Figure 1.
Serial Recombination with new rUGM.
A) First generation recombinant (H&E staining) generated with 1000 side population cells isolated from 9NT, micro-dissected piece of the recombinant and new rUGM used for serial recombination, and second generation growth (H&E staining and IHC analysis). B) First generation recombinant (H&E staining) generated with 400 non-side population cells isolated from 9NT, micro-dissected piece of the recombinant and new rUGM used for serial recombination, and second generation growth (H&E staining and IHC analysis). Ruler scale = mm; Scale bar = 50 µm.
Figure 2.
FACS isolation of the side population in human clinical specimens based upon DCV efflux.
A) Live cells are identified and gated based on 7AAD staining and a plot of forward scatter (FSC) vs. side scatter (SSC). B) Cells gated in (A) are plotted and gated as viable singlets based on FSC-Width (FSC-W) vs. FSC-Height (FSC-H). C,E,G,I,K,M,O,Q,S) The gated viable singlets (B) are plotted in a double scatter plot between red (650 nm) and blue (450/40 nm) emission and are isolated based upon efflux of DCV. D,F,H,J,L,N,P,R,T) ABCG2-mediated DCV efflux is inhibited in the presence of FTC to establish where the side population (SP) and non-side population (NSP) gates should be placed. Purple: viable single cells; Green: NSP; Blue: SP.
Table 1.
The percentage of total side population (SP) and non-side population (NSP) gated relative to total viable cells in tissue specimens from human benign prostate and prostate cancer specimens.
Table 2.
Incidence of human ductal growth in recombinants with equal number of sorted cells.
Table 3.
Incidence of human ductal growth in recombinants with representative number of sorted cells.
Figure 3.
Ductal Growth of Recombinants from Side and Non-Side Population Cells.
A) The percentage of recombinants with human ductal growth in the first generation with equal number of sorted cells side population n = 51 and non-sided population n = 52, analyzed by Fisher’s Exact two-sided test p = 0.017. B) The number of glands in recombinants with equal number of sorted cells derived from side population n = 19 and non-side population n = 6 was quantitated and analyzed by Exact Wilcoxon test p = 0.041. C) The percentage of recombinants with human ductal growth in the first generation with representative number of cells side population n = 42 and non-side population n = 45, analyzed by Fisher’s Exact two-sided test p = 1.000. D) The number of glands in recombinants with representative number of cells derived from side population n = 10 and non-side population n = 9 was quantitated and analyzed by Exact Wilcoxon test p = 0.161. E) Kaplan-Meier plot of recombinant survival rate in multiple generations, Logrank test p = 0.2692.
Figure 4.
Expression of Differentiation Markers in Recombinants.
Nonconsecutive serial sections of a recombinant derived from 250 side population cells from specimen NT6+ rUGM were stained. A) H&E staining, box represents area examined at higher power in D–H; B) p63 IHC; C) AR IHC; D) PSA IHC; E) ABCG2 IHC; F) Chromogranin A (ChrA) IHC arrows indicate positive cells; G) telomere (Telo) detection by FISH; and H) DAPI staining. B-H) Scale bar, 10 µm.