Creation and effectiveness of mammary stem cell medium / 中国组织工程研究

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.