Biomedical Engineering Reference
In-Depth Information
3.5.2
Definitive Proof of Mature Germ Cell Formation
Currently, more stringent criteria and assessments are needed for determining
whether an ES cell-derived germ cell truly is a functional mature germ cell, sperm
or oocyte. Ultimately, the real proof of concept and gold-standard for mature germ
cell identity is that the gamete-like cell can be transplanted
in vivo
to generate mature
viable oocytes or sperm and/or ultimately be used to fertilize and generate offspring.
Cell surface markers and mRNA and protein expression profiles characteristic of the
germ cell lineage have been used in numerous ES cell studies in mouse and human.
Formation of mature sperm from mouse ESCs has been relatively successful, as
demonstrated by genetic, meiotic, and morphological characterizations and trans-
plantation studies. Although “oocyte-like” cells have been differentiated from mouse
ES cells by several groups, their ultimate functionality or ability to generate off-
spring has not been demonstrated.
Previous reports and observations from our laboratory have indicated that
these oocyte-like cells are produced in limited quantity and are unable to prog-
ress through meiotic prophase I. Rigorous studies are needed to promote these
germ cells to undergo meiosis and acquire the potential to differentiate into
mature oocytes. To this end, our laboratory has aimed to differentiate mature and
functional female oocytes from mouse ES cells by using transplantation strate-
gies. Due to the critical importance of the ovarian somatic cell niche for primor-
dial germ cell commitment to an oocyte developmental program, meiotic
progression, and follicular maturation, Nicholas et al. (
2009
) developed an ovar-
ian tissue co-culture and transplantation system to achieve functional maturation
of ESC-derived oocytes (Fig.
3.7a
). Using the DPE-Oct4-GFP germ cell-specific
reporter and
Dazl
-null mESC lines it was demonstrated that germ cells formed
after 3 weeks of spontaneous EB differentiation and were subsequently isolated
via FACS. GFP-positive putative germ cells were sorted, and 100,000 were co-
aggregated with dissociated wild-type newborn ovarian tissue, and transplanted
under the kidney capsule of ovariectomized recipient female mice. The grafts
were harvested 3 weeks later, and the oocytes derived from DPE-Oct4-GFP
ESCs were confirmed by GFP expression using immunohistochemistry.
Excitingly, 23 ESC-derived oocytes were identified in sections of the trans-
planted ovarian tissue. Many oocytes were contained within follicles, with some
reaching the primary follicle stage. These results demonstrate that this
in vitro
gonadal co-culture and transplantation system may be an effective way to pro-
mote meiotic progression, maturation, and functional potential of ES-derived
oocytes (Fig.
3.7a
) (Nicholas et al.
2009
). This work is now being translated to
the human, with the goals of generating mature human oocytes and sperm from
human ES and iPS cells using similar gonadal transplantation strategies.
As illustrated above, mouse and human ES cells can differentiate to the germ cell
lineage and produce cells with characteristics of mature germ cells. However, in the
majority of studies to date, rigorous analysis of genomic remodeling or methylation
status, and assessment of meiosis and meiotic checkpoints, recombination and
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