Human epicult b

All primary human material was derived from five reduction mammoplasties at Addenbrooke’s Hospital under full informed consent and in accordance with the National Research Ethics Service, Cambridgeshire 2 Research Ethics Committee approval (08/H0308/178) as part of the Adult Breast Stem Cell Study. All tissue donors had no previous history of cancer and were premenopausal (ages 20 to 23). Mammary tissue was dissociated to single-cell suspensions as previously described (Eirew et al., 2010 (link)). Single-cell suspensions of human mammary cells were treated to detect the enzyme activity of aldehyde dehydrogenase (ALDH) using the Aldefluor Kit (StemCell Technologies) as per the manufacturer’s instructions. Antibodies for mammary progenitor cell isolation are listed in Supplemental Experimental Procedures. Cells were sorted using a Becton Dickinson Influx. Luminal progenitor populations were seeded as single cells into 96-well plates with 1 × 10⁴ irradiated NIH 3T3 feeder cells. Cultures were maintained in Human EpiCult-B (StemCell Technologies) supplemented with 5% fetal bovine serum (FBS) (StemCell Technologies) and 50 μg/ml gentamicin for 10 to 12 days.

Flow-sorted human luminal progenitor cells were seeded into 60 mm plates with 2.5 × 10⁵ irradiated NIH-3 T3 feeder cells. The cultures were maintained in Human EpiCult-B (StemCell Technologies) supplemented with 5% FBS (StemCell Technologies) and 50 μg/ml gentamicin for 48 h and then the media changed to serum-free conditions and maintained for an additional 12 days. Colonies were fixed with acetone to methanol (1:1), stained with Giemsa (Fisher Scientific) and enumerated under a microscope.