Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 69

Toyooka et al. ( 2003 ) found that co-culturing of BMP-producing cells, such as
isolated fetal gonadal cells, increased the number of PGCs formed from mouse ES
cells in vitro .
Similar to these studies in mice, our laboratory sought to develop methods to
more efficiently differentiate germ cells from human ES cells. Toward this goal,
Kee et al. ( 2006 ) differentiated human ES cells in the presence of human recombi-
nant BMP4, BMP7, and BMP8b and assayed the differentiation of germ cells by
genetic and immunohistochemical analyses. Human ES cells were differentiated
into embryoid bodies using the standard differentiation medium either with or with-
out various concentrations of human recombinant BMPs (1-100 ng/ml). The addi-
tion of BMP4, at 10 and 100 ng/ml, led to an approximate threefold increase in the
expression of the germ cell-specific markers VASA and SCP3 in embryoid bodies.
BMP7 and BMP8b showed synergistic effects on germ cell induction when used in
combination with BMP4. EBs differentiated in the presence of all three factors
had a 16-fold increase in VASA expression relative to undifferentiated hESCs.
Furthermore, the addition of BMPs to differentiating hESCs also increased the
percentage of cells in EBs that stained positively for VASA protein, with a fivefold
increase above that of EBs spontaneously differentiated in the absence of BMPs.
Although the effects of BMPs on the total putative germ cell population were mod-
est, the effects were significant and reproducible (Kee et al. 2006 ). These experi-
ments suggested that BMPs promote the differentiation of germ cells from human
ES cells.
A few subsequent studies demonstrated the derivation of germ cells from
human ES cells. Tilgner et al. ( 2008 ) developed a protocol that promoted the dif-
ferentiation of PGC-like cells from embryoid bodies using a simple fluorescence-
activated cell sorting (FACS) strategy. The isolated putative germ cells had greatly
increased expression of VASA , removal of parental imprints, and chromatin modi-
fications indicative of PGCs. In another study it was reported that small changes
to human ES cell growth conditions, including selecting for smaller ES cell colonies
(20-50 cells/colony) and decreasing the number of culture feeding cycles, rapidly
induced cells that were comparable to migratory PGCs (Bucay et al. 2009 ).
Interestingly, they found that expression of the chemokine receptor, CXCR4,
allowed the purification of this PGC-like population by FACS analysis. Upon dif-
ferentiation, these CXCR4-expressing cells increased their RNA and/or protein
expression of germ cell-specific markers including DAZL , PRDM1 ( PR domain
containing 1 , aka Blimp1 ), STELLAR , NANOG , TRA-1-60 , SSEA-4 , VASA , and
ACROSIN . They also identified Sertoli-like cells associated with these developing
PGC-like cells. These Sertoli-like cells showed expression of FSHR (follicle
stimulating hormone receptor) and SOX9 (SRY-sex determining region Y-box 9),
an autosomal gene necessary for Sertoli cell development. Further, electron
microscopy demonstrated morphological and ultrastructural characteristics remi-
niscent of Sertoli cells, including a highly invaginated nucleus and prominent
nucleolar complexes (Bucay et al. 2009 ). It is interesting to note that these male
germ- and Sertoli-like cells were obtained from XX chromosome-bearing female
ES cell lines (H9 and HSF-6).