172 PRIMORDIAL GERM CELL DIFFERENTIATION FROM ES CELLS IN VITRO IN MOUSE

Abstract

The primordial germ cells (PGC) in the genital ridge of the embryo are the progenitors of sperm and eggs. The goal of the present study was to derive PGC cells from embryonic stem (ES) cells and compare their gene expression with that of primary PGC cultures. R1 (Nagy A et al. 1990 Development 110, 815–821) and Oct4-GiP (Ying QL et al. 2002 Nature 416, 545–548) ES cell lines were differentiated into PGCs. For in vitro differentiation, the modified method of Geijsen N et al. (2004 Nature 427, 148–154) was used. In brief, ES cell suspension was put into hanging drops (400 cells per drop) for two days, where they formed embryoid bodies (EBs). The medium consisted of Iscove's Modified Dulbecco's Media (Gibco) supplemented with 10% FBS (Hyclone, Logan, UT, USA), 30 μg mL⁻¹ iron saturated transferrin (Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM 2-mercaptoethanol (Sigma, Hungary), non-essential amino acids (Sigma), 4.5 mM monothioglycerol (Sigma), 50 μg mL⁻¹ ascorbic acid (Sigma), 2 mM glutamine (Gibco), and antibiotics. The EB clumps were differentiated in suspension culture for 2 or 5 days, and then dissociated with collagenase treatment. Cells positive for SSEA-1 were isolated from dissociated EBs by immunomagnetic bead sorting and plated into gelatinized plates in the presence of 2 μM retinoic acid (Sigma). After 7 days of culture, individual PGC colonies were isolated and subcloned. The subcloned PGCs were cultured in PGC medium consisting of Dulbecco's Modified Eagle Media (Gibco) supplemented with 15% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 1000 U recombinant mouse leukaemia inhibitor factor (ESGRO^®, Chemicon International, Inc., Temecula, CA, USA), 20 ng mL⁻¹ basic fibroblast growth factor (Sigma), 60 ng mL⁻¹ soluble mouse stem cell factor (Sigma), and antibiotics. The gene expression profile was monitored using semi-quantitative RT-PCR. The gene expression of Oct4, Nanog, Stella, Piwil2, Rnf17, and Tex14 were analyzed during the differentiation. Primary PGC cultures were also isolated from (C57BL/6 × DBA)F1 embryos of age 8.5 and 11.5 days post-coitum, and differentiated in vitro. The previously described PGC medium was used to proliferate the isolated cells. The gene expression profile of PGCs and ES-derived PGC lines were compared. There were no great differences between the gene expression profiles of PGCs and ES cell-derived PGC cells. SSEA-1 and alkaline phosphatase staining of cells did not show differences between the two cell populations. We have shown here the two PGC populations do not differ from each other in gene expression of the selected genes. Further investigation is needed to differentiate the PGCs into gametes and to analyze the gene expression of other genes involved in gamete differentiation.

The authors would like to acknowledge Gyorgyi Kungl for the technical help. This research was supported by OTKA T046171 grant.