular carcinoma(HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition(EMT).METHODS In this study,we utilized a stem cell conditioned serumfree medium to enrich stem-like cells from mouse HCC and normal liver cell lines,Hepa 1-6 and AML12,respectively.We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating theRNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR(q RTPCR).Next,we examined the relationship between stem cells and EMT using q RT-PCR.RESULTS Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor,basic fibroblast growth factor and heparin sulfate.The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell(CSC) marker Cd44 compared to parental cells grown as adherent cultures.We report that epithelial markers E-cadherin and ZO-1 were downregulated,while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres.The 3-dimensional spheres also exhibited changes in expression of Snai,Zeb and Twist family of EMT transcription factors.CONCLUSION Our novel method successfully enriched stem-like cells which possessed an EMT phenotype.The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.
基金Supported by the National Natural Science Foundation of China(No.81400403)the International Science and Technology Cooperation Program of Jilin Province(No.20110733)the Technology Program of Soochow City(No.SYS201375)
文摘AIM： To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells（EPCs） in a mouse model of laser-induced retinal injury.METHODS： EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes： 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester（CFSE）, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein（Di I-Ac LDL）, and green fluorescent protein（GFP）. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo.RESULTS： EPCs labeled with CFSE and Di I-Ac LDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28 d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and Di I-Ac LDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina.CONCLUSION： The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and Di I-Ac LDL are suitable for short-term EPClabeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.
