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Additional Supporting Information may be found in the online version of this article.

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SC_11-1179_sm_supplFigure1.tif597KSupplementary Figure 1: Relative expression of SFRP-1 in CAF and NPF lines used in this study. Data represent patient-matched CAF and NPF expression (n=4) as fold change relative to BPH stromal cells collected from n=4 transurethral resection of the prostate (TURP) specimens. Mean ± SEM are represented by box and whisker plot including minimum and maximum values. * p>0.05, as analyzed by paired t test.
SC_11-1179_sm_supplFigure2.pdf785KSupplementary Figure 2: CAFs induced malignancy in α2β1integrinhi/CD133- BPH-1 basal cells regardless of variability in tumor potential across patient samples. Using a CAF line derived from a different patient that was proven to be less tumorigenic in recombination with unsorted (1x105) BPH-1 cells (80% tumor formation; A), we tested the effect on sorted CD133+ or CD133- BPH-1 cells (500 cells per graft) in tissue recombinants sub-renally grafted into SCID mice for 8-12 weeks. Photomicrographs showing immunolocalization of SV40 to identify BPH-1 cells, hematoxylin and eosin staining to determine structural morphology showed small benign cord structures with normal nuclear architecture (B), compared to CAF + CD133- grafts that showed larger squamous mass with abundant abnormal nuclei (C). Epithelial mass within grafts was determined using stereological analysis of SV40-positive cells (D); data were not significantly different between CAF + CD133+ and CAF + CD133- BPH-1grafts. (E): Using the 3-point criteria to define malignancy (and invasive BPH-1 to highlight aggressive tumors) we showed that in the presence of CAFs, only CD133- cells showed any tendency to form tumors similar to unsorted BPH-1 controls. Values represented as mean ± SEM; n=4-5 per group. Scale bar = 500μm (A-C left panel); 125μm (A mid panel); 50μm (A-C right panel).
SC_11-1179_sm_supplFigure3.pdf341KSupplementary Figure 3: Expression of CD133 in human embryonic stem (hES) cells. A representative scatter plot of CD133 immunoreactivity in primary culture of hES 3 cells showing single, live (propidium iodide negative) cells that were gated based on mouse IgG1-APC isotype control. 92.8% of hES 3 cells expressed CD133 using the CD133/1-APC antibody (Miltenyi Biotec).
SC_11-1179_sm_supplFigure4.pdf399KSupplementary Figure 4: Representative images obtained from one individual CAF line which consistently induced predominately mesoderm derived structures from hESCs. (A) Photomicrographs of hematoxylin and eosin staining in CAF + hESCs with this particular line. Whilst glandular structures were observed (arrow; A), the predominant differentiation of hESCs was into bone and cartilage morphology (indicated by *). (B) Photomicrographs showing co-immunolocalization of androgen receptor (AR) and prostate specific antigen (PSA). Similar to other lines, glands expressed AR but not PSA, neither of which were detected in the adjacent mesodermal structures. No evidence of malignancy was present in these grafts. Scale bar = 125μm except 1250μm in upper left panel of A.
SC_11-1179_sm_supplFigure5.pdf700KSupplementary Figure 5: Both CD133+ and CD133- BPH-1 cells survive in vivo when grafted with rat mesenchyme. (A-B): Photomicrographs showing immunolocalization of SV40 to identify BPH-1 cells, hematoxylin and eosin staining to determine benign nuclear morphology. (A): Benign cords with normal nuclear architecture from 500 CD133+ (top panels) and CD133- (lower panels) BPH-1 cells grafted with rat seminal vesicle mesenchyme in SCID hosts for 4 weeks. (B): Benign cords with normal nuclear architecture from 500 CD133+ (top panels) and CD133- (lower panels) BPH-1 cells grafted with rat urogenital sinus mesenchyme in SCID hosts for 16 weeks. Scale bar = 500μm (A-B left panels); 50μm (A-B right panels).

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