Creation and effectiveness of mammary stem cell medium

doi:10.3969/j.issn.2095-4344.2014.10.017

Abstract

Abstract:

BACKGROUND: Now in mammary stem cell research, no proper mammary stem cell medium is provided to culture mammary stem cells.
OBJECTIVE: To create a mammary stem cell medium 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^- cell populations 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, small-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⁺cell reached 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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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Key words: stem cells, breast neoplasms, fibroblasts, culture media, fluoroimmunoassay

CLC Number:

R394.2

Zhang Jun-hong, Yang Jing, Dou Xiao-wei, Wang Chun-hua, Li Qing, Zhao Chun-hua. Creation and effectiveness of mammary stem cell medium[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(10): 1585-1590.

Figures/Tables 3

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