Mammary stem cells have myoepithelial cell properties

Abstract

Contractile myoepithelial cells dominate the basal layer of the mammary epithelium and are considered to be differentiated cells. However, we observe that up to 54% of single basal cells can form colonies when seeded into adherent culture in the presence of agents that disrupt actin-myosin interactions, and on average, 65% of the single-cell-derived basal colonies can repopulate a mammary gland when transplanted in vivo. This indicates that a high proportion of basal myoepithelial cells can give rise to a mammary repopulating unit (MRU). We demonstrate that myoepithelial cells, flow-sorted using two independent myoepithelial-specific reporter strategies, have MRU capacity. Using an inducible lineage-tracing approach we follow the progeny of myoepithelial cells that express α-smooth muscle actin and show that they function as long-lived lineage-restricted stem cells in the virgin state and during pregnancy.

Figure 1. The vast majority of mouse basal cells express alpha-smooth muscle actin (αSMA). High EpCAM expression enriches for mammary stem cells and proliferating cells

(a) Flow cytometry plot showing stromal (black), luminal (blue) and basal (red) cell populations. The basal population has been subdivided into EpCAM^high (brightest 20%) and EpCAM^low (remaining 80%) subpopulations (b) Double-sorted basal EpCAM^high and EpCAM^low cells stained by immunocytochemistry for αSMA and isotype control inset. Mean (± SEM) of 5 independent experiments. Scale bars = 50 μm. (c) Relative mRNA transcript abundance of Acta2, Myh11 and Oxtr in basal EpCAM^high and EpCAM^low cells as detected by real-time PCR. Data normalised to Actb and Rplp0 reference genes. Mean (± SEM) of 4 independent experiments. (d) Relative abundance of αSMA protein in basal EpCAM^high and EpCAM^low cells as detected by Western blot (left). Data normalised to cytokeratin 14 (CK14) abundance. Mean (± SEM) of 3 independent experiments. A representative blot (right) showing protein standards (red), CK14 (55kDa) and αSMA (42kDa) bands (green). (e) Mammary repopulating unit (MRU) frequency of sorted basal EpCAM^high and EpCAM^low cells. Data for Basal EpCAM^high and Basal EpCAM^low pooled from 5 independent experiments. ** P = 0.0002. (f) Percentage of flow-sorted basal EpCAM^high and EpCAM^low cells positive for IdU/BrdU. Data is presented as the mean (± SEM) of 9 independent experiments. * P = 0.04. (g) Distribution of IdU/BrdU⁺ cells within the basal population.

Figure 2. Short-term culture increases MRU numbers by ~460-fold

(a) MRU frequency in freshly isolated and post-cultured basal cells with estimated number (± SEM) of MRUs per dish. Data pooled from 5 independent experiments for non-cultured basal and from 4 independent experiments for 7-day-cultured basal cells. (b) Representative engrafted fat pads derived from non-cultured basal cells and 7-day-cultured basal cells. Scale bar = 2 mm.

Figure 3. A high proportion of single-cell-derived basal colonies contain a MRU

(a) Table showing single-cell cloning efficiency of basal EpCAM^high and EpCAM^low cells and the proportion of single-cell-derived basal colonies that engrafted when transplanted into cleared mammary fat pads of NSG pups. Cloning efficiencies are presented as the mean ± SEM, with data pooled from 4 independent experiments. (b) Representative images of a GFP⁺ basal colony and a GFP⁺ engraftment from a transplanted basal colony (which was derived from a single basal EpCAM^high cell); scale bars = 500 μm. Images of sections through an engrafted fat pad stained for various markers by immunohistochemistry; scale bars = 100 μm.

Figure 4. Myoepithelial cells have MRU activity

(a) Sections of mammary glands from Acta2-GFP mice (C57BL/6J) stained for αSMA and GFP by immunohistochemistry. Representative image seen in 3 independent samples. Scale bar = 100 μm. (b) Flow cytometry dot plot showing GFP⁺ (αSMA⁺) events back-gated onto the EpCAM and CD49f plot. (c) CFE of basal αSMA⁺ and basal αSMA⁻ cells. Mean (±SEM) of 4 independent experiments. ** P = 0.0003. (d) MRU frequency in basal αSMA⁺ and basal αSMA⁻ cells. Data pooled from 3 independent experiments. ** P = 0.0002. (e) Wholemount (left) and sections (center and right) of X-gal-stained mammary glands from Myh11-Cre-GFP;Rosa26LacZ virgin (left and center) and 15-day-pregnant (right) mice. Images were observed in 8 (left panel) and 4 (central and right panels) independent samples. Scale bar for left panel = 1.8 mm. Scale bar for central panel = 120 μm. Scale bar for right panel = 50 μm. (f) Flow cytometry dot plot showing resolution of total basal and basal Myh11⁺ cells. Five independent samples analysed. (g) X-gal-stained colonies derived from flow-sorted basal and luminal cells. Scale bar = 5 mm. (h) Mammospheres derived from basal Myh11⁺ and total basal cells. Scale bar = 290 μm. (i) Mammosphere forming efficiency of basal Myh11⁺ and total basal cells. Data showing the mean sphere-forming efficiency from one of two independent experiments is presented. Data is derived from 3 technical replicates. (j) MRU frequency in basal Myh11⁺ and total basal cells. Data pooled from 3 independent experiments. (k) Wholemount and section of a primary engraftment (above) developed from basal Myh11⁺ cells in virgin (left and central) and 12-day-pregnant (right) hosts and a secondary engraftment (below) in a virgin host. Nineteen primary and three secondary outgrowths were x-gal stained in whole mount, of which three primary and three secondary outgrowths were sectioned. Scale bars for wholemounts = 1.8 mm. Scale bars for sections = 50 μm.

