Biomedical Engineering Reference
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Germ cells differentiated in these studies generally did not develop beyond the
primordial germ cell stage and did not enter meiosis. Further, there has been a lack
of available cell surface markers or fluorescent reporters to isolate reproducibly the
nascent human germ cell population and promote their maturation. Therefore, Kee
and co-workers sought to develop a system that would allow the isolation and
propagation of human germ cells differentiated from pluripotent human ES cells or
iPSCs (induced pluripotent stem cells). A germ cell-specific VASA-GFP reporter
system was constructed to enrich for germ cells from the complex population of
differentiated cell types derived from hESCs (Fig.
3.5
) (Kee et al.
2009
). In this
lentiviral vector pLVGV, enhanced GFP (eGFP) is flanked by 2.5 Kb of 5¢ promoter
upstream sequence and 1 Kb downstream 3¢ untranslated region (UTR) of human
VASA
inserted into the p2k7 lentiviral vector (Fig
3.5a
).
Fig. 3.5
VASA-GFP reporter system allows the isolation and characterization of germ cells dif-
ferentiated from human ES cells. (
a
) Simplified map showing the human VASA-GFP lentiviral
reporter vector, pLVGV. eGFP is flanked by 2.5 Kb upstream promoter sequence and 1 Kb down-
stream sequence of human VASA and inserted into the p2k7 lentiviral vector. (
b
) The percentage
of GFP-positive cells obtained by FACS analysis in three representative H9 ES cell populations
infected with the VASA-GFP reporter, including undifferentiated cells, spontaneously differenti-
ated cells, and cells differentiated with defined medium containing BMPs. The percentage of
GFP-expressing cells are circled. Note that the incubation with BMPs greatly improves the per-
centage of GFP-positive cells isolated in comparison to those spontaneously differentiated.
(
c
) Phase contrast image of a representative germ cell colony showing tightly packed morphology
after being isolated
via
FACS and replated and propagated on inactivated MEFs for 7 days (mag-
nification ×40; data provided by K. Kee)
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