title: Effect of long-term culture of mouse embryonic stem cells under low oxygen concentration as well as on glycosaminoglycan hyaluronan on cell proliferation and differentiation.
abstract: OBJECTIVE
  Maintaining undifferentiated stem cells in defined conditions is of critical importance to improve their in vitro culture. We have evaluated the effects of culturing mouse stem (mES) cells under physiological oxygen concentration as well as by replacing fibroblast feeder layer (mEF) with gelatin or glycosaminoglycan hyaluronan (HA), on cell proliferation and differentiation.
  
  
  METHODS
  After 3 days culture or after long-term cell culture under different conditions, levels of apoptotic cell death were determined by cell cycle and TUNEL (TdT-mediated dUTP nick end labelling) assays and levels of cell proliferation by CFSE (5-(and-6)-carboxyfluorescein diacetate succinimidyl ester) labelling. We assessed spontaneous differentiation into cardiomyocytes and mRNA expression of pluripotency and differentiation biomarkers.
  
  
  RESULTS
  After 3 days culture under hypoxic conditions, levels of proliferation and apoptosis of mES cells were higher, in correlation with increase in intracellular reactive oxygen species. However, when cells were continuously grown for 1 month under those conditions, the level of apoptosis was, in all cases, under 4%. Hypoxia reduced spontaneous differentiation of mES into cardiomyocytes. Long-term culture on HA was more effective in maintaining the pluripotent state of the mES cells when compared to that on gelatin. Level of terminal differentiation was highest on mEF, intermediate on HA and lowest on gelatin.
  
  
  CONCLUSIONS
  Our data suggest that hypoxia is not necessary for maintaining pluripotency of mES cells and appeared to be detrimental during ES differentiation. Moreover, HA may offer a valuable alternative for long-term culture of mES cells in vitro.
authors.affiliation: Departamento de Reproducción Animal INIA, Madrid, Spain. ramirez@inia.es
authors.forename: M Á
authors.initials: MÁ
authors.lastname: Ramírez
authors.forename: E
authors.initials: E
authors.lastname: Pericuesta
authors.forename: M
authors.initials: M
authors.lastname: Yáñez-Mó
authors.forename: A
authors.initials: A
authors.lastname: Palasz
authors.forename: A
authors.initials: A
authors.lastname: Gutiérrez-Adán
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism* / Q000502:physiology; D006820:Hyaluronic Acid / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D013997:Time Factors
chemical_list: D006820:Hyaluronic Acid; D010100:Oxygen