GFP expression occurs in cap cells of the TEBs at puberty. (A) TEBs (arrows) in carmine whole-mount-stained 5-wk mammary tissue. (B) GFP⁺ TEBs (green, arrows) and blood vessels (green, arrowheads) in 4-wk transgenic whole-mount mammary tissue. (C) One section of a 4-wk TEB. Cap cells are GFP⁺ (short arrows), and some GFP⁺ cap cells are among the GFP⁻ body cells (long arrows). (Red) Phalloidin-Alexa 594; (blue) DAPI. (D) GFP⁺ cap cells (green) are also p63⁺ (red); shown in enlargement (left panel; GFP and p63 only), and separately (middle two panels). Arrows mark the cap cell layer. (E) Cap cells (green) are SMA⁺ (red). (F) Most GFP⁺ cap cells are Ki67⁺ (red). (G) Some GFP⁺ cap cells were labeled with BrdU (red) within 4 h of BrdU injection. Bars: A,B, 1 mm; C,D,F,G, 100 μm; C (enlarged), E, 50 μm. (H) Histogram shows that 93.8% ± 2.4% of GFP⁺ cells in TEBs are Ki67⁺, and 34.6% ± 5.9% of GFP⁺ cells were labeled with BrdU during a 4-h exposure. Data represent mean ± SD; n = 20 TEBs.

Characterization of transplantation outgrowths from puberty GFP⁺ cap cells. (A) GFP⁺ TEBs (green, arrow) in an outgrowth from 40 GFP⁺ cap cells; enlarged TEBs are shown in the inset. (*) Approximate injection site. (B) Carmine alum-stained ductal outgrowths from four (three to eight) cells, or one single GFP⁺ cap cell (inset), respectively. (C) X-gal staining shows the TEBs (arrowheads) in a second serial outgrowth originated from eight (six to 10) primary GFP⁺LacZ⁺ cap cells. (D,E) Outgrowth TEBs consist of GFP⁺ SMA⁺ cap cells (D) and E-cad⁺ body cells (E). (F–I) Pregnancy-induced lobulo–alveolar formation in lactating outgrowths (lactation day 0.5) derived from 40 GFP⁺LacZ⁺ cap cells. (F) X-gal whole-mount staining shows lobulo–alveolar structures of outgrowth. (G) Frozen section of outgrowth lobulo–alveoli stained with X-gal (blue) and nuclear fast red (red). (H) Antibody staining for milk protein (arrows) in alveolar lumen. (Red) Milk; (green) phalloidin; (blue) DAPI. (I) Outgrowth lobulo–alveoli are composed of SMA⁺ myoepithelial cells (arrowheads) and E-cad⁺ alveolar luminal cells. Bars: A,B,F, 1 mm; B (inset), C, 0.25 mm; D,E,G–I, 50 μm.

Comparison of GFP⁺ mammary epithelial cells with previously identified lin⁻CD24^modCD49f^high and lin⁻CD24^modCD29^high MaSC phenotypes. (A) Mammary cells from puberty (4w + 4d), adult virgin (6m), and pregnancy (P8.5d, 6m) were stained and analyzed simultaneously. The left panels show that the distributions of lin⁻ mammary cells from different developmental stages based on CD24 and CD49f expression level are quite different. Unlike in virgin mammary cells, there are no clearly distinguishable CD24^highCD49f^low and CD24^modCD49f^high populations in puberty (4w + 4d) lin⁻ mammary cells. The right panels show that GFP⁺ epithelial cells account for 9.0% and 10.8% of the overlapped lin⁻CD24^modCD49f^high cells in puberty (4w + 4d) and in pregnancy (P8.5d), respectively. Lin⁻CD24^modCD49f^highGFP⁺ cells are not present in virgin transgenic mammary cells. (B) Mammary cells from puberty (4w + 1d), adult virgin (6m), and pregnancy (P8.5d, 6m) were stained and analyzed simultaneously. The left panels show the differences among the distribution of lin⁻ mammary cells from different stages according to CD24 and CD29 expression. Clearly distinguishable CD24^highCD29^low and CD24^modCD29^high populations do not exist in puberty (4w + 4d) lin⁻ mammary cells. The right panels show that GFP⁺ epithelial cells represent 22.4% and 4.0% of the overlapped lin⁻CD24^modCD29^high cells in puberty (4w + 1d) and pregnancy (P5.5d), respectively. (C) lin⁻CD24⁺CD49f^highGFP⁺ and lin⁻CD24⁺CD49f^highGFP⁻ cells were sorted from pubertal mammary glands of 5-w + 1-d-old transgenic mice (shown in the dot plot), and were cultured in Matrigel or transplanted into cleared fat pads. The histogram shows the colony-forming capability of lin⁻CD24⁺CD49f^highGFP⁺ cells is significantly higher than that of lin⁻CD24⁺CD49f^highGFP⁻ cells. Data represent mean ± SD of five independent experiments. The table shows lin⁻CD24⁺CD49f^highGFP⁺ cells possess significantly higher regenerative potential than lin⁻CD24⁺CD49f^highGFP⁻ cells upon transplantation. The latter GFP⁻ population may contain dormant MaSCs.

