Expanding Sca-1(+) mammary stem cell in the presence of oestrogen and growth hormone.
BACKGROUND
Sca-1 is controversial as a mammary stem cell marker in the literature, which may be due to the different isolation protocols and culture media used in different laboratories. The object of our study is to establish the Medium to promote the proliferation of mammary stem cell and explore the possibility of Sca-1 as mammary stem cell marker.


METHODS
We used BM medium supplemented with different concentration of 17Β-oestradiol and GH to find out MaECM medium which promoted the proliferation of mouse mammary epithelial cells and inhibited the growth of fibroblasts. Flow cytometry was used to isolate Sca-1(+) and Sca-1(-) cell populations from cultured mammary epithelial cells. Mammary fat pad transplantation and Mammosphere- forming assay were done to confirm the stem cell potential of Sca-1(+) cells. Differentiating culture was used to detect the differentiation potential of Sca-1(+) cells. Real-time PCR was carried out to analyse the expression of mammary stem cell-related genes in Sca-1(+) cells.


RESULTS
We first selected the medium suitable for mammary stem cell growth. Stem cell medium BM was used to culture mammary organoids, which generated many fibroblasts. We established MaECM medium supplemented with oestrogen and growth hormone (GH), in which oestrogen promoted mammary epithelial cell proliferation and inhibited fibroblast growth, and GH obviously enhanced the effect of oestrogen on mammary epithelial cell proliferation. Flow cytometry showed that 50% of cells were Sca-1(+) under the culture of MaECM medium. We confirmed that Sca-1(+) cells regenerated mammary outgrowths when transplanted in vivo, formed mammospheres in vitro and differentiated into luminal epithelial cells with milk-secreting function and myoepithelial cells under Matrigel culture. Furthermore, gene expression analysis by Real-time PCR revealed that Sca-1(+) cells expressed markedly higher levels of mammary stem cell-related genes in comparison to Sca-1(-) cells.


CONCLUSIONS
Our research demonstrates that Sca-1(+) mammary stem cells can be more easily isolated when cultured in the presence of oestrogen and GH.
Center of Excellence in Tissue Engineering, Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, 5 Dongdansantiao, Beijing 100005, China.
Xiaowei
X
Dou
Bin
B
Zhang
Rui
R
Liu
Jing
J
Li
Dan
D
Shi
Chunhua
C
Lu
Xishan
X
Zhu
Lianming
L
Liao
Zhijian
Z
Du
Robert Chunhua
RC
Zhao
D000818:Animals; D000950:Antigens, Ly / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D003470:Culture Media; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D013006:Growth Hormone / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D060888:Real-Time Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
D000950:Antigens, Ly; D003470:Culture Media; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D004958:Estradiol; D013006:Growth Hormone