Figure 5. Myoepithelial cell derived clones can undergo expansion within the basal layer of intact mammary glands and survive after multiple pregnancies

Experimental schedule for (b) and (c). (b) Sections of whole-mount x-gal stained mammary glands from Acta2-Cre-ER^T2;Rosa26LacZ mice injected with Tamoxifen at 4 weeks and dissected 1 and 6 weeks later, as indicated. Scale bar = 52 μm (c) A graph showing the percentage of myoepithelial cell clones containing 1, 2-4 and > 4 LacZ-positive cells in the glands dissected 1 and 6 weeks after Tamoxifen injection. The values shown are means+SEM from 4 mammary fat pads at each time point. *P < 0.01. For details see On Line Methods. (d) Section of whole-mount x-gal stained mammary gland from Acta2-Cre-ER^T2;Rosa26LacZ mouse injected with Tamoxifen at 4 weeks and dissected 5 weeks later, on day 8 of pregnancy. The arrows point to LacZ-positive cells in the basal layer of small lateral branches and emerging alveolar buds. Scale bar = 90 μm. (e) Experimental schedule for (f) and (g). (f) Fragments (upper panels) and sections (lower panels) of whole-mount x-gal stained mammary glands from 13-week-old virgin, 12-(P12) and 17-day-pregnant (P17) Acta2-Cre-ER^T2;Rosa26LacZ mice dissected 5 and 6 weeks after Tamoxifen injection, as indicated. Arrows point to LacZ-positive alveoli. D, mammary duct, BV, blood vessel. Scale bar = 0.35 mm for upper panels and 65 μm for lower panels. (g) A graph showing the percentage of myoepithelial cell clones containing 1, 2-4 and > 4 LacZ-positive cells in the ducts from virgin and pregnant Acta2-Cre-ER^T2;Rosa26LacZ mice. V1 and V2, two 13-week-old virgin mice; P12, P15 and P17, 1^st pregnancy days 12, 15 and 17, respectively. The values represent means+SEM of 3 counts performed in different gland areas (for details see On Line Methods).

Figure 6. The progeny of myoepithelial cells is restricted to the basal cell layer and survives after multiple pregnancies

(a) A graph showing the percentage of LacZ-positive cells in basal and luminal cell populations isolated from Acta2-Cre-ER^T2;Rosa26LacZ mouse and x-gal stained in suspension. V, 13-week-old virgin, P15 and P17, 1^st pregnancy days 15 and 17, respectively; 3^rdP6 and 3^rdP15, 3^rd pregnancy, days 6-7 and 15, respectively. Experimental schedule for V, P15 and P17 is shown in Figure 5e, for 3^rdP6 and 3^rdP15, in (g). Data shown for P15, P17 and 3^rdP15 were obtained with inguinal mammary glands from one mouse, in each case, for V and 3^rdP6, with pooled inguinal glands from two 13-week-old virgin and two 6-7-day-pregnant mice, respectively. (b) A mammary gland fragment from virgin Acta2-Cre-ER^T2;R26^mTmG mouse analyzed five weeks after Tamoxifen injection. Scale bar = 0.35 mm. (c) and (d) Flow cytometry dot plots showing GFP expression in luminal and basal cell populations isolated from virgin (c) and 1-day-lactating (d) Acta2-Cre-ER^T2;R26^mTmG mice. Experimental schedule for (b-d) was same as shown in Figure 5e. Red ovals indicate luminal (L) and basal (B) cell populations. (e) and (g) Experimental schedules for (f) and (h), respectively. (f) Fragment of whole-mount x-gal stained mammary gland from Acta2-Cre-ER^T2;Rosa26LacZ mice injected with Tamoxifen at 4 weeks and analysed 25 weeks later, on day 16 of the involution following 2^nd pregnancy (2^nd Inv16). Scale bar = 0.87 mm. In (e) and (g), 1^st and 2^nd P,L,Inv, first and second pregnancy, lactation and involution cycles, respectively. (h) A fragment (left) and a section (right) of x-gal stained mammary glands from Acta2-Cre-ER^T2;Rosa26LacZ mice injected with Tamoxifen at 4 weeks and analysed 23 weeks later, on day 15 of third pregnancy (3^rdP15). Scale bar = 0.87 mm, left panel and 50 mm, right panel. Arrows in (f) and (h) point to alveoli. BV, blood vessel; D, mammary duct.