title: Survival of primary human hepatocytes and death of induced pluripotent stem cells in media lacking glucose and arginine.
abstract: BACKGROUND
  Tumorigenicity is an associated risk for transplantation of hepatocytes differentiated from human induced pluripotent stem (hiPS) cells. Hepatocytes express the enzymes galactokinase and ornithine transcarbamylase (OTC) to aid in their own survival. However, hiPS cells do not express these enzymes, and therefore, are not be expected to survive in a medium containing galactose and ornithine and lacking glucose and arginine.
  
  
  METHODS
  Real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of galactokinase 1 (GALK1)1 and GALK2, ornithine carbamyltransferase, and phenylalanine hydroxylase (PAH). The hiPS cell line 201B7 was cultured in hepatocyte selection medium (HSM), which lacks glucose and arginine but contains galactose and ornithine. Furthermore, microscopic analysis of the cultured cells was performed after hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). The hiPS cells were immunostained to assess their pluripotency in HSM. In addition, the primary human hepatocytes were cultured with or without hiPS cells in HSM.
  
  
  RESULTS
  The expression levels of GALK1, GALK2, OTC, and PAH in 201B7 were 22.2±5.0 (average ± standard deviation), 14.2% ±1.1%, 1.2% ±0.2%, and 8.4% ±0.7% respectively, compared with those in the adult liver. The hiPS cell population diminished when cultured in HSM and completely disappeared after 3 days. The cultured cells showed condensation or fragmentation of their nuclei, thereby suggesting apoptosis. TUNEL staining confirmed that the cells had undergone apoptosis. The 201B7 cells were positive for Nanog, SSEA-4, and TRA-1-60. The primary human hepatocytes survived when cultured alone in HSM and when co-cultured with hiPS cells.
  
  
  CONCLUSIONS
  Therefore, HSM is and ideal medium for eliminating hiPS cells and purifying hepatocytes without inducing any damage.
authors: Department of Gastroenterology, National Hospital Organization, Shimoshizu Hospital, Yotsukaido City, Chiba, Japan. nihminor-cib@umin.ac.jp
authors: Minoru
authors: M
authors: Tomizawa
authors: Fuminobu
authors: F
authors: Shinozaki
authors: Takao
authors: T
authors: Sugiyama
authors: Shigenori
authors: S
authors: Yamamoto
authors: Makoto
authors: M
authors: Sueishi
authors: Takanobu
authors: T
authors: Yoshida
mesh_terms: D000328:Adult; D001120:Arginine / Q000032:analysis; D016923:Cell Death; D002454:Cell Differentiation; D002460:Cell Line; D002470:Cell Survival; D003470:Culture Media / Q000737:chemistry*; D005947:Glucose / Q000032:analysis; D022781:Hepatocytes / Q000166:cytology*; D006801:Humans; D039904:Pluripotent Stem Cells / Q000166:cytology*; D013194:Staining and Labeling
chemical_list: D003470:Culture Media; D001120:Arginine; D005947:Glucose
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