Biomedical Engineering Reference
In-Depth Information
11.3.8
Definitive Autologous SSC Transplants in Primates
As discussed above, in an autologous monkey-to-monkey SSC transplant paradigm,
detecting successful engraftment of transplanted stem cells is impossible if the cells
are not marked to allow their discrimination from endogenous cells. In our experience,
significant biological variability between out-bred monkeys causes some amount
inconsistency in the degree of spermatogenic depletion following high-dose chemo-
therapy [(Hermann et al.
2007
) and (Hermann and Orwig, unpublished)]. Thus, it is
not adequate to assume that recovering spermatogenesis in treated animals following
SSC transplants necessarily arises from transplanted cells. To address this problem,
donor SSCs can be genetically marked with lentiviral vectors. The value of using len-
tiviral vectors to mark SSCs is their ability to genetically modify non-dividing cells
(SSCs are thought to rarely divide). Vectors carrying marker genes (e.g., eGFP,
dsRED) under the control of ubiquitous transcriptional regulatory sequences (e.g., the
UBC
promoter) are particularly useful for future evaluation of donor cell engraftment.
Viral, marker, and promoter sequences can be detected in DNA prepared from ejacu-
lated sperm of transplant recipients to provide confirmation of successful engraftment.
Further, fluorescent reporter proteins can be used to identify sites and extent of donor-
derived spermatogenesis in recipient testes. The efficiency of marking primate SSCs
with lentivirus vectors, however, is unknown. Lentivirus transduction of SSCs can be
as high as 40% in mouse and rat testis cell suspensions (Ryu et al.
2007
), and so it is
likely that a majority of primate SSCs would not be genetically marked for identifica-
tion of donor cell engraftment. Alternatively, donor primate testis cells could be pre-
loaded with a vital fluorescent dye (e.g., PKH26 or CFDA) prior to transplantation in
recipient testes (Honaramooz et al.
2002
; Maki et al.
2009
). The fluorescent loading
approach would mark most donor cells, but analysis is limited to 2-3 weeks after
transplantation to minimize the dilution of the fluorescent dye through cell divisions.
11.3.9
Allogeneic SSC Transplants
To circumvent the limitation of efficient donor cell marking, it may be possible to
utilize an allogeneic transplant paradigm where donor testis cells from an unrelated
individual animal are transplanted into the recipient's testis. A potential drawback to
the allogeneic transplant approach is that some or all donor cells could be immuno-
logically rejected in allogeneic recipient primates. However, there are reports that
transplantation of allogeneic testis cells are tolerated in large animal models allowing
engraftment of unrelated donor SSCs (Honaramooz et al.
2002, 2003b
; Kim et al.
2008
). In primates, if the donor's cells are tolerated by the recipient's immune system,
engraft, and produce spermatogenesis that results in ejaculated sperm, it is possible
to discriminate sperm originating from donor and recipient germ cells using genetic
techniques such as microsatellite DNA fingerprinting, SNP genotyping, or HLA
allotyping. Microsatellite fingerprinting or SNP genotyping of ejaculated sperm DNA
Search WWH ::
Custom Search