Isolation, Culture And Evaluation Of Mammary Epithelial Stem Cells In Mice

Mammary gland is a unique organ, which can postnatally regenerate, terminally differentiate, and involute upon successive cycle of pregnancy, lactation, and involution, respectively, and provides an optimal model for the study of organogenesis, differentiation, apoptosis, and pattern formation involved in development. This impressive renewal capacity is due to the function of a multipotent mammary gland stem cell population. The research of mammary epithelial stem cells has many significant meaning such as rebuilding woman breasts after surgical sectioning because of the breast tumors or cancers, enlarging lady breast for more beautiful, regenerating transgenic mammary glands secreting medical or tonic proteins for theraping genetic deficient diseases or for healthier in human, and establishing models for organogenesising, cell differentiation and regeneration, and so on.Thus, in order to investigate the developmental pathways not only involved in the regulation of growth and patterning, but also in the determination of cell lineages and differentiation, we utilized the fluorescent immunohistochemical methods, flow cytometry analysis sorting (FACS) and molecular methods to investigate the developmental law of mammary gland at the different developmental stages , distribution of the stem cells in mammary gland, the methods of isolation, culture and evaluation for the stem cells, the multipotent abilities in vivo and in vitro, and the efficient cultural system for stem cells enriched in vitro.The results showed below:1. The optimal stage of clearing the mammary gland from fat pad is the 3-week-old in balb/c mice.2. There are stem cells in the mammary glands at any stage of postnatal development.3. The best stages of gaining the stem cells from the mammary glands are when the mice are 6 to 8 weeks old or in pregnant stage.4. The mammary epithelial stem cells, sorted with anti-Scal-1 antibody by the FACS, have multipotent abilities because they could be induced to mimic the function of mammary gland to produce the milk component B-casein in vitro and could regenerate the functional mammary glands as injecting into the cleared fat pads.5. Firstly in mammary epithelial stem cells, we investigated that there were some cells to express the specific genes, defined in embryonic stem cells or other known stem cell lineages, such as Oct-4, Rex-1, IntegrinB1, bcrp1/acbg 2 and CD 34.6. Estabished an optimal cultural system for enriched mammary epithelial stem cells, firstly. After being cultured in optimal cultural system in vitro for 72 hours, we could sort 30 % of mammary stem cells (Scal-1+) in the cells derived from the mammary orgnoids and the percentages of stem cells was higher than that of the reference, but less that of the cells cultured in vitro for 22 hours.7. We sorted several percentages of CD34+ cells derived from the cultured mammary gland orgnoids and the percentages of CD34+ cells in 22-hour cultural system was lower than that of the 72-hour cultural system. These results need to be investigated further.In summary, we should investigate the more efficient cultural system for mammary orgnoids in vitro for enriched more mammary epithelial stem cells, and to screen the expression profiles in mammary epithelial stem cells to discover the specific marker for mammary epithelial stem cells in order to utilize the model of mammary epithelial stem cells to regenerate transgenic mammary glands in cow or sheep for human health, to uncover the mechanism of organogenesis, and differentiation.