Methods for assaying PCSCs

Although, a SC in any type of adult tissue has the common self-renewal and differentiation abilities, it will be wrong to generalize the results obtained from one tissue while defining a SC in another tissue. SCs in different tissues can differ significantly from one another. The actual assay to identify a CSC that has self-renewal and tumor progression capability is an in vivo model known as the serial transplantation in animal models. Other assays are usually generated in an in vitro environment and to be ideal, they have to full-fill the following criteria: they should be quantitative, highly specific in measuring only the cells of interest, sufficiently sensitive to measure candidate stem cells even at low frequencies, and fast [37].

For SC studies, human primary cells are the optimal tools to mimic and represent the original characteristics of tissues; however, it is quite difficult to get primary cell cultures from PC tissues due to limited access. Furthermore, cell lines can serve as a resource for CSC studies, but there are several disadvantages in utilization of this in vitro model: it cannot replicate exact in vivo conditions during the long-term culture process and some cell property changes might take place like gene alterations; the in vitro cultured cells often lose their original differentiated function; and it cannot stably maintain the exact properties of the original organ. Nevertheless, primary PC cells, established PC cell lines, xenograft and animal models have all been utilized to identify PCSCs with different surface markers.

4.3.1. In vivo systems

Gerald R. Cunha and Ben Lung have developed tissue recombination of a rodent model for the growth of normal epithelial cells in 1978 [68]. In this system, tissue fragments of fetal urogenital sinus mesenchyme were used to support the growth of normal prostate epithelial tissue fragments when implanted in collagen under the renal capsule of immunodeficient mice. This system was later modified to evaluate the growth activities of different prostate cell subpopulations using mechanical and enzymatic digestion to dissociate both, the urogenital sinus mesenchyme and adult murine prostate tissue into single cell suspensions [69]. Dissociated prostate epithelia regenerate ductal structures that histologically resemble normal murine prostate. Matrigel transplantation method was described that provides a reconstitution assay of prostatic cells. It was shown that the prostate contains stem cells capable of reconstituting the whole prostate and this method can be used to analyze prostate stem cells, epithelial mesenchymal interactions, and prostate cancer stem cells [70]. Ken Goto and colleagues performed serial transplantation that was analogous to the serial reconstitution method to investigate PSCs self-renewal [71]. They showed that regenerated prostate tissue could be dissociated and transplanted to regenerate prostate tissue at least three times.

4.3.2. In vitro culture systems and assays

There are two types of culture system to study CSCs: primary cell cultures and cell lines. Primary cell cultures are directly established from human tissues and have the advantage that their cells represent the original features of the tissue. However, difficulties including the limited access to biopsy materials, the need for the exclusion of contamination by cancer or normal cells, their limited lifespan, and the small population of the putative SCs are its disadvantages [72]. Cell lines are permanent cell cultures with unlimited proliferation capacity. They are widely used in many aspects of research as the most common in vitro culture model, because they have a big advantage in being easy to handle for their infinite reproducible quantities. So far, most of the human PC cell lines have been established from metastatic lesions or from xenograft tumors.

Prostate colony assay: The clonal and population analyses of mammalian stem cells was first accomplished by using two dimensional culture conditions [73]. Co-culture with irradiated fibroblast feeder layer is now also used to cultivate human prostate epithelial cells. In this assay, the feeder layer contains serum free medium (but, growth factors added) and low cal-

cium [74]. Under these conditions, murine prostate epithelial cells form colonies of cells that express epithelial cytokeratins when cultured with irradiated 3T3 feeder cells [28].

Prostate sphere assay: Colonies that are derived from primitive cells cannot be passaged efficiently, since culture conditions promote cell differentiation. The three dimensional sphere is a non-adherent culture system that has been used as a useful model to elucidate stem cell characteristics [75]. A suspension culture system like this is thought to keep cancer stem cells in their undifferentiated state facilitating their enrichment; like for AR-negative and AR-positive PC cell lines that both can form prostaspheres [76]. Actually, all PC cell lines can form prostaspheres; but, because heterogeneity exists only a subpopulation of cells in each cell line can form these prostaspheres. The expression of stem cell markers, such as CD133 and CD44, is also significantly enhanced in a prostasphere.

In contrast to the suspension sphere culture systems 3-D culture in Matrigel, which is a widely used commercially available basement membrane, has been demonstrated to promote the differentiation of PSCs. It was possible to induce morphological and phenotypical differentiation in normal and malignant prostate epithelial cell lines with Matrigel [72].

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