Fig. 1.
Analysis of mammospheres generated by LA7 cells. (A) Self-renewing mammospheres generated by LA7 cells at passages 8 (p8) and 47 (p47). (Magnification: p8, ×20; p47, ×32.) (B) Mammosphere from a single LA7 cell at passage 13 (d0), plated onto collagen over a 10-day time period, generates morphologically differentiated cells (d1–d9) and forms tubular-like structures at days 5–9 (d5–d9, red arrowheads). (Magnification: d0, ×40; d1 and d3, ×20; d5, d7, and d9, ×10.) (C) Mammosphere-generated outgrowths on collagen express K18. (C1–C3) Images show K18 staining on cells derived from the disaggregated mammosphere before outgrowth generation (C1), a mammosphere outgrowth generated from a single LA7 cell at passage 13 (C2), and a mammosphere outgrowth generated from a single LA7 cell at passage 36 (C3). (C4) A negative control stained using the secondary antibody only. (Magnification: ×40.) Specifically, a single outgrowth from a single-cell-generated mammosphere was sectioned into portions, and individual portions were used for marker expression analysis for C2–C4 and D2–D4 and for E (lane 2). (D) Mammosphere-generated outgrowths on collagen express K14. Shown is K14 staining on cells derived from the disaggregated mammosphere before outgrowth generation (D1), a section of the mammosphere outgrowth generated from a single LA7 cell at passage 13 (D2), and a section of the mammosphere outgrowth generated from a single LA7 cell at passage 36 (D3). (D4) A negative control stained with the secondary antibody only. (Magnification: D2, ×32; D1, D3, and D4, ×40.) (E) Mammosphere-generated outgrowths express β-casein. After incubation using lactogenic hormones, the remaining portion of the 10-day outgrowth at early passage, used in Fig. 2 C and D, was used to test for the expression of β-casein with Western blot analysis. Lanes: 1, Cells derived from mammospheres; 2, outgrowth from a mammosphere at passage 13 after hormone induction; 3, outgrowth from a mammosphere at passage 13 without induction.