Figure 4

Effect of growth factors on organ regeneration. (a) Whole gland immunostaining reveals the development of K5⁺ basal mammary epithelial cells (Magenta) and K8/18⁺ luminal epithelial cells (Cyan) in pregnant mice generated after transplantation of tdTomato + mammary organoids in the left side. The un-injected control showed only the presence of K5 basal epithelial cells and complete absence of tdTomato staining (Scale bar = 100 µm). Anti-DsRed was used to detect td-Tomato which is a derived mutant of DsRed. (b) Whole gland imaging for endogenous tdTomato fluorescence to check the effect of growth factors on their ability to generate mammary repopulating units (MRUs). Organoids grown in EGF, FGF2, FGF10 or in a combination of growth factors were partially digested and 10⁴ cells were transplanted into the left mammary fat pad after surgically removing the endogenous mammary epithelium. Arrow heads mark the terminal end buds (TEBs). (c) Quantification showing the significant reduction in the formation of TEBs after transplantation of organoids grown in different mammary specific growth factors. (scale bar = 1 mm, n = 3 mice per condition). (d) Representative brightfield images of organoids grown in the presence of single GFs for 7 days. “All GFs” containing EGF, FGF2, FGF10 were added to the single GF-grown organoids from 7th to 14th day. A counter panel of organoids were grown in the presence of “individual GFs” as a control. Organoids grown in the cocktail of GFs from day 0 were also used a control. Scale bar = 1000 µm. (e) Organoid size were measured from individual organoids and each dot represents average size of organoids from each replicate (n = 2 biological replicates containing 3 technical replicates each). One way ANOVA was used to measure statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).