Biomedical Engineering Reference
In-Depth Information
Both female (XX-bearing) and male (XY-bearing) undifferentiated hESC lines
were stably transduced with this reporter and subsequently cultured in adherent
differentiating conditions. As noted, previous studies found that spontaneous
human germ cell differentiation in EBs was inefficient and comprised a relatively
low percentage of the overall cell populations. Therefore, adherent differentiation
of ES cells cultured on Matrigel in the presence of BMPs was selected for these
studies. Conditioned medium (bFGF-containing medium collected after overnight
incubation on irradiated MEFs) was used to maintain the undifferentiated cells
when drug selection was required for lentiviral transduction. For hESC differentia-
tion, cells were incubated in differentiation medium supplemented with 50 ng/ml
recombinant BMP4, BMP7, and BMP8b, cultured for 0, 7, or 14 days, and analyzed
by FACS.
BMPs reproducibly increased the number of PGCs formed, which displayed the
expression patterns of mRNAs and proteins characteristic of germ cells. Following
the addition of BMPs for 14 days, the percentage of cells that were positive for GFP
increased to ~5% of the total differentiated cell population, compared to ~0.8% of
cells differentiated in the absence of BMPs (Fig.
3.5b
). The germ cell-specific
markers
DAZL
,
VASA
, and
STELLAR
were highly enriched in the GFP-positive
population, as was expression of the germ cell genes
PRDM1
(
Blimp1
) and
NANOG
, and the meiotic gene,
SCP3
, which is essential for synaptonemal complex
formation in meiosis. In addition, due to the importance of erasure of methylation
at imprinted loci in early germ cell development, the methylation status of imprinted
loci as well as global DNA methylation levels in GFP-positive and GFP-negative
cell populations was examined. Bisulfite genomic sequencing and 5-methyl-cytosine
nuclear staining indicated that the VASA-GFP germ cell population was generally
hypomethylated and likely undergoing global imprinting erasure.
To further investigate the properties of these isolated GFP-positive germ cells
and their ability to propagate
in vitro
, their alkaline phosphatase (AP) activity, a
marker for pluripotency, was tested. After isolation of germ cells by FACS, GFP-
positive cells were replated onto MEFs and examined for morphological character-
istics and AP activity. After 7 days the GFP-positive cells gave rise to tight colonies
of cells resembling those of embryonic germ cells (Fig.
3.5c
), while no colony was
found in the replated GFP-negative cells. Further, the VASA-GFP-expressing germ
cells showed intense AP activity, similar to that seen in EGCs. Therefore, the isolated
cells had all the hallmarks of developing human male and female germ cells and
could be propagated
in vitro
(Kee et al.
2009
). Moreover, this VASA-GFP reporter
system mediated the efficient isolation of the human PGC population and pro-
vided a mechanism to propagate and characterize developing germ cells in order
to probe their morphological changes, epigenetic reprogramming, and genetic
requirements.
In addition to using human ES cells to generate PGCs
in vitro
, current work is
focused at deriving PGCs from iPS cells. Two studies using specific germ cell
reporter systems have already demonstrated that germ cells can be derived from iPS
cells (Park et al.
2009
; Panula et al., unpublished). Further, several iPSC lines are
being established from fibroblasts of normal men and women and females with
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