Figure 3.

CRIPTO Promotes Maintenance of fMaSCs

(A) Sphere-bisecting confocal cross sections of day 5, fMaSC-derived colonies grown in Matrigel and stained with KRT8 (green) and KRT14 (red). Colonies were grown in differentiation media or maintenance media with soluble CRIPTO (0.33 μg/ml) or ALK4^L75A-Fc (10 μg/ml) as indicated. Yellow arrows indicate KRT8⁺KRT14⁺ double-positive cells. Red arrows indicate lineage-committed KRT14 single-positive cells. Control represents the same isotype. Scale bar represents 100 μm.

(B) KRT8 and KRT14 staining was quantified from images and is represented as the percent KRT8⁺KRT14⁺ double-labeled area per organoid. The percentage of organoids containing KRT8⁺KRT14⁺ double-labeled cells is also indicated. Results of three independent representative experiments are shown.

(C) Mammary gland repopulation efficiency of first-generation colonies grown in Matrigel-containing maintenance media culture with soluble CRIPTO and/or ALK4^L75A-Fc. Each circle represents one recipient mammary gland. Shaded circles represent ducts extending through >50% of the fat pad.

(D) Colony-forming efficiencies (>100 μm) at first, second, and third passage for fMaSCs grown for 7 days per passage in differentiation or maintenance media with CRIPTO and/or ALK4^L75A-Fc. Results of three independent experiments (maintenance media) and a representative experiment (differentiation media) were quantified. Triplicate wells were quantified in each. Error bars represent SDs. ^∗p ≤ 0.01.

See also Figure S3.