Normal Human Breast Contains Distinct Basal and Luminal Stem Cells that Express EMT and MET Markers, Respectively
(A) Cells isolated from reduction mammoplasty tissue were immunostained with lineage markers (Lin−), EPCAM, and CD49f antibodies, and were first gated based on viability (DAPI−) and lineage markers. The sorted cells (Lin−EPCAM+CD49f+ and Lin−EPCAM−CD49f+ cells) were injected into the No. 4 mammary gland fat pads, which were previously humanized with normal human breast fibroblasts. After about 2 months, the mice were sacrificed and the outgrowths that were generated in the fat pads were assessed by hematoxylin and eosin staining. Scale bar, 100 μm.
(B) Cells were immunostained with EPCAM and CD49f antibodies followed by CD24/CD44 or ALDEFLUOR. Inset displays the negative control: cells incubated with DEAB, the specific inhibitor of ALDH, were used to establish the baseline fluorescence of these cells.
(C) Cells were immunostained with Lin−, EPCAM, and CD49f antibodies, and subsequently with ALDEFLUOR. The cells were first gated based on viability (DAPI−) and lineage markers. The sorted cells (Lin−EPCAM+CD49f+ALDH+ cells and Lin−EPCAM+CD49f+ALDH− cells) were grown in differentiating conditions on collagen-coated plates for 12 days and the number of colonies generated was assessed.
(D) Sorted cells (Lin−EPCAM+CD49f+ALDH+ and Lin−EPCAM+CD49f+ALDH− cells) were cultured in 3D Matrigel culture for 3 weeks and the number of branched structures generated was assessed.
(E) Localization of CD24 (magenta), CD44 (green), ALDH1 (red), and DAPI (blue) in normal breast tissue as assessed by immunofluorescence staining. Yellow arrow, CD24−CD44+ cells; white arrow, ALDH1+ cells. Scale bar, 100 μm.
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