Epithelial Stem Cells Endogenous To Human Breastmilk Give Rise To Three-Dimensional Differentiated Milk-secreting Units In Vitro

Human breastmilk has previously been shown to contain a cellular complement that can generate differentiated epithelial lineages in primary culture. We have identified the generative population giving rise to these cultures in human milk based on the qualified stem cell marker p63, and shown that this population is enriched for the CD49f+/CD29hi/CD24hi phenotype shown to give rise to mammary outgrowths in vivo. The total cell population isolated from expressed breastmilk samples from 20 lactating women was immunofluorescently labeled for stem and differentiated epithelial cell markers prior to cultivation and at three day intervals for 21 days of growth in two‐ dimensional culture. We show that expansion of the stem cell population occurs in the floating cell population within the first five days of growth and depletes reciprocally with emergence of an adherent population homogenously expressing pan‐epithelial markers. In the proceeding 16 days of growth sub‐populations of adherent cells express myoepithelial and luminal epithelial markers in mutually exclusive colonies, indicating that lineage commitment occurs prior to adherence and differentiation. Further, we have used cells harvested from primary culture to cultivate alveolar milk‐secreting structures (mammospheres) in a three‐dimensional biomatrix. We also demonstrate using live‐cell confocal imaging that tissue architecture in three dimensions is highly dependant on plating density, and that cultivation of cells at high density results in rapid (<12 hours growth) self‐assembly into dutal structures that later develop terminal lumen‐containing alveoli whilst plating at low density results in slow formation of single mammospheres. This approach provides a platform to study regulators of milk synthesis and mammary gland differentiation in an integrated parencymal/extra‐ cellular matrix system, and for the first time allows experimentation on cells isolated non‐invasively from the fully differentiated lactating epithelium.