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. 2003 May 15;17(10):1253-70.
doi: 10.1101/gad.1061803.

In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells

Gabriela Dontu  1 , Wissam M Abdallah, Jessica M Foley, Kyle W Jackson, Michael F Clarke, Mari J Kawamura, Max S Wicha
Affiliations

In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells

Gabriela Dontu et al. Genes Dev. .

Abstract

Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.

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Figures

Figure 1
Figure 1
(A) Mammosphere morphology and optimal growth conditions. Mammosphere after 15 d of cultivation. (B) The sphere-formation efficiency is considerably higher in serum-free medium in the presence of EGF and/or bFGF than in the absence of growth factors. Data are presented as means ± S.E.M.
Figure 2
Figure 2
Cellular and ECM composition of secondary mammospheres. (A) Nomarski DIC (differential interference contrast) image of an unstained mammosphere. Fifteen-day-old secondary mammospheres immunostained in suspension (B–I), after centrifugation on slides (J–L), or as paraffin-embedded sections (N–R). (M) H&E-stained section through a paraffin-embedded sphere. The primary antibodies used are indicated under each picture. FITC-labeled or Texas-red-labeled secondary antibodies were used. Laminin immunostaining was positive in ∼20% of mammospheres. (P) A representative positive-staining sphere is shown.
Figure 3
Figure 3
Experimental design for assessing the differentiation and self-renewal potential of cells grown as mammospheres. (A) Differentiation is tested by plating cells at clonogenic densities. (i) Differentiation into ductal and myoepithelial cells is assessed on cells cultivated on collagen in the presence of serum. (ii) Differentiation into ductal, myoepithelial, and alveolar cells is tested in the same conditions as in i with the addition of prolactin and Matrigel as an overlayer. (iii) The ability to generate complex ductal-alveolar structures is tested in 3D Matrigel culture. (B) Self-renewal is tested by evaluating the ability of mammosphere-derived cells to form new spheres, containing multipotent cells.
Figure 4
Figure 4
Mammospheres contain multipotent cells, capable of differentiating along all three lineages in the mammary gland. (A) Monolineage ductal epithelial colony immunostained for ESA. (B) Monolineage myoepithelial colony immunostained for CD10. (C) Bilineage colony (ESA, red; CD10, purple). The percentage of cells with bilineage differentiation potential is considerably higher in primary mammospheres compared with dissociated tissue. (D) Secondary mammospheres contain virtually only bipotent progenitors. (E) Triple-lineage colony (ESA, red; CD10, purple). (F) The same colony immunostained for β-casein (FITC, green) to identify the third lineage, alveolar cells. (G) Triple-lineage colony (ESA, brown; CD10, purple; β-casein, red). (H) Distribution of colony types generated by mammospheres derived cells in conditions that allow differentiation along all three lineages. (I) Ductal-acinar structure generated by a single cell in 3D Matrigel culture. (J) Acinar structure generated by a single cell in 3D Matrigel culture. (K) Acinar structure, immunostaining for myoepithelial lineage (CD10, FITC, green) and ductal epithelial lineage (ESA, Texas red). (L) Acinar structure, immunostaining for β-casein (red).
Figure 5
Figure 5
Secondary and later generation mammospheres contain multipotent progenitor cells. (A) Colony generated clonally by a secondary mammosphere-derived cell; immunostaining with ESA (red) and CD10 (purple). (B) Detail of the colony presented in A; ESA-positive cells (black arrows), CD10-positive cell (white arrows), cells expressing both ESA and CD10 (black arrowheads), and cells negative for both markers (white arrowheads). (C) Clonality of secondary mammospheres generated in mixed culture. HMECs grown as primary mammospheres were transfected with a retrovirus encoding for EGFP or DsRed2. Secondary mammospheres formed by the EGFP- and DsRed2 mammosphere-derived cells, mixed in equal ratio in suspension culture. The vast majority of the secondary mammospheres are monochromatic.
Figure 6
Figure 6
Hoechst 33342 staining of HMECs: uncultured cells from dissociated tissue (A,B) and mammosphere-derived cells (C,D). A low Hoechst staining population (SP) can be identified in both uncultured mammary cells (gated R2; A) and cells grown as mammospheres (gated R2; C). The majority of cells stain intensely with Hoechst (gated R4). (B,D) The exclusion of the Hoechst dye is blocked by Verapamil. Only the sorted SP cells (gated R2) generate mixed lineage colonies on collagen (E) and mammospheres in suspension culture (Table 2). Data are presented as means ± S.E.M.
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References

    1. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumori-genic breast cancer cells. Proc Natl Acad Sci. 2003;100:3983–3988. - PMC - PubMed
    1. Alvi A, Clayton H, Joshi C, Enver T, Ashworth A, Vivanco MM, Dale T, Smalley M. Functional and molecular characterisation of mammary side population cells. Breast Cancer Res. 2002;5:E1. - PMC - PubMed
    1. Blau HM, Brazelton TR, Weimann JM. The evolving concept of a stem cell: Entity or function? Cell. 2001;105:829–841. - PubMed
    1. Bocker W, Moll R, Poremba C, Holland R, Van Diest P, Dervan P, Burger H, Wai D, Ina DR, Brandt B, et al. Common adult stem cells in the human breast give rise to glandular and myoepithelial cell lineages: A new cell biological concept. Lab Invest. 2002;8:737–746. - PubMed
    1. Brown AMC. Wnt signaling in breast cancer: Have we come full circle? Breast Cancer Res. 2001;3:351–355. - PMC - PubMed

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