Biomedical Engineering Reference
In-Depth Information
6.1
Parallelism Between the Results of
In Vitro
and
In Vivo
SSC Assays
As described in Chap. 5, when SSCs are cultured on a feeder layer in serum-free
medium, supplemented with glial cell line-derived neurotrophic factor (GDNF), a
secreted form of its receptor (GFRA1), and fibroblast growth factor 2 (FGF2), they
form morphologically distinct, three-dimensional aggregates of spermatogonia
(Fig.
6.1
) (Kubota et al.
2004
; Kanatsu-Shinohara et al.
2003
; Yeh et al
.
2007
).
Here, we call these aggregates “clusters” to distinguish them from “colonies” of
spermatogenesis that emerge in recipient testes after transplantation (Chap. 5).
These clusters can be serially passaged every 6-7 days and maintained for an
extended period of time. Transplantation of clusters results in the regeneration and
maintenance of complete spermatogenesis, indicating that clusters contain SSCs.
In the definitive SSC assay technique, spermatogonial transplantation, the estab-
lishment of colonies 2 months after transplantation qualitatively demonstrates SSC
activity, while the number of colonies found in a recipient testis indicates the num-
ber of functional SSCs (Nagano et al.
1999
; Zhang et al.
2003
). Likewise, in the
CFA assay (Yeh et al.
2007
), the number of clusters indicates the relative SSC activ-
ity. Since clusters form within 6 days of culture, the CFA assay generates quantita-
tive data in a far shorter period of time than the transplantation assay.
It is of note that, although the concept of this
in vitro
technique is simple and
straightforward, it appears to contradict the functional definition of SSCs: i.e., the
ability to regenerate and support the long-term maintenance of spermatogenesis.
As spermatogenesis cannot be reproduced
in vitro
at present, the CFA assay is not
based on cells' regenerative capacity. It is thus logical to raise two critical questions
(1) Can SSCs indeed be detected on the basis of cluster formation without demon-
strating completion of spermatogenesis and (2) Does an assay that is completed in
only 6 days reliably detect long-term stem cell function? The following evidence
provides answers to these questions (Yeh et al.
2007
).
Figure
6.1b
depicts the proliferation kinetics of clusters and SSCs
in vitro
over
the period of 12 weeks. Clusters were derived initially from pup testis cells and,
during the study period, counted visually after one cycle of culture (6-7 days) peri-
odically. Proliferation kinetics of clusters were determined using the cluster number
Fig. 6.1
(continued) continue over the extended culture period, demonstrating that cluster
numbers faithfully reflect colony numbers. (
c
) Numbers of colonies (
filled bars
, measured with
spermatogonial transplantation) and clusters (
open bars
, measured with the CFA assay) derived
from mouse pup testis cells that were exposed to a hypotonic solution for indicated times. Pup
testis cells were first enriched for SSCs, and then incubated in the hypotonic solution for indicated
times (Yeh et al.
2007
). The numbers are expressed as a normalized value using the result of
control (no exposure to the hypotonic solution) as a denominator. A significant decline in numbers
of colonies and clusters is seen after a 20-min exposure. Note that both numbers are nearly identi-
cal under each experimental condition, indicating the faithfulness of the CFA assay to spermatogo-
nial transplantation. (
b
,
c
) were reproduced and modified from (Yeh et al.
2007
) with permission
from the Society of Study for Reproduction
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