14 CD133 was also found to represent a small subpopulation in human tumor tissue, and it was absent in normal liver tissue. The subsequent analysis of CD133 expression in human liver cell lines revealed a positive correlation between CD133 expression and tumorigenic potential in vivo. Sorted CD133+ and CD133- fractions from HCC cell lines (i.e. PLC8024, Huh7 and HepG2) were then subjected to functional analyses in vitro and xenograft transplantation in vivo to study the exhibition of properties representative of both stem cells and cancer cells. The CD133+ cells were

found to be more tumorigenic than the CD133- cells, as evidenced by a greater colony-forming ability, higher proliferative potential and the ability to initiate tumor formation. Moreover, the CD133+ cells were characterized by properties of normal stem/progenitor cells, including the increased expression of “stemness”-associated genes and the abilities to check details self-renew and differentiate into non-hepatocyte lineages.14 Recently, our studies have been extended with the use of HCC clinical specimens, and a similar phenomenon has been observed.15 The clinical significance

of CD133 in HCC was also similarly reported by Song and colleagues.16 Aldehyde dehydrogenase (ALDH), a molecular metabolic mediator, was first identified as conferring resistance to cyclophosphamide in normal hematopoietic stem/progenitor cells.17 Recent studies have suggested that high ALDH activity can confer chemoresistance in CSCs.18–20 In colon cancer, higher ALDH activity has been observed in EpCAMhigh/CD44+ colon CSCs.21 ALDH was also Selleck Inhibitor Library Calpain found to be able to predict a poor clinical outcome in CSC-driven breast cancer patients.19 In the subsequent analysis of the characterization of liver CSCs marked by a CD133 phenotype, our group identified ALDH to be preferentially expressed in the CD133+ population in HCC, and the use of a combination of these markers was shown to more accurately define liver CSCs.20 A hierarchical organization of cells that differentially express CD133 and ALDH exhibit an ascending tumorigenic potential in the order of CD133+ALDH+ > CD133+ALDH- > CD133-ALDH-.20

In the following year, another CD surface protein was used for the identification of liver CSCs. Yang and colleagues found a significant positive correlation of CD90 expression with tumorigenicity and metastatic potentials in the panel of liver cell lines tested.22,23 In the clinical specimens, all of the tumor tissues and almost all of the blood samples contained a CD45-CD90+ subpopulation. The CD45-CD90+ cells isolated from both the tumor tissues and blood samples was shown to initiate and maintain tumor formation when injected intrahepatically into SCID/Beige mice in the first and the subsequent serial transplantation experiments.22,23 The existence of a CD45-CD90+ population in blood samples from HCC patients suggests the presence of CSCs in the systemic circulation.