ABSTRACT
BACKGROUND:Now in mammary stem cellresearch, no proper mammary stem cellmedium is provided to culture mammary stem cells. OBJECTIVE:To create a mammary stem cellmedium and validate its application by isolating Sca-1+mammary stem cells. METHODS:We first used BM medium to culture mammary organoids, and after 6 days, the expression of Sca-1 and vimentin was detected in fibroblasts by immunofluoresence method. Then, we established MaECM medium which arrested fibroblasts growth. After 6 days culture of mammary organoids by MaECM medium, Sca-1+and Sca-1-cellpopulations were sorted out by magnetic sorting and the purity was analyzed by flow cytometry. Sorted 1×104 Sca-1+or Sca-1-cells were transplanted into the bilateral mammary fat pads of four mice, and after 6-8 weeks, the fat pads were harvested for whole-mount immunohistochemical analysis and hematoxylin-eosin staining. RESULTS AND CONCLUSION:After 6 days culture of mammary organoids under BM medium, smal-sized colonies were generated around lots of fibroblasts. Immunofluoresence staining detected strong expression of vimentin and Sca-1 in fibroblasts, indicating that the BM medium is not suitable to isolate Sca-1+mammary stem cell. The MaECM medium promoted the proliferation of mammary epithelial cells whereas arrested fibroblasts growth. After 6 days culture of mammary organoids under MaECM medium and magnetic sorting, the flow cytometry showed that the purity of Sca-1+cellreached 92%and 5%in the Sca-1+and Sca-1-population, respectively. The results from transplantation test showed that six mammary outgrowths were regenerated out of eight injected fat pads in the Sca-1+cells transplantation, but in the Sca-1-transplantation population, one mouse died and the other transplants failed to produce outgrowths. We developed the MaECM medium which promoted the proliferation of mammary epithelial cells whereas arrested Sca-1+fibroblasts growth. Using the medium, we confirmed that Sca-1+mammary cells have capacity of isolating mammary stem cells.
ABSTRACT
Studies show that the Notch signal transduction pathway involves in the regulation and control of cell proliferation,differentiation and apoptosis.Aberrant Notch signaling transduction pathway can induce breast cancer in transgenic mice.High expressions of either Notch receptors or their ligands have been linked to poor prognosis in patients with breast cancer.Inhibition of Notch signal transduction pathway may be beneficial for breast cancer treatment.
ABSTRACT
Mammary stem cell is the subpopulation in mamnlary gland,capable of self-renewal and dif-ferentiating into ductal,alveolar and myoepithelial cells.Recently,several laboratories have employed various methods identifying mammary stem cells,including Sca-1,Hoechst dye efflux,CD24,CD3 1-CIM5-Terl19- CD24+CD29hi and so on.which offer a useful tool to elucidate the mechanism of mammary slem cell self-renew- al and design more effective therapies of mammary cancer.
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.