Biomedical Engineering Reference
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many of them to progress through meiotic prophase I (Bowles et al.
2006
; Farini
et al.
2005
; Koshimizu et al.
1995, 1996
). Based on these observations, several
groups have attempted to drive mouse ES cells towards the germ cell lineage and
produce mature gametes
in vitro
. These pioneering investigations in mice have
clearly demonstrated that mouse ES cells can give rise to the germ cell lineage,
postmeiotic germ cells, and more mature gametes (Nayernia et al.
2006
; Geijsen
et al.
2004
; Hubner et al.
2003
; Qing et al.
2007
; Toyooka et al.
2003
). Mouse ES
cells have been differentiated
in vitro
into EBs and subsequently into male sperm-
like and female egg-like cells using various defined media, conditions, and germ
cell-specific markers or genetic reporters. Cells with transcriptional profiles, pro-
tein expression, and morphologies similar to that of sperm and oocytes, have now
been derived
in vitro
.
In 2003, it was first demonstrated that oocyte-like cells could be generated by
spontaneous differentiation of mouse ESCs in adherent cultures (Hubner et al
.
2003
). Germ cell formation was identified by using a reporter construct containing
the germ cell-specific
Oct4
promoter (
Oct4
∆
PE
) to drive the expression of Green
Fluorescent Protein (GFP) in the germ cells exclusively. GFP-positive cells were
found associated with follicular-like structures that expressed high levels of known
oocyte- and germ cell-specific genes and exhibited profiles of steroidogenesis simi-
lar to that of the somatic cells of the gonad. Apparently, the cells resembling
oocytes were able to “recruit” adjacent ES cells, which differentiated in parallel
into gonadal somatic-type cells. When these follicular-like structures were cultured
further
in vitro
, the oocytes underwent spontaneous parthenogenesis into blasto-
cyst-like structures, which then degenerated. A second study also identified oocyte-
and follicular-like cells by co-culturing the differentiating mouse EBs with medium
that had been conditioned by testicular cultures from newborn male mice (Lacham-
Kaplan et al.
2006
). These mature germ cells resembling oocytes were identified by
germ cell-specific marker analysis and morphological characteristics. Presumably,
certain secreted factors and hormones from this testis-conditioned medium induced
female gamete differentiation.
In other studies attempting to differentiate sperm from ES cells, Toyooka et al.
(
2003
) used a germ cell-specific reporter construct containing the
mouse VASA
homolog
(
mvh
) promoter and
GFP
. Mvh is specific for differentiating mouse germ
cells from the late migration stage to the postmeiotic stage (Toyooka et al.
2000
).
The putative male germ cells were isolated from EBs that had been co-cultured with
isolated fetal mouse gonadal cells. After these ES-derived GFP-expressing cells
were isolated and transplanted into the mouse testis, remarkably, they underwent
meiosis and differentiated into cells resembling elongated spermatids (Toyooka
et al.
2003
). Another study used the stage-specific embryonic antigen 1 (SSEA1),
a marker of pluripotency, as a cell surface marker in the isolation of mouse PGCs
from EBs cultured in RA (Geijsen et al.
2004
). Genomic reprogramming was
examined in these cells and it was found that these mature male germ cells had the
expected erasure of methylation at the sex-specific imprinted
Igf2
(
Insulin-like
growth factor 2
) and the
H19
locus. Further, these cells underwent meiosis and
formation of a haploid genome and when injected into oocytes they restored the
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