Biomedical Engineering Reference
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somatic diploid chromosome complement and induced development to the blastocyst
stage (Geijsen et al.
2004
).
While these studies were innovative and the findings impressive, significant
unanswered questions remain regarding the functionality of these
in vitro
-derived
“gametes.” However, in 2006 Nayernia and co-workers were the first to generate
what resembled spermatids
in vitro
that were indeed capable of fertilization.
Notably, the full functional ability of these gametes was shown by using them in
intracytoplasmic sperm injection (ICSI) to fertilize normal mouse oocytes and
generate live viable offspring (Nayernia et al.
2006
). In these pioneering studies,
the male gametes were isolated by using two germ cell-specific fusion genes,
Stra8-
eGFP
(
stimulated by retinoic acid gene 8
with
enhanced GFP
) and
Prm1-DsRed
(
protamine 1
with
Red Fluorescent Protein
). Mouse ES cells expressing the
Stra8
fluorescent reporter were induced with RA, cultured without RA for 2 months, then
transfected with the
Prm1
fluorescent reporter. Using this strategy two transgenic
cell lines were established, that subsequently formed EB-like structures and
expressed
mvh
. After subsequent induction with RA for 72 h the cell lines produced
dsRed-positive cells that arose from the GFP-positive cells. When these spermatid-
like cells containing structures resembling immature flagella were released into the
supernatant medium, they were reportedly motile as observed with phase contrast
microscopy. These cells fertilized oocytes, supported development to embryos, and
of the 65 embryos transferred into the oviducts of pseudopregnant females, 12 ani-
mals were born. Of these, six of the seven animals generated from these ES cell-
derived sperm developed into adult mice. It was noted that there was a relatively
low fertilization rate; of 210 oocytes microinjected only 65 oocytes developed to
two-cell embryos. This may have been due to ICSI techniques or the heterogeneity
and quality of the
in vitro
-derived male gametes.
3.4.4
Generation and Isolation of Human Germ Cells In Vitro
Although great progress has been made in the generation of mature gametes from
mouse ES cells, comparable studies in the human have been more challenging.
However, recent investigations, outlined below, have provided accumulating evi-
dence and valuable tools to isolate, quantitate, and enhance the maturation and
meiotic progression of human germ cells
in vitro
.
Determination of the essential growth factors and signaling molecules is impor-
tant for studies attempting to generate germ cells from human ES cells. It is critical
to establish whether factors known to be required for mouse PGC development both
in vivo
and
in vitro
might also induce the differentiation of human germ cells
in vitro
. Previous work indicates a central role of BMPs in germ cell specification
and maintenance in the mouse. As outlined in Sect.
3.2
of this chapter, Bmp4,
Bmp7, and Bmp8b play critical roles in the initial stages of germ cell development.
Additional studies have found that when mouse epiblast explants were cultured
with BMP4 and BMP8b
in vitro
they formed PGCs (Ying et al.
2001
). Further,
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