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

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.

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.

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.

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.