title: Efficient enrichment of hepatic cancer stem-like cells from a primary rat HCC model via a density gradient centrifugation-centered method.
abstract: BACKGROUND
  Because few definitive markers are available for hepatic cancer stem cells (HCSCs), based on physical rather than immunochemical properties, we applied a novel method to enrich HCSCs.
  
  
  METHODS
  After hepatic tumor cells (HTCs) were first isolated from diethylinitrosamine-induced F344 rat HCC model using percoll discontinuous gradient centrifugation (PDGC) and purified via differential trypsinization and differential attachment (DTDA), they were separated into four fractions using percoll continuous gradient centrifugation (PCGC) and sequentially designated as fractions I-IV (FI-IV). Morphological characteristics, mRNA and protein levels of stem cell markers, proliferative abilities, induced differentiation, in vitro migratory capacities, in vitro chemo-resistant capacities, and in vivo malignant capacities were determined for the cells of each fraction.
  
  
  RESULTS
  As the density of cells increased, 22.18%, 11.62%, 4.73% and 61.47% of primary cultured HTCs were segregated in FI-FIV, respectively. The cells from FIII (density between 1.041 and 1.062 g/ml) displayed a higher nuclear-cytoplasmic ratio and fewer organelles and expressed higher levels of stem cell markers (AFP, EpCAM and CD133) than cells from other fractions (P<0.01). Additionally, in vitro, the cells from FIII showed a greater capacity to self-renew, differentiate into mature HTCs, transit across membranes, close scratches, and carry resistance to chemotherapy than did cells from any other fraction; in vivo, injection of only 1×10(4) cells from FIII could generate tumors not only in subcutaneous tissue but also in the livers of nude mice.
  
  
  CONCLUSIONS
  Through our novel method, HCSC-like cells were successfully enriched in FIII. This study will greatly contribute to two important areas of biological interest: CSC isolation and HCC therapy.
authors.affiliation: PLA Center of General Surgery, General Hospital of Chengdu Army Region, Chengdu, Sichuan Province, China.
authors.forename: Wei-hui
authors.initials: WH
authors.lastname: Liu
authors.forename: Xing
authors.initials: X
authors.lastname: Wang
authors.forename: Nan
authors.initials: N
authors.lastname: You
authors.forename: Kai-shan
authors.initials: KS
authors.lastname: Tao
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Li-jun
authors.initials: LJ
authors.lastname: Tang
authors.forename: Ke-feng
authors.initials: KF
authors.lastname: Dou
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000139:chemically induced / Q000473:pathology*; D002454:Cell Differentiation; D002465:Cell Movement; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D004052:Diethylnitrosamine; D004195:Disease Models, Animal; D008099:Liver / Q000378:metabolism / Q000473:pathology*; D008113:Liver Neoplasms / Q000139:chemically induced / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000145:classification / Q000378:metabolism / Q000473:pathology*; D061251:Primary Cell Culture; D051381:Rats; D011916:Rats, Inbred F344; D014357:Trypsin / Q000378:metabolism
chemical_list: D015415:Biomarkers; D004052:Diethylnitrosamine; D014357:Trypsin