Biomedical Engineering Reference
In-Depth Information
cells and recipient animals to avoid immunological rejection, the CFA assay
eliminates such a consideration all together. Finally, the variation of data obtained
is less (approximately 50%) with the CFA assay than with the transplantation assay
(Yeh et al.
2007
), implying that the CFA assay can generate a consistent dataset
with fewer samples than the transplantation assay.
6.3
Cautionary Issues
6.3.1
Does the CFA Assay Assess the Full Range
of Stem Cell Characteristics?
As described above, the CFA assay relies solely on the self-renewing ability of SSCs
and does not detect the regenerative capacity of SSCs. This
in vitro
technique there-
fore does not stand as an unequivocal SSC assay on its own. It is prudent to combine
both the CFA and transplantation assays for efficiently generating convincing
results. For example, when the effect of a given growth factor on SSCs needs to be
investigated, one may need to examine a wide range of factor doses. Such a task is
labor-intensive and time-consuming if the transplantation assay is the only means to
derive data. Using the CFA assay, one can promptly evaluate the factor's effects
across varied doses, taking advantage of the procedural simplicity of the assay; then,
based on the outcome, the effect of the factor on SSCs can be confirmed with the
transplantation assay only in a defined and limited number of factor doses. This is
perhaps an ideal approach for the use of the CFA assay while circumventing its
weakness, i.e., the inability to measure the regeneration activity of SSCs.
6.3.2
Do Clusters Arise Only from SSCs?
We do not yet have a definitive answer to this question. A possibility was raised
recently that there is a subpopulation of spermatogonia that have initiated differentia-
tion but still retain stem cell activity (Nakagawa et al.
2007
). If such cells indeed exist,
they may well form clusters in an
in vitro
environment. It is also possible that com-
mitted progenitors do have the ability to form clusters, at least for a short period of
time. This is known to be the case in the assay of neural stem cells (NSCs) (Reynolds
and Rietze
2005
; Singec et al.
2006
; Seaberg and van der Kooy
2002
). NSCs can be
maintained and propagated
in vitro
in the form of free-floating cell aggregates, called
neurospheres. Importantly, NSCs are not the only cells that can form neurospheres,
but progenitors committed to differentiation can also do so. Whether or not a similar
situation applies to SSCs and clusters is unknown at present; however, the parallelism
between the CFA and transplantation assays (Fig.
6.1
) indicates that even if committed
cells can produce clusters, it does not significantly interfere with the faithful measure-
ment of relative SSC activity with the CFA assay.
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