Bone Morphogenetic Protein 4 Mediates Human Embryonic Germ Cell Derivation

Abstract

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). Unlike embryonic stem cells, virtually little is known regarding the factors that regulate EGC survival and maintenance. In mice, the growth factor bone morphogenetic protein 4 (BMP4) has been shown to be required for maintaining mouse embryonic stem cells, and disruptions in this gene lead to defects in mouse PGC specification. Here, we sought to determine whether recombinant human BMP4 could influence EGC derivation and=or human PGC survival. We found that the addition of recombinant BMP4 increased the number of human PGCs after 1 week of culture in a dose-responsive manner. The efficiency of EGC derivation and maintenance in culture was also enhanced by the presence of recombinant BMP4 based on alkaline phosphatase and OCT4 staining. In addition, an antagonist of the BMP4 pathway, Noggin, decreased PGC proliferation and led to an increase in cystic embryoid body formation. Quantitative real-time (qRT)-polymerase chain reaction analyses and immunostaining confirmed that the constituents of the BMP4 pathway were upregulated in EGCs versus PGCs. Downstream activators of the BMP4 pathway such as ID1 and phosphorylated SMADs 1 and 5 were also expressed, suggesting a role of this growth factor in EGC pluripotency.

Techniques and Conditions for Embryonic Germ Cell Derivation and Culture

“…In agreement with mouse, transmembrane SCF expression of the feeder layer promoted human PGC growth (Kerr et al, 2006). It is also possible to derive and maintain human EGCs without serum using 15-20% KSR (Invitrogen) (Turnpenny et al, 2003;Pan et al, 2005;Hua et al, 2009;Hiller et al, 2011). Whereas serum provides essential nutrients also provides some factors that promote differentiation of EGCs and make the analysis of the experiments difficult.…”

“…Recently it has been reported that the addition of 5-20 ng/ml recombinant BMP4 (R&D Systems) increases, in a dose-responsive manner, the efficiency of EGC derivation and maintenance. The results revealed an increase in EGC derivation of 5-fold with 20 ng/ml BMP4 and, after 3 weeks the survival efficiency increased by 50-fold in the presence of BMP4 (Hiller et al, 2011). Survival appeared to be in part related to the ability of BMP4 to inhibit spontaneous differentiation in these cultures, an inherent problem with long-term maintenance of human EGC cultures.…”

“…The cell suspension is plated on the previously prepared feeder layer, culturing each gonad on 4-10 wells of a 96 well plate, keeping in mind that derivation efficiency could be affected by the plating density (Kerr et al, 2006). To get rid of accompanying somatic cells, PGCs could be sorted before plating Hiller et al, 2011). PGCs are isolated using magnetic cell sorting technology and an indirect labeling of cells with magnetically tagged goat anti-mouse IgM antibodies towards a mouse-anti-SSEA1 antibody (Miltenyi Biotech).…”

“…Culturing a pure population of PGCs allows ruling out possible effects of the somatic cells of the gonads in EGC derivation. When PGCs are counted after sorting, approximately 50 PGCs are seeded in each well of a 96-well plate (Hiller et al, 2011). The plate is incubated at 37ºC in 5% (or 8% (Shamblott et al, 1998) CO 2 with 95% humidity for 7 days.…”

“…These included different concentrations of hbFGF, 20 µM retinoic acid, 1000 U/ml hLIF, 10 ng/ml hSCF (Cell Signaling), 100 µM forskolin (Sigma), 20 ng/ml stromal cell-derived factors & ( , -SDF), and 50 ng/ml BMP-4. BMP-4 was previously used to improve human EGC culture (Hiller et al, 2011) and retinoic acid has been shown to replace bFGF for mEGC derivation (Koshimizu et al, 1996). Three different protocols have been tried: 10 ng/ml hbFGF, LIF, hSCF and forskolin; 20 ng/ml hbFGF, LIF, hSCF, , -SDF and BMP-4; and retinoic acid, LIF, hSCF, , -SDF and BMP-4.…”