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. 2019 Mar;82(1):e65.
doi: 10.1002/cpcb.65. Epub 2018 Sep 28.

Isolation, Culture, and Differentiation of Mammary Epithelial Stem/Progenitor Cells from Fresh or Ex Vivo Cultured Human Breast Tissue

Guang Chen  1   2 , Hakim Bouamar  1 , Lu-Zhe Sun  1
Affiliations

Isolation, Culture, and Differentiation of Mammary Epithelial Stem/Progenitor Cells from Fresh or Ex Vivo Cultured Human Breast Tissue

Guang Chen et al. Curr Protoc Cell Biol. 2019 Mar.

Abstract

In vivo transplantation is the gold standard method for characterization of stem/progenitor cell self-renewal, tissue regeneration, and tumorigenesis. The method requires an enriched population of stem cells that represent a small fraction of a given tissue. An enriched population of stem/progenitor cells increases the likelihood of engraftment and reduces the number of recipient animals needed for in vivo transplantation. Methods for mammosphere formation by mammary epithelial stem and progenitor cells have been widely adopted for enriching stem/progenitor cells, allowing researchers to study genetic and epigenetic properties, interaction with other cell types, and differentiation and oncogenic transformation. The generation of mammospheres is complex, however, involving many steps and requiring particular skill. Here we describe a detailed mammosphere protocol, including isolation and culture of human primary mammary epithelial stem/progenitor cells and their differentiation and passage in 3D organoid culture. We also describe a protocol for ex vivo culture of fresh human breast tissue for use in assays of clinical treatment. Step-by-step instructions detail tissue handling through passage of the stem/progenitor cell-generated 3D organoids, which can be used to assess the properties, function, and neoplastic transformation of mammary stem/progenitor cells. © 2018 by John Wiley & Sons, Inc.

Keywords: mammosphere; primary epithelial cells; progenitors; stem cells.

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Figures

Figure 1
Figure 1
Ex vivo culture of fresh breast tissue. The tissue was cut into small pieces and cultured on the sponge in a well of a 6-well plate as shown in the figure.
Figure 2
Figure 2
Flow cytometer sorting profile of input human mammary cell preparations showing the gates used to isolate fractions of stroma cells, mature luminal cells, luminal and basal cells containing progenitor/stem cells.
Figure 3
Figure 3
Mammospheres formed by luminal progenitor cells and basal stem cells after 6 days of cultivation. Scale bar represents 25 μm.
Figure 4
Figure 4
3D organoids formed by luminal progenitor and basal stem cell spheres after 7 days of cultivation. Scale bar represents 50 μm
See this image and copyright information in PMC

References

    1. Deome KB, Faulkin LJ, Jr, Bern HA, Blair PB. Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res. 1959;19(5):515–520. - PubMed
    1. Dong Q, Wang D, Bandyopadhyay A, Gao H, Gorena KM, Hildreth K, … Sun LZ. Mammospheres from murine mammary stem cell-enriched basal cells: clonal characteristics and repopulating potential. Stem Cell Res. 2013;10(3):396–404. doi: 10.1016/j.scr.2013.01.007. - DOI - PMC - PubMed
    1. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003;17(10):1253–1270. doi: 10.1101/gad.1061803. - DOI - PMC - PubMed
    1. Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha MS. Stem cells in normal breast development and breast cancer. Cell Prolif. 2003;36(Suppl 1):59–72. - PMC - PubMed
    1. Dontu G, Wicha MS. Survival of mammary stem cells in suspension culture: implications for stem cell biology and neoplasia. J Mammary Gland Biol Neoplasia. 2005;10(1):75–86. doi: 10.1007/s10911-005-2542-5. - DOI - PubMed

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