Murine amniotic fluid stem cells contribute mesenchymal but not epithelial components to reconstituted mammary ducts

Abstract

Introduction: Amniotic fluid harbors cells indicative of all three germ layers, and pluripotent fetal amniotic fluid stem cells (AFSs) are considered potentially valuable for applications in cellular therapy and tissue engineering. We investigated whether it is possible to direct the cell fate of AFSs in vivo by transplantation experiments into a particular microenvironment, the mammary fat pad. This microenvironment provides the prerequisites to study stem cell function and the communication between mesenchymal and epithelial cells. On clearance of the endogenous epithelium, the ductal tree can be reconstituted by the transfer of exogenously provided mammary stem cells. Analogously, exogenously provided stem cells from other tissues can be investigated for their potential to contribute to mammary gland regeneration.

Methods: We derived pluripotent murine AFSs, measured the expression of stem cell markers, and confirmed their in vitro differentiation potential. AFSs were transplanted into cleared and non cleared fat pads of immunocompromised mice to evaluate their ability to assume particular cell fates under the instructive conditions of the fat-pad microenvironment and the hormonal stimulation during pregnancy.

Results: Transplantation of AFSs into cleared fat pads alone or in the presence of exogenous mammary epithelial cells caused their differentiation into stroma and adipocytes and replaced endogenous mesenchymal components surrounding the ducts in co-transplantation experiments. Similarly, transplantation of AFSs into fat pads that had not been previously cleared led to AFS-derived stromal cells surrounding the elongating endogenous ducts. AFSs expressed the marker protein α-SMA, but did not integrate into the myoepithelial cell layer of the ducts in virgin mice. With pregnancy, a small number of AFS-derived cells were present in acinar structures.

Conclusions: Our data demonstrate that the microenvironmental cues of the mammary fat pad cause AFSs to participate in mammary gland regeneration by providing mesenchymal components to emerging glandular structures, but do not incorporate or differentiate into ductal epithelial cells.

Figure 1

Isolation, characterization, and differentiation potential of fetal stem cells derived from murine amniotic fluid. (a) Amniotic fluid stem cells (AFSs) were isolated at E13.5 and obtained by using a two-step culture model [9] from C57/B6-TG (ACTB-EGFP) and Rosa26 mice. (b) The freshly isolated and cultured AFSs exhibit pluripotency markers and exhibit no spontaneous differentiation in culture. (c) Histogram analysis of cell-surface markers showed that AFSs express embryonic and mesenchymal stem cell markers but lack the expression of hematopoietic cell markers. Grey line, isotype control. (d) Cultured AFSs exhibit an epithelial phenotype, as demonstrated by the co-expression of keratins and vimentin. (e) Differentiation of AFSs into mesodermal cell types after specific stimulation for 14 days is marked by the appearance of lipid granules (adipogenic) or mineralized matrix (osteogenic). The epithelial-stimulation protocol further potentiates the epithelial nature of AFSs (epithelial). Insets show higher magnification of the treated cells. Scale bars, 50 μm.

Figure 2

Amniotic fluid stem cells (AFSs) form spheres and tubular structures in 3D cultures. AFSs alone or equal numbers of AFSs and MECs derived from GFP transgenic C57/B6-TG (ACTB-EGFP) mice were subjected to 3D differentiation and monitored for up to 10 days after plating. (a) AFS-derived progeny form spherical structures 5 days after plating and extend to a branched tubular network in 3D Matrigel culture. (b) These structures are positive for α-SMA and vimentin. Inset shows a section through a spherical structure. (c) We observed the emergence of tubular structures formed by AFSs surrounding GFP⁺ MECs (upper panel) or spheroids formed from GFP⁺ MECs with a hollow central lumen surrounded by a layer of cells contributed by AFSs (middle panel) and side branches surrounding GFP⁺ MECs (lower panel). (d) AFSs closely associated with GFP⁺ MECs express α-SMA. Scale bars, 50 μm, and for inset, 30 μm.

Figure 3

Cell-fate tracing of transplanted amniotic fluid stem cells (AFSs) into cleared fat pads. (a) The 2 × 10⁵ AF-derived cells (rapidly adherent within 5 days) and AFSs (slowly adherent, after 5 days of culture) derived from various donors were transplanted into 3-week-old recipient mice and analyzed 8 weeks later as whole mounts. Only the slowly adhering cells selected in the second culture step are able to engraft and can be classified as AFSs. (b) Histologic analysis confirms that the GFP⁺ AFSs survived and differentiated into adipocytes and connective tissue. Scale bar for whole mounts and tissue sections is 50 μm, (Blue) X-Gal stain or hematoxylin, (Pink) Nuclear fast red, and (Brown) DAB staining.

Figure 4

Cell-fate tracing of amniotic fluid stem cells (AFSs) in co-transplantation with MECs into cleared fat pads. (a) AFSs were transplanted in a 1:1 ratio with supporting MECs (10⁵ each) and analyzed 8 weeks later. X-Gal-stained whole mounts from virgin mice show that AFS-derived progeny surround the developing ductal structures. (b) Histologic analysis of whole mounts inoculated with AFSs and MECs confirms that AFSs form a stromal cell layer around the ductal outgrowth but do not integrate into the myoepithelial or luminal cell layer in virgin mice. (c) Histologic analysis of whole mounts at pregnancy day 18.5 inoculated with GFP⁺ AFSs and MECs shows that the stromal cell layer derived from GFP⁺ AFSs exhibit no adverse effects on alveologenesis. Insets show higher magnification. Scale bar for whole mounts and tissue sections is 50 μm. A few α-SMA⁺ and GFP⁺ AFS-derived cells can be detected within the alveolar unit, as shown by laminin β1 staining. The circles indicate alveolar units. Scale bar is 10 mm. (Blue) X-Gal stain or hematoxylin, (Pink) Nuclear fast red, and (Brown) DAB staining.

Figure 5

Amniotic fluid stem cells (AFS)-derived cells migrate to other organs. The 10⁵ AFSs derived from a Rosa26 donor were injected into cleared fat pads of 3-week-old recipient mice and analyzed 14 weeks later. X-Gal-stained whole mounts demonstrate the presence of LacZ⁺ cells in several organs. Tissue sections show the localization of AFS-derived LacZ⁺ cells between hepatocytes, adjacent to airway epithelial cells, and in the mucosa of the gastrointestinal tract. Scale bar for whole mounts is 500 μm, and for sections, 50 μm, (Blue) X-Gal stain and (Pink) Nuclear fast red staining.

Figure 6

Amniotic fluid stem cells (AFS)-derived cells contribute stromal components for extending ducts. The 10⁵ AFSs derived from a Rosa26 donor were injected into noncleared fat pads of 3-week-old recipient mice and analyzed 2 weeks later. (a) Whole-mount analysis and tissue sections of X-Gal-stained whole mounts demonstrate the presence of LacZ⁺ cells in the stroma of extending ducts. (b) AFS-derived LacZ⁺ cells retain the expression of α-SMA but did not integrate into the myoepithelial cell layer. Scale bar for whole mount is 1,000 mm, and for tissue sections, 50 μm; (*) injection site, (Blue) X-Gal stain, (Pink) Carmine alum or Nuclear fast red staining and (Brown) DAB staining.