Side Population Phenotype Enriched From Hepatocellular Carcinoma Displays Cancer Stem-Like Property And May Correlate With ATP-Binding Cassette Transporter ABCG2

Stem cell biology has come of age.A stem cell is typically characterized by its capacity for self-renewal and ability to give rise to multiple differentiated cellular populations(often termed cellular plasticity).The field of stem cell biology has exploded with the study of a wide range of cellular populations involving endodermal,mesenchymal,and ectodermal organs.Despite progress in characterizing select stem cell populations from distinct organs(e.g.bone marrow,liver,the nervous system,etc.),a pattern of differences have become evident.These distinctions are associated with the host and/or the injury model involved with isolating a specific cellular population.An additional and of tentimes greater challenge when working with stem cells is the lack of unique markers to identify these cells.While some markers are observed in stem cell subsets,(e.g.the efflux of certain dyes appears to be a common property of hematopoietic stem cells);stem cells are routinely characterized by the absence rather than the presence of specific lineage-related markers.Found by Coodell in 1996,the detection of side population(SP) cells,which have the ability to strongly efflux Hoechst33342 fluorescence dye,has attracted attention as a method of stem cell isolation.This population almost completely disappeared after treatment with the calcium channel blocker verapamil.Side populations(SP),as defined by Hoechst exclusion in flow cytometry,have been described a few years ago.While they represent only a small fraction of the whole cell population,their properties confer an important place in several investigations.SP cells express high levels of various members of ABC transporters family,such as MDR1 and ABCG2,which are responsible for drug resistance.Targeting SP could improve cancer therapy by blocking these transporters.In addition,SP appear to be enriched in stem cells,cells that play a pivotal role in normal development and cancer biology.Recently it is considered that there is a small population of cells with stem cell property not only in cancer cell lines but also in solid cancer. Current advances in stem cell biology enable us to identify cancer stem cells in solid tumors as well as putative stem cells in normal solid organs.These cells show the ability of self-renewal and multi-potential differentiation, and can initiate and maintain a tumor.The origin of cancer stem cells might be their normal stem cells,progenitor cells,or bone marrow-derived cells.It is still difficult to isolate cancer stemcells in solid cancer.Thus, they could provide a useful tool and a readily accessible source for stem cell studies in both the normal and cancerous settings.However,these cells are poorly defined and pose challenges in their identification and isolation, particularly since they are few in number.Briefly,There are three possible methodologies to isolate or identify cancer stem cells;the use of a surface marker,use of cells cultured in a specific condition(sphere),or the use of side population cells identified by FACS.SP cells were mainly localized in the epidermis,with a few in the dermis.The gold standard assay that fulfills the definition of cancer stem cell may be serial transplantation in animal models.The NOD-Scid is a useful model for cancer research due to increased tumor incidence.Our experiments firstly aimed to isolate and establish hEFMSCs,one kind of Mesenchymal Stem Cells originated from human early foetus,used in the following SP project as a stem cell research model.This part of study examined whether hEFMSCs was mesenchymal stem cells and whether were able to differentiate into hepatocyte-like cells in vitro.MSCs were from human early foetus,and the surface phenotype and the mesodermal multilineage differentiation potentials of hEFMSCs,such as into adipocyte-like and neurocyte-like cells,were characterized and tested.Facs shows hEFMSCs' phenotype is CD13+,CD29+,CD90+,CD106+,SH2+,CD3-,CD4-,CD5-,CD11b-,CD14-,CD15-,CD34-,CD45-,CD45RA-,CD133-,HLA-DR-.After 3 weeks of induction under conditioned culture medium containing a variety of cell growth factors, cuboidal morphology,which is characteristic of hepatocytes,was observed,and cells also expressed marker genes specific of liver cells in a time-dependent manner.Hence,hEFMSCs may serve as a cell source for tissue engineering and cell therapy of hepatic tissues.Secondly,we applied side population(SP)cell analysis and sorting to established hepatocellular carcinoma(HCC) cell lines to detect subpopulations that function as cancer stem cells and to elucidate their roles in tumorigenesis.Among 8 cell lines analyzed,SP cells were detected in PLC,Huh7,Hep3B,SMMC7721 cells and Primary HCC specimens,and the ratio is by 0.8%,0.3%,0.6%,0.3%,0.4%respectively.Notablely,we also did not find SP in HepG2,whose difference from other cancer cell lines seems to be its origin from blastoma,which consistent with the report by Tetsuhiro.The stem cell line hEFMSCs'SP was also be dectected by 0%.Analysis of cell cycles SP cells demonstrated SP cells have no G2-M phases.In vitro cultured SP showed high colony-forming and proliferative potential and compared with those of non-SP cells.NOD-SCID subcutaneouly transplant experiments showed that after injection by 10~3 SP cells,PLC-SP were 100%sufficient for tumor formation, SMMC-SP were 83.3%sufficient for tumor formation,whereas an injection of 2×10~4PLC-Non-SP cells and 2×10~4 SMMC-Non-SP cells did not initiate tumors.Thirdly,based on the closely relationship between ABCG2 and the SP cell phenotype,we want to know if the expression of ABCG2,which participates in dye efflux in many stem cells cases was expressed at high levels of mRNA in sorted SP cells(data are not shown because of the absence of SP RNA).We conclude that HCC SP stem cells are closely related to ABCG2.Our data shows ABCG is expressed in a variety of cell types including normal tissues,stem cells,HCC cell lines and liver tumors by RT-PCR assay.We also explore to find if there are differences between clinical tumors and peritumoral tissue by measuring ABCG2 mRNA expression levels using Real-time PCR.Thus,better characterization of SP will advance our understanding of stem cells and will provide us an accessible target for drug resistance in cancer therapy.So,we conclude that cancer stem cells are likely to be responsible for disease relapse or metastasis,and also for resistance to radiation or conventional chemotherapy.The novel promising therapies against cancer stem cells are considered,including antibody-based therapy,signal inhibitors, overcoming radiation and drug resistance,or differentiation therapy.