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mass expressed
STELLAR
,
OCT4
, and
NANOS1
, but unlike ES cells, did not
express the germ-cell specific marker
DAZL
and the somatic marker
NCAM1
(
Neural Cell Adhesion Molecule 1
) (Clark et al.
2004a
). These results indicate that
in the absence of the somatic cell environment of the developing embryo, a subset
of the inner cell mass population embarks on the germ cell lineage pathway.
In these same investigations, when human ES cells were differentiated
in vitro
there was a shift in RNA and protein expression from that of immature/premeiotic
germ cells to those of mature germ cells and gametes. Methods for spontaneous
differentiation included taking undifferentiated hESCs and pooling them into a
starting homogeneous population before distributing into individual wells of an
ultralow-attachment plate, which facilitates the growth of embryoid bodies in sus-
pension (without bFGF). Human ES cells maintained in these culture conditions for
up to 21 days differentiated into embryoid bodies, which expressed several markers
of later germ cell differentiation including, the gonocyte marker
VASA
.
Because VASA-expressing putative germ cells were detected in EBs the expres-
sion of other germ cell markers were also assayed. The meiotic markers
Synaptonemal Complex Protein 1
and
3
(
SCP1
or
SYCP1
and
SCP3
or
SYCP3
) and
BOULE
, and the post-meiotic gamete markers
Growth Differentiation Factor 9
(
GDF9
) and
Tektin1
(
TEKT1
), were expressed at day 14 of EB differentiation
(Clark et al.
2004a
). VASA-expressing cells occurred in clusters of cells at the
edges and throughout small sections of EBs. STELLAR and DAZL expression on
the other hand, was more predominant in cells of EBs, occurring in clusters, lining
the edges, and throughout EB sections. It was also noted that with increasing dif-
ferentiation of EBs, expression of mRNA markers of the somatic lineages also
increased, including
NCAM1
(differentiated ectoderm),
Alphafetoprotein
(
AFP
;
differentiated mesoderm), and
Tyrosine Kinase Receptor
(
KDR
; differentiated
endoderm). As expected, somatic cell differentiation occurred in subpopulations of
cells in parallel with germ cell differentiation. Taken together, this work was the
first to clearly demonstrate that a subset of human ES cells can differentiate toward
the germ cell lineage and produce cells with markers characteristic of gonocytes
(Fig.
3.4
). Isolation of these ES cell-derived germ cells, extended differentiation,
characterization at the single-cell level, and subsequent functional assays were the
next steps of these pioneering investigations.
3.4.3
Generation of Mouse Germ Cells and Gametes In Vitro
Several previous studies have shown that a cocktail of soluble growth factors,
including kit ligand (KL), leukemia inhibitory factor (LIF), stroma-derived factor 1
(SDF1), BMP4, and bFGF, and compounds including
N
-acetyl-L-cysteine, forsko-
lin, and retinoic acid (RA), are able to sustain the survival and self-renewal of
mouse germ cells, after being removed from their somatic cell support in the
gonads and cultured
in vitro
. The culture conditions used minimized apoptosis
(programmed cell death) in germ cells, enhanced their proliferation, and allowed
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