Characterization of GFP⁺ cap cells in puberty. (A) Histogram shows the percentage of lin⁻GFP⁺CD49f^high cap cells in total lin⁻ puberty mammary cells. Results represent mean ± SD of four mice for each group. (B–E) Flow cytometric analysis shows that GFP⁺CD49f^high cap cells are CD29^high (B), Sca-1^−/low (C), CD133⁻ (D), and CD61⁺ (E). (F) Side population analysis shows that puberty GFP⁺ cells are depleted in the side population. (G) RT–PCR shows s-SHIP mRNA in sorted GFP⁺ cells but not in GFP⁻ cells. (H,I) Single GFP⁺CD49f^high cap cells form alveolar-like colonies in Matrigel; photographed with a Nikon microscope in situ in Martrigel (phase and GFP) (H), or removed from Matrigel and examined for GFP by confocal microscopy (I). (J) K14⁺ cells (red) comprise the outer layer of the colony (DAPI-stained for nuclei; blue). Bars: H,J, 20 μm; I, 50 mm. (K) Histogram shows the colony-forming capacity of lin⁻GFP⁺CD49f^high and lin⁻GFP⁻ cells in Matrigel. Data represent mean ± SD of five independent experiments.

Identification and characterization of GFP⁺ cells in alveolar buds at pregnancy. (A–D) Alveolar buds in P11.5 transgenic mammary tissues. Carmine whole-mount staining shows alveolar buds along the ductal network (A), GFP expression (green) in the apical alveolar buds, and some blood vessels (B). Frozen sections show that GFP is expressed mainly in the peripheral cells, especially the tips of the alveolar buds (C,D), and some GFP⁺ cells are seen in the inner layer of the alveolar buds (D). (E) GFP⁺ cells in P2.5 mammary tissue are SMA⁺ (red). (F) Most GFP⁺ cells in P7.5 alveolar bud are Ki67⁺ (red). (G) Histogram shows that 91.5% ± 5.9% of GFP⁺ cells at pregnancy are Ki67⁺, and 27.7% ± 10.9% of GFP⁺ cells were labeled with BrdU within 4 h of injection. Data represent mean ± SD; n = 20 alveolar buds. (H) Flow cytometric analysis of lin⁻ mammary cells from P5.5 mammary tissues. GFP⁺ cells consist of CD49f^high alveolar bud cells (gate) and CD49f ^−/low blood vessel cells. (I) Histogram shows the percentage of GFP⁺CD49f^high alveolar bud cells in the total lin⁻ population during pregnancy. Results represent mean ± SD of four mice for each group. (J) Carmine alum staining of an outgrowth from 100 pregnancy GFP⁺ cells shows the TEBs (arrowheads) at the ductal ends. (K) GFP⁺ TEBs in the outgrowth of 50 pregnancy GFP⁺ cells. (*) Approximate injection site. Bars: A,B, 200 μm; C,D,F, 20 μm; E, 50 μm; J,K, 1 mm.

GFP expression in breast tumors. (A,B) GFP is expressed in Wnt1 tumors (A) and GFP⁺ cells are SMA⁺ (B). (C,D) No GFP expression was detected in ErbB2 tumors (C), and no SMA⁺ cells were detected in ErbB2 tumors (D). Bars, 50 μm.