Characteristics of human embryonic stem cells cultured on human- and mouse- origin feeder layers / 中国组织工程研究

ABSTRACT

BACKGROUND: Whether human embryonic stem cells (hESCs) cultured on different feeder layers can maintain identical or similar characteristics remains unclear.OBJECTIVE: To compare the characteristics of hESCs cultured on human- and mouse- origin feeder layers.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Kunming Institute of Zoology, Chinese Academy of Sciences between September 2007 and February 2009.MATERIALS Two ICR pregnant mice with 12.5-13.5 embryonic days were provided by Animal Center of Kunming Medical College. Immortalized human adult fibroblast (HAFi) cell line was presented by School of Medicine, Johns Hopkins University (USA). hESCs line was provided by Kunming Institute of Zoology, Chinese Academy of Sciences.METHODS: Mouse embryonic fibroblasts (MEF) were harvested from ICR mice by trypsinization method. HAFi were conventionally cultured. After γ ray treatment, two kinds of cells were incubated on 6-well gelatin-coated plate with density of 2.5×10~4/cm~3. hESCs were cultured on HAFi or MEF feeder cells containing β-mercaptoethanol DMEM/F12 and basic fibroblast growth factor.MAIN OUTCOME MEASURES: Morphology, expressions of specific molecular markers, Oct-4 positive rate, as well as cell doubling time of hESCs cultured by two methods were compared.RESULTS: ①BG02 cells on MEF and HAFi shared the similar morphology and characteristic pluripotency markers, which expressed SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct-4, but not SSEA-1. However, the proportion of positive Oct-4 cells in hESCs colonies maintained on MEF was lower than that of HAFi (P < 0.05) with shorter doubling time (P < 0.05).CONCLUSION: hESCs cultured on MEF and HAFi represent some differences in the growth and pluripotency characteristics.