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Identification and isolation of human prostate epithelial stem cells based on ₂ß₁-integrin expression

¹ Prostate Research Group, Department of Surgery, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK
² Prostate Research Group, Department of Oncology, Western General Hospital, University of Edinburgh, Edinburgh, EH4 2XU, UK
³ YCR Cancer Research Unit, Department of Biology, University of York, PO Box 373, York, YO1 5YN, UK

View larger version (26K): [in a new window]   Fig. 1. Frozen sections of prostate were labelled with anti 2-integrin antibody, directly conjugated to fluorescein isothiocyanate (FITC), and viewed by confocal microscopy. The intensity of fluorescence was measured along a line drawn through the lateral cell borders. (A) Section of human prostate stained with a FITC-conjugated antibody to the 2-integrin subunit. (B) Fluorescence was measured along basolateral cell borders, with each numbered peak corresponding to one cell border. Peaks 3-4 shows a brightly stained cell surrounded by weakly stained cells (peaks 1-2, 5-6).

View larger version (14K): [in a new window]   Fig. 2. Basal cells were plated onto type I collagen (open bars), type IV collagen (closed bars) and laminin-1 (grey bars) for 5 and 20 minutes and the nonadherent cells were washed off and plated onto fresh ECM-coated plates (marked as 20 min+ and 60 min+). Cells were counted before the addition of irradiated feeders and were then cultured for up to 21 days. Controls were plated with no pre-selection. Colonies containing 32 or more cells were scored. Colony forming efficiency (CFE) was calculated as the number of colonies formed per number of adherent cells x100%. CFEs are expressed as the ratio of the control CFE. Results shown are the means±s.e.m. of five experiments.

View larger version (92K): [in a new window]   Fig. 3. Basal cells that adhered to type I collagen within 5 minutes stained with antibodies 34ßE12, anti-CK18, CK19, PAP and PSA.

View larger version (12K): [in a new window]   Fig. 4. Percentage of freshly isolated basal cells expressing CK18 (closed bars) after 5, 20, 60 minutes and overnight (marked as unselected) attachment to type I collagen. Results shown are the means±s.e.m. of 10 experiments.

View larger version (13K): [in a new window]   Fig. 5. Basal cells were labelled with FITC-conjugated antibodies to the 2-, 3- and 6-integrin subunits and analysed by flow cytometry. (A) Analysis of 2-integrin expression. Bold solid line, unselected basal cells; solid line, cells that adhered to type I collagen within 5 minutes. (B) Analysis of 3-integrin expression. Bold solid line, unselected basal cells; solid line, cells that adhered to type I collagen within 5 minutes. (C) Analysis of 6-integrin expression. Bold solid line, unselected basal cells; solid line, cells that adhered to type I collagen within 5 minutes.

View larger version (70K): [in a new window]   Fig. 6. Colony types founded by basal cells that adhered within 5 minutes to type I collagen. Type I (A) and type II (B) colonies are shown at 21 days growth.

View larger version (102K): [in a new window]   Fig. 7. Xenografts of prostate acini formed by transplantation of basal cells selected by adhesion to type I collagen for 5 minutes and stained with antibodies 34ßE12, anti-PAP, anti-PSA, anti-AR, and smooth muscle actin (SMA). Grafts were also stained with propidium-iodide (PI). Basal and secretory luminal cells are present.