ABSTRACT
BACKGROUND: The mostly accepted standpoints regarding adult stem cell plasticity are that adult stem cells existing in various organs contain primitive pluripotent stem cells. However, the origin, identification methods and biological characteristics of pluripotent stem cells remain unclear. OBJECTIVE: To investigate if a kind of pluripotent stem cells with more primitive character can be isolated from adult tissues. DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Center of Tissue Engineering, Basic Medical Department of Peking Union Medical College from March 2007 to January 2008. MATERIALS: Pregnant BALB/C rats on gestation day of 12.5-14.5 d were adopt in the study. METHODS: The fetal perisome samples were attained from pregnant BALB/C rats under sterile conditions, and made into cell suspension by trypsin digestion method. Immunomagnetic beads were used to sort Sca-1+CD117-Lin-cells. When 70%-80% cells were mixed, the cells were subcultured, and the 5 passage cells were taken in the experiment. MAIN OUTCOME MEASURES: Microstructure, ultrastructure, growth characteristics, immunophenotype and embryonic stem cell associated antigen were analyzed by inverted phase contrast microscope, scanning electron microscope, flow cytometry and RT-PCR respectively. Histochemical and immunofluorescence staining were used to investigate the potentiality of the Sca-1+CD117-Lin-cells differentiated into trilaminar germ disc. RESULTS: Sca-1+CD117-Lin-cells adhering to the wall with spindle or short fusiform shapes, which could amplification fast and stablely to 50 passages. Cells were found logarithmic growth on days 3-7, after that cell growth reached platform stage. There were about 32 h for doubling time, 90.43% cells were at the G0/G1 phase, and 9.57% at the G2/M+S phase. The cells expressed high levels of Sca-1, no levels of CD117, CD14, CD19, CD31, CD34, CD45 and Flk-1, and moderate levels of CD44. The phenotype of human BMSCs was stabilization. Embryonic stem cell associated antigen Sox2, Oct-4 or Nanog were showed positive expression. RT-PCR indicated that the cells were able to differentiate into osteoblasts, adipocytes, chondrocyte, hepatocyte-like cells and neuroglia cells in vitro. CONCLUSION: The Sca-1+CD117-Lin-cells isolated from adult tissue are primitive multiple potential adult stem cells, which can differentiate into trilaminar germ disc.