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. 2014 Oct 15;28(20):2205–2218. doi: 10.1101/gad.245142.114

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© 2014 Cai et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 5. — Hormonal treatment induces a collaborative niche for MaSC expansion in culture. (A) Coculture experiments of basal and luminal cells were established as illustrated. (B) A representative picture of colonies formed in the coculture system. Bar, 20 μm. (C) Quantification of the numbers of basal DsRed-marked colonies that were maintained in the described culture conditions for three passages. The numbers of primary colonies were similar. The numbers of secondary and tertiary colonies were increased with hormonal treatment. The increasing colony numbers were inhibited by Fz8CRD in each passage. (***) P < 0.01. (D) Coculture was maintained for 10 passages. Colony numbers were continuously increased with hormonal treatment. One of three similar independent experiments is shown. (E–G) Tertiary basal DsRed-marked colonies maintained in the described culture conditions were transplanted into cleared fat pads by limiting dilution. Recipient fat pads were harvested at 8-wk post-transplantation. (E) DsRed outgrowth was detected in a higher percentage when cultured with hormones, whereas Fz8CRD inhibited the DsRed-marked basal reconstitution rate. Results are combined from three independent experiments. (F) Representative pictures in virgin recipients are shown in whole mount with live DsRed and keratin marker expression in tissue sections. (G) The reconstituted mammary gland displayed normal alveogenesis during pregnancy and produced abundant milk protein. Bars: in the whole mount, 2 mm; in the tissue sections, 20 μm. (H) Model of the differentiated luminal cells synergistically regulating the self-renewal of MaSCs. Hormone stimulates the expression of Wnt4 in PR⁺ luminal cells and Rspo1 in PR⁻ luminal cells. Rspo1 and Wnt4 collaborate to induce basal cell Wnt response, enabling MaSC property maintenance or their expansion in number.