Epithelial stem cells are of interest due to their capacity for organ replenishment and for their potential role in cancer-initiation. During the normal life span of an adult organ, stem cells operate to replace lost or damaged tissue to ensure proper organ function1
. More recently, epithelial stem cells have also been demonstrated as a target population for cancer2
. Due to their long-lived nature and inherent self-renewal capacity, adult stem cells are a likely cell-of-origin for many cancers3
. The isolation of these cells and investigation into their properties will be useful for understanding their basic biological processes and for designing new therapies.
The prostate represents an ideal model system to investigate the properties of adult stem cells due to the seemingly unlimited ability of the rodent prostate gland to undergo cycles of involution after androgen-depletion and subsequent organ regeneration in response to androgen add-back4
. Perhaps the most important reason to study prostate stem cells (PrSCs) is that they share the property of androgen-independence (or castration-resistance) with the subset of prostate cancer cells that survive in an androgen-deprived environment and can drive the lethal phase of the disease, termed hormone-refractory or castration-resistant prostate cancer (CRPC)5,6
. Identifying critical self-renewal pathways in PrSCs may provide new therapeutic targets for the treatment of CRPC.
Several methods have been described for the isolation of primitive cells from the mouse prostate7,8,9,10,11,12,13
. While genetically engineered mouse models can be useful for stem cell isolation, they limit the widespread use of such an approach. We have previously reported the isolation of PrSCs from wild-type mice capable of self-renewal and tri-lineage differentiation in vivo9
. PrSCs can be reproducibly isolated by fluorescence activated cell sorting (FACS) using the antigenic profile Lin- Sca-1+ CD49f+ (LSC)10
or Lin- Sca-1+ CD49fhi Trop2hi (LSCT)9
. These cells possess a basal phenotype and primarily reside in the region of the gland proximal to the urethra. Primitive cells with basal characteristics and an antigenic profile similar to PrSCs (LSC/LSCT) can be reproducibly isolated from un-fractionated prostate when primary cells are cultured in the prostate sphere assay10,14
. Here we will describe our methods to isolate PrSCs from primary prostate tissue, culture PrSCs in vitro, and measure PrSC activity using quantitative in vitro and in vivo assays.
Epithelial cells quickly lose their self-renewal potential when they are cultured in two-dimensions15
. We have developed a three-dimensional culture system to maintain and expand primitive prostate cells that retain the capacity for both self-renewal and differentiation14
. Cells are suspended in a semi-solid matrix comprised of prostate epithelial growth medium (PrEGM) and Matrigel. Matrigel is comprised primarily of laminin, collagen, fibronectin and other extracellular matrix (ECM) components. This in vitro environment closely mimics the ECM-rich basement membrane where basal cells reside in the gland. More importantly, basal cells express high levels of ECM-binding integrins that promote proper cell signaling and likely keep them in an undifferentiated state16
. The culture system is supplemented with selected growth factors and cytokines including EGF and FGF in the PrEGM media.
Three assays (the colony-forming assay, the sphere-forming assay and the in vivo prostate-regeneration assay) have been developed to measure primitive prostate cell activity10
. Similar to other epithelial stem cell assays, the colony-forming assay is performed in a two-dimensional culture system and measures both proliferative colony-forming activity and differentiation. Colonies are clonal in origin, express basal and luminal keratins, and can be induced to undergo differentiation upon addition of androgen10,14,15
. The sphere-forming assay is performed under three-dimensional conditions, as described above. Spheres are also clonal in origin, are comprised of several hundreds of cells, and can be dissociated and re-plated to measure self-renewal activity10,14,15
. Sphere cells undergo spontaneous differentiation with the most primitive cells residing around the outside and the more mature cells oriented towards the lumen, similar to the architecture of the native gland14
. Finally, the in vivo prostate-regeneration assay17,18
measures the ability of PrSCs to form prostatic tubules when combined with inductive stroma and implanted under the kidney capsule or skin (subcutaneous) of immunodeficient mice. Regenerated tubules are indistinguishable from primary prostate tubules, with an outer basal layer, an inner luminal layer and rare neuroendocrine cells9
. The subrenal regeneration assay can be used to study self-renewal in vivo by implanting prostate cells from transgenic mice harboring a probasin promoter driven luciferase, and performing androgen cycling15
. While the in vitro colony-forming and sphere-forming assays take 7–10 days, the in vivo prostate-regeneration assay takes considerably longer (6–10 weeks). We have functionally defined PrSCs based on their ability to generate colonies, spheres and prostatic tubules in these assays.