Spermatogonial Stem Cell Transplantation and Culture - Male Germline Stem Cells Developmental and Regenerative Potential - page 118

Fig. 5.5 Diagrammatic representation of the establishment of a typical rodent SSC culture using
MACS selection. (a) Testes from transgenic mouse pups are enzymatically digested resulting in a
heterogeneous cell population. (b) Cells are immunologically labeled with MACS conjugated
antibodies for a specific germ cell surface antigen (Thy-1; mouse or EpCam; rat). (c) The labeled
cells are then placed over a separation column within a magnetic field. Cells that are not bound to
the specific MACS conjugated antibody flow through the column. (d) After removal of the mag-
netic field, the retained positively labeled cells flow through the column. (e) These enriched cells
can then be used for SSC culture and maintained long-term in the presence of GDNF, GFRa1, and
bFGF
signal transduction molecules, it was demonstrated that the mechanism of GDNF
activation of these genes is through SRK family kinase signaling mechanisms and
that GDNF activation of AKT is important for SSC survival (Oatley et al. 2007 ;
Braydich-Stolle et al. 2007 ; Lee et al. 2007 ). For this type of experiment to study
regulation of self-renewal, SSCs should be used as soon as contaminating somatic
cells are absent from the culture, about 4-6 weeks after initiation. A scheme for
initiation of SSC cultures is outlined in Fig. 5.5 . Although culture will theoretically
maintain SSCs indefinitely, cells can become modified, especially during long-term
culture and lose nearly all testis colonizing and spermatogenic ability without an
apparent change in culture phenotype. Therefore, it is critical to verify stem cell
content of cultures during experimental procedures.
Culture of SSCs in a three-dimensional environment would more closely mimic
the niche environment of the seminiferous epithelium, and the ability to induce
germ cell differentiation and meiosis may be facilitated in a three-dimensional
environment. A recent report describes the utilization of a three-dimensional soft
agar culture system containing somatic testis feeder cells (Stukenborg et al. 2008 ).