Making Germ Cells from Human Embryonic Stem Cells - Male Germline Stem Cells Developmental and Regenerative Potential - page 64

mouse Nanog ), STELLAR ( Stella-Related , or Germ and embryonic stem cell
enriched protein STELLA ), and GDF3 ( Growth Differentiation Factor 3 ) were
cloned, and similar to OCT4 their expression was exclusive to both undifferentiated
hESCs and human germ cells. Upon differentiation of hESCs the expression of all
three genes decreased, while the reduction in NANOG activity specifically led to a
loss of hESC pluripotency (Clark et al . 2004b ). Underscoring the importance of the
proteins in common to both ES cells and germ cells is the observation that mutant
phenotypes are often associated with both ES cell maintenance (or early embryonic
development) and germ cell development (i.e., Oct4 and Tial1 ) (Beck et al. 1998 ;
Kehler et al. 2004 ).
The early differentiation of human and mouse germ cells is marked by formation
of distinct translational protein complexes, which form a “germ cell particle.” This
germ cell particle includes homodimers and heterodimers of DAZ gene family
members as well as many germ plasm homologs, such as the PUM and NANOS
genes (see Fig. 3.3B ) (Fox et al. 2007 ; Moore et al. 2003, 2004 ). Although human
ES cells and germ cells express several of these same critical proteins, the spatial
and temporal patterns of protein-protein interactions and signaling complexes
apparently differ between germ cells and ES cells as resolved by Fluorescence
Resonance Energy Transfer (FRET) (Fox et al. 2007 ).
In light of much recent work it has become evident that in addition to similar
ultrastructural and developmental characteristics the genes expressed in ES cells
and early germ cells form a largely overlapping set, suggesting that these cell
types may be closely related or identical or may share common regulatory pathways
(Clark et al. 2004a, b ; Fox et al. 2007 ; Moore et al. 2003, 2004 ).

3.4.2 Can Human ES Cells Give Rise to Germ Cells?

There are clear morphological, developmental, and genetic similarities between
undifferentiated germ cells and embryonic stem cells. Many investigations have
shown that mouse and human ES cells can be differentiated into multiple tissue
lineages in vitro . Therefore, it was logical to test whether human ES cells could also
give rise to the germ cell lineage. Our laboratory first investigated whether human ES
cell lines, like those of mice, were capable of forming germ cells in vitro . Clark et al.
( 2004a ) used quantitative polymerase chain reaction (PCR) to examine the transcrip-
tional profiles of three pluripotent human ES cell lines, HSF-6 and H9 (karyotype 46;
XX), and HSF-1 (karyotype 46; XY) before and after differentiation.
Undifferentiated cultures of human ES cells were maintained on irradiated
mouse embryonic fibroblast (MEF) cells, as human ES cells grow best when in
contact with fibroblast cells of either mouse or human origin. All cultures were
grown at 37°C with 5% CO 2 in a standard medium containing recombinant human
basic fibroblast growth factor (bFGF). Similar to previous work, OCT4 was
expressed at high levels in all undifferentiated ES cell lines tested. Other genes
examined included those previously reported in undifferentiated human ES cells,