The ability to isolate prostate stem cells is essential to explore their role in prostate development and disease. In vitro prostate colonyand sphere-forming assays were used to quantitatively measure murine prostate stem/progenitor cell enrichment and self-renewal. Cell surface markers were screened for their ability to positively or negatively enrich for cells with enhanced growth potential in these assays. Immunohistochemical and FACS analyses demonstrate that specific cell surface markers can be used to discriminate prostate stromal (CD34 ؉ ), luminal epithelial (CD24 ؉ CD49f ؊ ), basal epithelial (CD24 ؉ CD49f ؉ ), hematopoietic (CD45 ؉ , Ter119 ؉ ), and endothelial (CD31 ؉ ) lineages. Sorting for cells with a CD45 ؊ CD31 ؊ Ter119 ؊ Sca-1 ؉ CD49f ؉ antigenic profile results in a 60-fold enrichment for colony-and sphere-forming cells. These cells can self-renew and expand to form spheres for many generations and can differentiate to produce prostatic tubule structures containing both basal and luminal cells in vivo. These cells also localize to the basal cell layer within the region of the gland that is proximal to the urethra, which has been identified as the prostate stem cell niche. Prostate stem cells can be isolated to a purity of up to 1 in 35 by using this antigenic profile. The remarkable similarity in cell surface profile between prostate and mammary gland stem cells suggests these markers may be conserved among epithelial stem cell populations.CD49f ͉ integrin ␣6 ͉ Sca-1 ͉ CD24 heat-stable antigen ͉ stem cell niche S tem cells are of interest clinically because of their potential to repair damaged tissues, treat degenerative diseases, and because of their purported role in tumor initiation. The ability to identify and isolate stem cells is necessary to study their specialized biology. Enrichment for many types of tissue stem cells has been achieved by using cell surface markers. Murine hematopoietic stem cells can be enriched by sorting Lin Ϫ Thy-1 lo Sca-1 ϩ ckit ϩ cells from the bone marrow (1). Recent studies suggest that even better purity can be achieved by further sorting based on expression of the SLAM family receptors CD150 and CD48 (2). Bronchioavelolar stem cells can be isolated from their niche at the bronchioalveolar duct junction (BADJ) by sorting cells with a CD45 Ϫ CD31 Ϫ Sca-1 ϩ CD34 ϩ profile (3). Data from two recent reports show that mouse mammary stem cells possess a Lin Ϫ Sca-1 ϩ CD140a Ϫ CD24 ϩ CD49f ϩ CD29 ϩ cell surface profile and can be isolated to a purity of up to 1 in 20 by using subsets of these markers (4, 5).The presence of stem cells in the prostate first was proposed to explain the seemingly inexhaustible capacity of the organ to regenerate during androgen cycling experiments (6). The identification of side-population cells and replication quiescent BrdU label-retaining cells further suggests that stem cells exist in the gland (7,8). Several studies have enriched for primitive prostate cells by using cell surface markers. Richardson et al. (9) demonstrated that th...

ABSTRACT/SUMMARY The successful isolation and cultivation of prostate stem cells will allow us to study their unique biological properties and their application in therapeutic approaches. Here we provide step-by-step procedures on the basis of previous work in our laboratory for: the harvesting of primary prostate cells from adolescent male mice by a modified enzymatic procedure; the isolation of an enriched population of prostate stem cells through cell sorting; the cultivation of prostate stem cells in vitro; and characterization of these cells and their stem-like activity, including in vivo tubule regeneration. Normally it will take approximately 8 hours to harvest prostate cells, isolate the stem cell enriched population, and set up the in vitro sphere assay. It will take up to 8 weeks to analyze the unique properties of the stem cells, including their regenerative capacity in vivo.

Murine prostate stem cells express integrin ␣6, which modulates survival, proliferation, and differentiation signaling through its interaction with the extracellular protein laminin. When plated in vitro in laminin containing Matrigel medium, 1 of 500 -1,000 murine prostate cells can grow and form clonogenic spheroid structures that we term prostate spheres. Prostate spheres can be serially passaged individually or in bulk to generate daughter spheres with similar composition, demonstrating that sphere-forming cells are capable of self-renewal. Spheres spontaneously undergo lineage specification for basal and transit-amplifying cell types. P63-expressing cells localized to the outer layers of prostate spheres possess higher self-renewal capacity, whereas cells toward the center display a more differentiated transitamplifying phenotype, as demonstrated by the expression of the prostate stem cell antigen. When dihydrotestosterone is added to the medium, the androgen receptor is stabilized, is imported to the nucleus, and drives differentiation to a luminal cell-like phenotype. A fraction of sphere cells returned to an in vivo environment can undergo differentiation and morphogenesis to form prostate tubular structures with defined basal and luminal layers accompanied by prostatic secretions. This study demonstrates self-renewal and multilineage differentiation from single adult prostate stem/ progenitor cells in a specific in vitro microenvironment.

SUMMARY The Polycomb group transcriptional repressor Bmi-1 is often up-regulated in prostate cancer, but its functional roles in prostate stem cell maintenance and prostate cancer are unclear. Loss- and gain-of-function analysis in a prostate sphere assay indicates that Bmi-1 expression is required for self-renewal activity and maintenance of p63+ stem cells. Loss of Bmi-1 blocks the self-renewal activity induced by heightened beta-catenin signaling, suggesting that Bmi-1 is required for full activity of another self-renewal pathway. In vivo, Bmi-1 expression is necessary for normal prostate tubule regeneration. Altered self-renewal and proliferation through Bmi-1 modulation diminishes the susceptibility of prostate cells to transformation. In an in vivo prostate regeneration system, Bmi-1 inhibition protects prostate cells from FGF10 driven hyperplasia and slows the growth of aggressive Pten-deletion induced prostate cancer. We conclude that Bmi-1 is a crucial regulator of self-renewal in adult prostate cells, and plays important roles in prostate cancer initiation and progression.

