Fig. 2: Mammary organoid generation.

Step-by-step process for generating mammary organoids from three different sources: induced pluripotent stem cells (iPSCs), breast tissue, and milk-derived cells. Each approach follows a distinct isolation and differentiation protocol using key growth factors and signaling molecules to promote lineage specification. Cocktail 1 contains parathyroid hormone for mammary lineage differentiation; hydrocortisone, insulin, FGF10, hepatocyte growth factor for branch and alveolar formation, and hydrocortisone, insulin, FBS for lactation. Cocktail 2 contains R-spondin, neuregulin, FGF7, FGF10, EGF, noggin, A83-01, Y27632, SB202190, B27, NAC, nicotinamide, and Cocktail 3 contains R-spondin, neuregulin, FGF7, FGF10, EGF, noggin, A83-01, Y27632, SB202190, B27, NAC, nicotinamide. Milk-derived organoids do not need prolactin as they are already lactogenic. The final organoids, cultured in a supportive extracellular matrix, exhibit heterogeneous cellular composition, including luminal, basal, and adipocyte populations, mimicking the mammary gland microenvironment.