Bovine CD49 positive-cell subpopulation remarkably increases in mammary epithelial cells that retain a stem-like phenotype

•We report the immunophenotyping of bovine mammary epithelial precursors and differentiated cells in long-term culture•We show an enrichment of CD49f positive cell along the culture•We show the regenerative potential of CD49f positive cells after 35 days of culture•We show the patter of expression of terminally differentiated epithelial markers in SF7 medium

We previously proved that adult stem cells reside in the bovine mammary gland and possess an intrinsic potential to generate a functional mammary outgrowth. The aim of this study was to investigate on the immunophenotyping features retained by mammary stem-like cells detected in long term culture. Flow cytometry analysis showed different subpopulations of mammary epithelial cells emerging according to the timing of cell culture. CD49f+-cells significantly increased during the culture (p < 0.01) and a similar trend was observed, even if less regular, for CD29+ and ALDH1 positive cell populations. No difference during the culture was observed for CD24 positive cells but after 35 days of culture a subset of cells, CD49f positive, still retained regenerative capabilities in in vivo xenotransplants. These cells were able to form organized pseudo-alveoli when transplanted in immunodeficient mice. These results prove the presence of a multipotent cell subpopulation that retain a strong epithelial induction, confirmed in in vivo xenotransplants with a presumable in vitro expansion of the primitive population of adult mammary stem cells.