FIGURE 2. Development of primordial germ cells in the differentiating EB.

a, Effects of treatment with retinoic acid (RA) on EB-derived cells. Retinoic acid differentiates ES cells (top panel) but supports PGCs¹² (middle panel). Culture of EB-derived SSEA1⁺ cells in the presence of retinoic acid selects for PGCs that retain germ cell marker expression (bottom panel). b, SSEA1⁺ cells were isolated from EBs of different ages by immunomagnetic beads, cultured in retinoic acid for 7 days, and SSEA1 expression was analysed by flow cytometry. The percentage of GFP⁺ ES and EB-derived cells expressing SSEA1 is plotted (n = 3) c, Alkaline phosphatase (AP) expression on ES cells (left panel) and day 7 EB-derived cells (right panel) after 5 days of culture in retinoic acid. The day 7 EB-derived cells formed large colonies of cells positive for alkaline phosphatase that resembled PGCs on gross inspection¹⁷. Cells positive for alkaline phosphatase migrated out of the large colony (arrowheads). d, Imprint erasure of Igf2r and H19/Igf2 loci. Top panel, DMR2 region of Igf2r locus. Southern analysis of genomic DNA from individual ES and EB-derived embryonic germ clones digested with PvuII and MluI is shown. Bottom panel, H19/Igf2 locus. The same clones as in the top panel were digested with SacI and HhaI. The day of EB development at which individual embryonic germ clones were derived is indicated. Control lanes include J1 ES cells (somatic methylation profile), methylation-deficient DNMT1^null ES cells³⁰ and parthenogenetic (parthen.) ES cells (showing a maternal methylation pattern on both Igfr2 alleles).