Extended Data Fig. 2: Establishment of mini gland culture system.

a, Brightfield images of organoids without feeder cells for 24 days in branching media. b, Immunofluorescence staining of organoid in branching media for indicated markers at d24. c, Representative FACS plots showing mammary cell population from digested organoids without feeder cells. d, Immunofluorescence staining of organoids for indicated markers without feeder cells after pseudo estrus cycles at d20. e, Representative FACS plots showing basal and luminal cell populations from digested organoids without feeder cells after pseudo estrus cycles. f, qRT-PCR analysis of gene expression of mammary gland markers in mammary organoids, compare with primary mammary glands. n = 3, biological replicates. Data are represented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. g, Tracking of the size of the mini gland during one month culture. n = 10, biological replicates. Data are presented as mean ± SEM. h, Immunofluorescence staining for indicated markers from the adult primary mammary gland (left) and mammary mini gland (right). i, Comparison of the TEB widths of the mammary mini-glands and the primary mammary glands. n = 10, biological replicates. Data are presented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. j, Comparison of the duct widths of the mammary mini glands and the primary mammary glands. n = 4, biological replicates. Data are presented as mean ± SEM. P values were calculated using two-sided unpaired Student’s t-test. k, Bright-field images of organoids cultured for 6d in the indicated medium. l, Bright-field images of mini glands cultured in the indicated medium, showing the effect of Fgf10 and Fgf2 in mini gland culture. The scale bar represents 500 μm in a, k, l, 50 μm in b, d, h.

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