1. Comparative evaluation of different in vitro systems that stimulate germ cell differentiation in human embryonic stem cells 
Mark Richards, Ph.D., Chui-Yee Fong, Ph.D., Ariff Bongso, D.Sc., Ph.D. 
Fertility and Sterility 
Volume 93, Issue 3, Pages (February 2010)
DOI: /j.fertnstert
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Montage of undifferentiated hESC colonies, hESC EBs, POF fibroblasts, and differentiating EB POF cultures. (A) Undifferentiated bulk-cultured HES-3 colonies on MEF feeders; (B) hESC EBs in suspension culture; (C) early fourth-passage porcine ovarian fibroblasts; (D) hESC EB outgrowths after 15 days on mitotically inactivated POFs. All scale bars = 200 μm.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
HES-3 qRT-PCR data relative to 15-day untreated EBs. A clear increase in VASA, BOULE, and other germ cell marker expression is detectable in some of the treatment arms. Residual AFP and Nestin expression were also detectable across almost all treatments. CM = conditioned medium.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Increased VASA reactivity in 30-day HES-3 EBs cocultured with POFs. (A) Phase contrast image of 15-day-old differentiating HES-3 EB on POF; (B) 6-diamino-2-phenylindole (DAPI) counterstain; (C) VASA immunoreactivity; (D) DAPI-FITC overlay; (E) phase contrast image of 30-day-old differentiating HES-3 EB on POF; (F) DAPI counterstain; (G) VASA immunoreactivity; (H) DAPI-FITC overlay. CM = conditioned medium. All scale bars = 200 μm.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Semiquantitative RT-PCR confirmation of germ cell marker gene expression in 30-day differentiating EB cultures on POF. L = 100-bp DNA ladder; lane 1: VASA (239 bp); lane 2: BOULE (260 bp); lane 3: DAZL (232 bp); lane 4: GDF3 (435bp); lane 5: GDF9 (252 bp); lane 6: MLH1 (247 bp); lane 7: SCP1 (221 bp); lane 8: PUM1 (436 bp); lane 9: PUM2 (200 bp); lane 10: POU5F1 (247 bp).
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

6. Figure 5
(A) Quantitative RT-PCR gene expression analysis for 30-day HES-3 EBs on POF relative to 15-day HES-3 EBs on POF. Relative to the 15-day sample, all germ cell marker genes show corresponding elevated expression in the 30-day sample. Grey bars, 30-day EBs; white bars, 15-day EBs. (B) Quantitative RT-PCR gene expression analysis in VASA-positive population relative to VASA-negative population in 30-day HES-3 EB-POF cocultures. Out of the total cell population, 6.5% was VASA positive as determined by FACS. All germ cell markers show elevated expression in the VASA-positive cell population. RQ values for VASA gene expression in the VASA-positive population were more than 20-fold higher than in the VASA-negative population. Grey bars, VASA-positive cells; white bars, VASA-negative cells.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions