The mammary microenvironment alters the differentiation repertoire of neural stem cells

Abstract

A fundamental issue in stem cell biology is whether adult somatic stem cells are capable of accessing alternate tissue sites and continue functioning as stem cells in the new microenvironment. To address this issue relative to neurogenic stem cells in the mouse mammary gland microenvironment, we mixed wild-type mammary epithelial cells (MECs) with bona fide neural stem cells (NSCs) isolated from WAP-Cre/Rosa26R mice and inoculated them into cleared fat pads of immunocompromised females. Hosts were bred 6-8 weeks later and examined postinvolution. This allowed for mammary tissue growth, transient activation of the WAP-Cre gene, recombination, and constitutive expression of LacZ. The NSCs and their progeny contributed to mammary epithelial growth during ductal morphogenesis, and the Rosa26-LacZ reporter gene was activated by WAP-Cre expression during pregnancy. Some NSC-derived LacZ(+) cells expressed mammary-specific functions, including milk protein synthesis, whereas others adopted myoepithelial cell fates. Thus, NSCs and their progeny enter mammary epithelium-specific niches and adopt the function of similarly endowed mammary cells. This result supports the conclusion that tissue-specific signals emanating from the stroma and from the differentiated somatic cells of the mouse mammary gland can redirect the NSCs to produce cellular progeny committed to MEC fates.

Fig. 1.

NSCs isolated from WAP-Cre/Rosa26R mice are kept in an undifferentiated state (A) with NSCs expressing nestin (green) and Sox2 (red) or allowed to differentiate (B) as determined by the expression of markers specific for neurons (TUJ1, green), astrocytes (GFAP, pink), and oligodendrocytes (CNPase, red).

Fig. 2.

NSC-derived cells are present in chimeric outgrowths. (A) X-gal-stained whole mammary fat pad from a 16-day postlactation host mouse reveals that LacZ⁺ cells are represented throughout the entire chimeric outgrowth. (B) Regeneration of chimeras in secondary transplants demonstrates that LacZ⁺ cells are capable of replicating and contributing to all portions of the regenerated mammary outgrowth. Insets are histological sections of whole mounts demonstrating that only epithelial cells are LacZ⁺ in (A) and (B). (C) Tissue section of the gland in A demonstrating the distribution of LacZ⁺ epithelial cells. Note that the surrounding stroma remains LacZ⁻. (D) Tissue section of the gland in B reacted for smooth muscle actin (brown), a myoepithelial marker. Arrows indicate LacZ⁺ myoepithelial cells. Magnifications: 20 × in A and B, 100 × in the insets. (Scale bars: C, 15 μm; D, 10 μm.)

Fig. 3.

NSC-derived progeny may express myoepithelial or luminal epithelial cell markers. (A) Colocalization of smooth muscle actin (SMA, red) and β-galactosidase (green) in chimeric outgrowth. Arrows indicate myoepithelial cells that express both SMA and β-galactosidase. (B) NSC progeny expressing both casein (red) and β-galactosidase in a mammary chimera in a pregnant host. Arrows indicate cells expressing both casein and β-galactosidase. (C and D) Second-generation transplant sections demonstrating that β-galactosidase-positive (green) NSC-derived progeny express (C) ERα (red) and (D) PR (red). Arrows indicate select cells that co-express β-galactosidase and the nuclear steroid receptor of interest. (Scale bars: 20 μm.)

Fig. 4.

Assessment of DNA content within individual cells by propidium iodide staining followed by flow cytometric cell cycle analysis. A histogram plotting total cellular fluorescence (FL2 area) on the x-axis versus cell number on the y-axis shows the DNA content in each of 20,000 cells from the two starting populations (NSCs and MECs) and single cell suspensions prepared from an intact host mammary gland and an NSC/MEC-injected chimeric outgrowth. The relative G₀/G₁ (2N) and G₂/M (4N) peaks are prominent, with the less frequent S-phase cells accounted for by the valley between the 2N and 4N peaks.

Fig. 5.

LacZ+ cells within chimeras occasionally express NSC markers. Cross-sections of involuted chimeric mammary glands were probed for (A) nestin (red, arrow) and β-galactosidase (green), (B) Sox2 (pink, arrows), or (C) Oct4 (red, arrows) and β-galactosidase (green). (D) Cross-section of second transplantation generation demonstrating the persistence of neural markers nestin (red, arrow) and double-labeled with Sox2 (pink, arrows). All sections were stained with DAPI (blue) for nuclear visualization. (Scale bars: A, 10 μm; B–D, 20 μm.)

Fig. 6.

NSCs can be recovered from injected glands after ductal development, pregnancy, lactation, and involution. Chimeric mammary glands composed of NSCs and wild-type MECs were dissociated, and the resulting cell suspension was placed in neural culture conditions. (A) Recovered cells from a mammary chimera in NSC culture conditions. The cells demonstrate a NSC morphology and also positive expression of nestin (red) and Sox2 (green). (B) Recovered cells from a second-generation transplant mammary chimera in NSC culture conditions. No cells display any NSC morphology or express Sox2; occasional cells express low levels of nestin. (C) Morphology of freshly isolated wild-type MECs grown in NSC culture conditions. (D) No fluorescent antibody detection of nestin and Sox2 was present in these cultures (C), indicating that no cells with NSC characteristics could be recovered from intact wild-type mammary glands. (Scale bars: 10 mm.)