Many cancers and leukemias are associated with strong dominant oncogenic mutations that activate tyrosine kinases and other classes of molecules, including transcription factors and antiapoptotic mechanisms. Some of these events can be targeted with small molecules or antibody-based therapeutics, but many remain intractable. In addition, cancer-related enzyme targets can often mutate, and drug-resistant variants are selected. Therapies directed at the mRNA encoding dominant oncogenes could provide a more global set of technologies for cancer treatment. To test this concept, we have used the model of transformation of hematopoietic cells by the chimeric Bcr-Abl oncogene, a highly activated tyrosine kinase. Our results show that tandem arrays of miRNA mimics, but not single miRNA mimics, directed against the Abl portion of the mRNA and introduced by lentiviral vectors can effectively alter the leukemogenic potency when the degree of suppression of expression of Bcr-Abl is reduced >200-fold from control levels. Only methods capable of such dramatic sustained reduction in the level of expression of highly activated kinase oncogenes are likely to be effective in controlling malignant cell populations.

The steroid hormone signaling axis is thought to play a central role in initiation and progression of many hormonally regulated epithelial tumors. It is unclear whether all cancer-initiating signals depend on an intact hormone receptor signaling machinery. To ascertain whether cell autonomous androgen receptor (AR) is essential for initiation of prostate intraepithelial neoplasia (PIN), the response of AR-null prostate epithelia to paracrine and cell autonomous oncogenic signals was assessed in vivo by using the prostate regeneration model system. Epithelial-specific loss of AR blocked paracrine FGF10-induced PIN, whereas the add back of exogenous AR restored this response. In contrast, PIN initiated by cell-autonomous, chronicactivated AKT developed independent of epithelial AR signaling. Our findings demonstrate a selective role for AR in the initiation of PIN, dependent on the signaling pathways driving tumor formation. Insights into the role of hormone receptor signaling in the initiation of epithelial tumors may help define this axis as a target for chemoprevention of carcinomas.tissue recombination | tumor promoter | tumor suppressor P harmacologic agents targeting hormone receptor signaling are widely used in therapy of carcinomas arising from the breast (estrogen receptor; ER), prostate (androgen receptor; AR) and endometrium (ER, and progesterone receptor; PR) (reviewed in refs. 1 and 2). It is unclear whether the hormone receptor signaling axis plays a causative role in initiation of these malignancies or whether it can be bypassed by oncogenic signals. Loss of PR has a protective effect in initiation of mammary tumors caused by 7,12-dimethylbenz(a) anthracene (DMBA) in mice (3). These results suggest that initiation of breast cancer via DMBA depends on PR signaling. Conversely, estrogen receptor knockout mice developed mammary tumors similar to controls in response to Wnt-1 expression (4), suggesting that initiation of these tumors is independent of ER signaling. The benefits of androgen ablation in treating prostate cancer were demonstrated decades ago (5; reviewed in ref. 6). However, the roles of autonomous and extrinsic AR in initiation of prostate cancer remain unknown.

Understanding prostate stem cells (PSCs) may provide insight for the design of therapeutics for prostate cancer. We have developed a quantitative in vivo colony-forming assay and have demonstrated that the Sca-1 antigen is present on the surface of a prostate cell subpopulation that possesses multiple stem cell properties. Immunofluorescent analysis demonstrates that Sca-1 is expressed by both basal and luminal cells in the proximal region of the adult prostate, but is not expressed by either lineage in more distal regions. The proximal region has been suggested as the PSC niche based on BrdU label-retention studies and the presence of distinct smooth-muscle cells that produce high levels of TGF-β. Sca-1 is also expressed by nearly all cells within fetal prostate epithelial chords, suggesting Sca-1 may be conserved on PSCs throughout development. Malignant epithelial cells from TRAMP mice, as well as normal prostate cells with lentiviral-mediated alteration of the PTEN/AKT signaling pathway, give rise to PIN lesions and prostate cancer in vivo. Alteration of PTEN/AKT signaling in Sca-1-enriched PSCs also results in PIN lesions, suggesting that PSCs can serve as one target for prostate carcinogenesis.

Prostate cancer is a leading cause of cancer-related death in adult men. It can regress dramatically upon antihormonal therapy, but it often recurs in a more aggressive, androgen-independent form. Defining the prostate tissue stem cells (PrSCs) and their involvement in cancer initiation and maintenance may lead to better therapeutics. Using a tissue-regeneration model in which dissociated prostate epithelial cells mixed with inductive mesenchyme give rise to prostatic tubules, we have identified a small population of prostate cells that contains multiple stem cell characteristics. In this system, prostate cancer can be initiated by autocrine or paracrine growth factor signaling and intracellular overexpression of genes often found mutated in human prostate cancer. Using an in vitro prostate sphere assay, we further defined the PrSC population and demonstrated their selfrenewal and multilineage differentiation capabilities. Microarray analyses of the stem-and non-stem-cell populations have assisted us in finding and evaluating additional markers that can better define the PrSC population and further delineate the different cell types of the prostate, including those that serve as the target cell for tumor initiation.