Macrophage-Secreted CSF1 Transmits a Calorie Restriction-Induced Self-Renewal Signal to Mammary Epithelial Stem Cells.

Calorie restriction enhances stem cell self-renewal in various tissues, including the mammary gland. We hypothesized that similar to their intestinal counterparts, mammary epithelial stem cells are insulated from sensing changes in energy supply, depending instead on niche signaling. The latter was investigated by subjecting cultures of mammary epithelial stem cells for 8 days to in vivo paracrine calorie-restriction signals collected from a 4-day-conditioned medium of individual mammary cell populations. Conditioned medium from calorie-restricted non-epithelial cells induced latent cell propagation and mammosphere formation-established markers of stem cell self-renewal. Combined RNA-Seq, immunohistochemistry and immunofluorescence analyses of the non-epithelial population identified macrophages and secreted CSF1 as the energy sensor and paracrine signal, respectively. Calorie restriction-induced pStat6 expression in macrophages suggested that skewing to the M2 phenotype contributes to the sensing mechanism. Enhancing CSF1 signaling with recombinant protein and interrupting the interaction with its highly expressed receptor in the epithelial stem cells by neutralizing antibodies were both affected stem cell self-renewal. In conclusion, combined in vivo, in vitro and in silico studies identified macrophages and secreted CSF1 as the energy sensor and paracrine transmitter, respectively, of the calorie restriction-induced effect on mammary stem cell self-renewal.

Intramammary rapamycin administration to calves induces epithelial stem cell self-renewal and latent cell proliferation and milk protein expression.

Mammary epithelial stem cells differentiate to create the basal and luminal layers of the gland. Inducing the number of differentiating bovine mammary stem cells may provide compensating populations for the milk-producing cells that die during lactation. Inhibition of mTOR activity by rapamycin signals self-renewal of intestinal stem cells, with similar consequences in the mouse mammary gland and in bovine mammary implants maintained in mice. The implementation of these results in farm animals for better mammary development and production was studied in 3-month-old calves. mTOR activity decreased by ~50% in mammary epithelial cells subjected to 3-week rapamycin administration, with no negative consequences on mammary morphology or ß-casein expression. Subsequently, stem cell self-renewal was induced, reflected by a higher propagation rate of cultures from rapamycin-treated glands compared to respective controls and higher expression of selected markers. Followed by 4-day estrogen and progesterone administration, rapamycin significantly induced proliferation rate. Higher numbers of basal and luminal PCNA+ cells were detected in small ducts near the elongating sites as compared to large ducts, in which only luminal cells were affected. Rapamycin administration resulted in induction of individual milk protein genes' expression, which was negatively correlated to their endogenous levels. The inductive effect of rapamycin on luminal cell number was confirmed in organoid cultures, but milk protein expression decreased, probably due to lack of oscillation in rapamycin levels. In conclusion, intramammary rapamycin administration is an effective methodology to reduce mTOR activity in bovine mammary epithelial cells and consequently, induce stem cell self-renewal. The latent positive effect of rapamycin on epithelial cell proliferation and its potential to improve milk protein expression in calves may have beneficial implications for mature cows.

Newly characterized bovine mammary stromal region with epithelial properties supports representative epithelial outgrowth development from transplanted stem cells.

Limited outgrowth development of bovine mammary epithelial stem cells transplanted into de-epithelialized mouse fat pads restricts advanced studies on this productive organ's development and renewal. We challenged the mouse-bovine incompatibility by implanting parenchymal adjacent or distant bovine stromal layers (close and far stroma, respectively) into the mouse fat pad to serve as an endogenous niche for transplanted stem cells. The close stroma better supported stem cell take rate and outgrowth development. The diameter of these open duct-like structures represented and occasionally exceeded that of the endogenous ducts and appeared 8.3-fold wider than the capsule-like structures developed in the mouse fat pad after similar cell transplantation. RNA-Seq revealed lower complement activity in this layer, associated with secretion of specific laminins and WNT proteins favoring epithelial outgrowth development. The close stroma appeared genetically more similar to the parenchyma than to the far stroma due to epithelial characteristics, mainly of fibroblasts, including expression of epithelial markers, milk protein genes, and functional mammary claudins. Gene markers and activators of the mesenchymal-to-epithelial transition were highly enriched in the epithelial gene cluster and may contribute to the acquired epithelial properties of this stromal layer.