Isolation, incubation and identification of mouse embryonic hepatic stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: Fetal liver cells can have stronger abilities to proliferation and differentiation and lower immunogenicity compared to bone marrow stem cells. However, there are few studies on direct isolation and culture of embryonic hepatic stem cells (EHSCs). OBJECTIVE: To isolate and cultivate EHSCs in vitro and to identify their biological features. DESIGN, TIME AND SETTING: The cytology in vitro controlled study was performed at the Chongqing Key Laboratory of Neurology from March to June 2008. MATERIALS A total of 9 SPF Kunming fetal mice aged 13.5 days were obtained from Animal Experimental Center of Chongqing Medical University. METHODS: Collagenase + EDTA digestion and differential adherence method were used to isolate EHSCs, which were then incubated at 2?108 /L. Cells were digested and passaged when 80%-90% cells were confluent. Using streptavidin-biotin-peroxidase complex technique, adhered cells following 5 days of incubation were labeled with various EHSC surface marker. MAIN OUTCOME MEASURES: Morphology, passage, amplification and surface marker surface of EHSCs were measured. RESULTS: The isolated EHSCs adhered to the culture plastic and presented pykno-round cells and distinct borderline 24 hours after cultivation in vitro. Cells grew spindle-shaped 3 days. After 7 days they grew like epithelium. Cell amplified speed following passage did not have significant changes. Cells still presented epithelium-like shape at the passage 5. The adhered cells at day 5 following primary incubation were positively for human stem cell factor receptor and alpha fetoprotein, and negatively for albumin and cytokeratin 19. CONCLUSION: EHSCs were positively for human stem cell factor receptor and alpha fetoprotein, and negatively for albumin and cytokeratin 19 in early primary culture. This indicated that the cultivated cells are proved to be primordial progenitor cells and still in undifferentiated early phase.