We have recently identified in human breastmilk early‐stage stem cells capable of multi‐lineage differentiation. To elucidate the potential of these cells to differentiate into functional lactocytes in vitro, freshly expressed breastmilk was collected, and cells shown by qPCR to express stem cell markers were cultured in mammary differentiation medium for up to four weeks. qPCR was then used to detect and quantify mRNA of milk proteins (lactoferrin, LF; α‐lactalbumin, LA) in cultured cells, whereas immunofluorescence microscopy was used to detect them at the protein level. Culture supernatants were harvested at regular time points and Western Blotting was used to examine expression of milk proteins and their glycosylation patterns. Plated breastmilk cells attached to the substrate and proliferated, forming epithelial colonies. Cells from the colonies gradually differentiated into functional lactocytes producing LF and LA, which were detected in harvested cultured cells both at the mRNA and protein levels. LF and LA were also secreted into the culture supernatants over the culture period of 4 weeks. Glycosylation of secreted milk proteins was observed at the molecular weights of LF and LA. It is concluded that breastmilk cells are able to differentiate in culture into functional lactocytes producing and secreting milk proteins, a number of which are glycosylated.Grant Funding Source : Women and Infants Research Foundation, Medela AG