Introduction
In the human embryo, the germ cells originate from PGCs, which evolve from the epiblast-cell layer. Human pluripotent stem cells may be differentiated into PGCs as a first step of a potential novel treatment of infertility, related to lack or low quality gametes.
Aim
To direct the differentiation of human pluripotent stem cells into PGCs and further on to meiotic germ cells.
Materials and Methods
Our methodology relied on identifying the key factors and recapitulating the steps in human embryonic gamete differentiation. The experimental system was human embryonic stem cells, grown on Laminin under specific feeder-free culture system which enabled their maintenance as epiblast-like cells.
Subsequently, molecular factors which are involved in PGCs-specification were used to induce early germ-cell differentiation. The detection of PGC-like fate was performed using a reporter cell line and using real-time PCR and immunofluorescent stainings.
Results
We established for the first time a human epiblast-like culture system. The cells in this culture system expressed markers of pluripotent epiblast cells and were subsequently used for further differentiation.
Following Bone-morphogenic-protein (BMP4) and retinoic-acid-based differentiation, the cells expressed early PGC-markers as confirmed by molecular methods and immunofluorescent stainings.
Conclusions
The initial step of PGC differentiation in the embryo, which is the formation of epiblast cells was recapitulated in culture. The epiblast-like culture system showed the potential to give rise to PCG- like cells. These results may serve as the basis for further differentiation into later developmental stages of gametes using factors of more advanced stages.
