Biomedical Engineering Reference
In-Depth Information
ability to efflux Hoechst 33342 was first observed using bone marrow aspirates
by Goodell et al. (1996). While simultaneously displaying the Hoechst fluores-
cence at two emission wavelengths (675 and 450 nm) they identified a small
subpopulation of whole bone marrow cells that were unstained; they termed the
SP. They further determined that these cells contained SC characteristics and
had a 1000-fold increase in the ability to repopulate the bone marrow.
The SP was shown to exist in prostate epithelial cells isolated from men
undergoing surgery for bladder outflow obstruction as a result of benign
prostate disease (Bhatt et al. 2003). The SP accounted for approximately 1%
of prostate epithelial cells. Furthermore Bhatt et al. showed that this SP was
significantly reduced in the presence of verapamil, an inhibitor of ABC trans-
porters, and that the SP was comprised of both cells in G 0 and those entering G 1
of the cell cycle. It was the conclusion of Bhatt et al. that the SP contains both
the prostatic SCs and the TA cells. The SCs were identified by (1) the efflux of
Hoechst 33342, (2) their quiescent nature (i.e., cells in G 0 ), and (3) the expres-
sion of the basal marker integrin-a2, while the transit-amplifying cells could be
identified by (1) the efflux of Hoechst 33342, (2) cycling cells (i.e., cells entering
G 1 ), and (3) expression of both the basal marker integrin-a2 and the luminal
marker K8.
In prostate cancer cell lines, LAPC-9 xenograft tumors contained a detect-
able SP but there was no detectable SP in DU145, LNCaP, PC3, and PPC-1 cell
lines (Patrawala et al. 2005). It was also determined that the SP cells of LAPC-9
were more tumorigenic than the non-SP cells, with as few as 100 SP cells giving
rise to tumors (25% incidence), whereas 300,000 non-SP cells were needed to
generate a tumor. Since the ABCG2 transporter was implicated as responsible
for the SP phenotype, Patrawala et al. isolated ABCG2 + and ABCG2 cells
from the DU145 prostate cancer cell line and showed that they were similarly
tumorigenic. Furthermore ABCG2 + cells did not show increased expression of
genes known to play a role in SC maintenance (i.e., Notch, smoothened, Oct-3/
4,and b-catenin). Thus, they concluded that the SP is enriched for CSCs, but
that it contains not only the most primitive of stem cells but also the more
differentiated TA cells.
Currently, most investigators agree that the SP represents an enrichment of
CSCs but that it is not a pure population (reviewed in Hadnagy et al. 2006). It is
widely believed that this population contains not only the more primitive SC
but also TA cells or cells further along the differentiation pathway.
3.2 Enrichment of Prostatic CSCs by Culturing Non-adherent
Spheres
A second biological approach to isolate prostatic CSCs is based on their ability
to form spheres when plated in clonal numbers. This takes advantage of several
principles that were identified in other normal and cancer stem cells. For
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