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Purification and direct transformation of epithelial progenitor cells from primary human prostate

Nature Protocols volume 6, pages 656–667 (2011)Cite this article

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Abstract

Epithelial cell transformation has been demonstrated in numerous animal models for the study of solid tumor biology. However, little evidence exists for human epithelial cell transformation without previous immortalization via genetic influences such as SV40 T-antigen, thus limiting our knowledge of the events that can transform naive human epithelium. Here we describe a system developed in our laboratory to directly transform freshly isolated primary human prostate epithelial cells without previous culture or immortalization. Prostate tissue is obtained from patients and benign tissue is separated from malignant tissue. Benign and malignant tissues are mechanically and enzymatically dissociated to single cells overnight, and immune cells and epithelial subsets are isolated on the basis of differential expression of surface antigens. Epithelial progenitor cells are transduced with lentiviruses expressing oncogenes and combined with inductive stroma for in vivo studies. At 8–16 weeks after transplantation into immune-deficient mice, the development of lesions, histologically classified as benign prostate, prostatic intraepithelial neoplasia and adenocarcinoma, can be evaluated.

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Figure 1: Tissue preparation to obtain single cells.
Figure 2: Gating strategy to purify basal, luminal and immune cell subsets.
Figure 3: Schematic of third-generation lentiviral and packaging vectors.
Figure 4: Regeneration of benign and transformed human prostate glands in vivo.

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Acknowledgements

We thank members of the Witte lab for helpful comments and testing of the protocol. We thank I. Verma (Salk Institute for Biological Studies) for generously providing lentiviral vectors. A.S.G. is supported by the Warsaw Family Research Fellowship and a Dissertation Year Fellowship from the Graduate Division at the University of California, Los Angeles (UCLA). J.M.D. is supported by the UCLA Tumor Biology Program (US Department of Health and Human Services, Ruth L. Kirschstein Institutional National Research Service Award no. T32 CA009056). D.L.B. is supported by a Postgraduate Scholarship from the Natural Sciences and Engineering Research Council of Canada. J.H. is supported by the American Cancer Society, the Department of Defense (DOD) Prostate Cancer Research Program and the UCLA Specialized Program of Research Excellence in Prostate Cancer (principal investigator, R. Reiter). J.H. and O.N.W. are supported by a Prostate Cancer Foundation Challenge Award. O.N.W. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Andrew S Goldstein and Justin M Drake: These authors contributed equally to this manuscript.

Authors and Affiliations

  1. Molecular Biology Institute, University of California, Los Angeles, California, USA

    Andrew S Goldstein, Robert E Reiter & Owen N Witte

  2. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California, USA

    Justin M Drake & Owen N Witte

  3. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, California, USA

    Daina L Burnes & Owen N Witte

  4. Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, California, USA

    David S Finley & Robert E Reiter

  5. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, California, USA

    Hong Zhang, Jiaoti Huang & Owen N Witte

  6. Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California, USA

    Robert E Reiter & Jiaoti Huang

  7. Department of Pathology and Laboratory Medicine, University of California, Los Angeles, California, USA

    Jiaoti Huang

  8. Howard Hughes Medical Institute, University of California, Los Angeles, California, USA

    Owen N Witte

Authors
  1. Andrew S Goldstein
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Contributions

A.S.G., D.L.B., H.Z., J.H., R.E.R. and O.N.W. contributed to protocol development. A.S.G., J.M.D., D.L.B. and H.Z. generated the figures. A.S.G., J.M.D., D.S.F. and O.N.W. wrote the paper.

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Correspondence to Owen N Witte.

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The authors declare no competing financial interests.

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Goldstein, A., Drake, J., Burnes, D. et al. Purification and direct transformation of epithelial progenitor cells from primary human prostate. Nat Protoc 6, 656–667 (2011). https://doi.org/10.1038/nprot.2011.317

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