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Endocrine and epigenetic control of trophectoderm (TE) differentiation from human embryonic stem cells (hESCs): the role of bone morphogenic protein (BMP4) and histone deacetylases (HDACs)
medium and the other was cultured in SSCs-proliferation medium (StemPro34 medium SFM +bFGF, +GDNF and +LIF). After 6 days of culturing, cultured cells were assessed for PGC-specific marker (VASA) and SSC-specific markers (integrin b1, Thy-1 and GFR a-1) by using immunostaining, FACS and RT-PCR. RESULTS: First of all, the specification-rate of PGCs (VASA-positive) was increased in both CHA-hES4 and H1 lines treated with BMP-4 and RA. And, the incubation in SSCs-proliferation medium increased the ratio of SSC-specific markers-positive cells. Also, SSC-like cells derived from hESCs has shown similar characters to SSCs obtained from human testis. CONCLUSION: These results showed that our three-step culture system could induce primordial germ cells in EB population and differentiate from PGC to SSCs-like cells. Also, this study would be a useful tool for germ cell biology and application of hESCs.
P-513 Wednesday, October 27, 2010 ENDOCRINE AND EPIGENETIC CONTROL OF TROPHECTODERM (TE) DIFFERENTIATION FROM HUMAN EMBRYONIC STEM CELLS (hESCs): THE ROLE OF BONE MORPHOGENIC PROTEIN (BMP4) AND HISTONE DEACETYLASES (HDACs). T. M. Erb, S. E. Mucko, C. Schneider, P. J. Sammak. Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh Medical Center, Pittsburgh, PA; Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA. OBJECTIVE: Compared to spontaneous embryos, E3.5 ART embryos exhibit epigenetic variability, delayed development, and dysregulated differentiation in part due to in vitro media composition. hESCs provide a platform for evaluating endocrine and epigenetic effects on early human development to trophectoderm. State of the art media for TE-directed differentiation of hESCs are undefined, complicating mechanistic studies of early human placental development. We describe a novel, chemically defined essential media for TE derivation and evaluate whether HDACs regulate human TE differentiation. DESIGN: TE-directed hESC differentiation in defined media, phase contrast and confocal immunofluorescence microscopy, HDAC inhibition. MATERIALS AND METHODS: Pluripotent hESCs were maintained on mouse embryo fibroblasts (epiblast-ESCs) or in defined StemPro (ICMESCs). TE-directed differentiation media contained DMEM/F12 with insulin, heparin, BMP4 and the Activin A inhibitor SB431542. RESULTS: TE was produced from preimplantation ICM-ESCs, not post implantation epi-ESCs, both derived from the same source hESCs. TE forms within 48hrs in Erb’s Minimal Pluripotency Media (EMPM) with 100ng/mL of BMP4 treatment. BMP4 is sufficient, has a dose response effect, and is synergistic when combined with SB431542 for TE-directed differentiation of hESCs. Terminally differentiated syncytiotrophoblasts appeared by day 8. Neurectoderm and extraembryonic endoderm were suppressed. Tricostatin A, an HDAC inhibitor, slows differentiation in a dose dependent manner. CONCLUSION: Our in vitro model enables mechanistic studies of endocrine and environmental effects on human embryo development. We show that early placental development depends partly on highly pluripotent precursor hESCs, BMP4 and on HDAC activation. Since blastocyst development is synchronized to uterine receptivity, kinetic effects of epigenetic regulators on TE differentiation might produce delays that adversely affect implantation rates. Supported by: FAMRI 6-3401-2150, TME; R01 EB006161, PJS.
P-514 Wednesday, October 27, 2010 PRIMORDIAL GERM CELL DERIVATION FROM MOUSE iPS CELLS AND ASSOCIATED EPIGENETIC CHANGES. X. Li, J. Deng, L. Sun, D. Diep, K. Zhang, B. Beutler. Department of Genetics, The Scripps Research Institute, La Jolla, CA; Obstetric & Gynecology Department, Wayne State University, School of Medicine, Detroit, MI; Bioengineering Department, University of California, San Diego, La Jolla, CA. OBJECTIVE: Induced pluripotent stem cells (iPS cells) have the pluripotency that is similar to that of ES cells, and provide an accessible tool to study the germ cell development. However, in vitro germ line differentiation from iPS cells has not been reported. In this study, we aimed to derive the primordial germ cells (PGC) and more mature germ cells in vitro from iPS cells, and use this differentiation system as a platform for studying the poorly understood process of epigenetic reprogramming experienced by PGCs, which is
FERTILITY & STERILITYÒ
important for the erasure of imprinting and epimutations, and the return to pluripotency. DESIGN: Develop the putative PGCs and more mature germ cells from mouse iPS cells carrying an Oct4-GFP reporter, and use the targeted bisulfite resequencing technology to study the DNA methylation changes involved in PGCs and germ cell line formation. MATERIALS AND METHODS: We differentiated the mouse iPS cells into EBs, subsequently isolated PGCs and continuously grew germ cell lines. The isolated Oct4-GFP+ cells mixed in tissue from embryonic gonads, as well as more mature germ cells at various stages of differentiation were transplanted into the testes of germ cell-depleted mice to study the efficiency of gametogenesis from iPS cells. We utilized bisulfite padlock sequencing to determine the DNA methylation status of 33,000-39,000 CpG sites in mouse imprinted genes, miRNA genes and genes on X and Y chromosomes, and compared the global methylation patterns in iPS cells and PGCs. RESULTS: iPS cells support the earliest stages of germ lineage formation in cell culture, as demonstrated by the formation of PGCs and subsequent more mature germ cells. Differentially methylated region and associated genes involved in early reprogramming in PGCs were identified. CONCLUSION: iPS cells sustain the early gametogenesis and can be employed as a potential source of gametes. This in vitro differentiation system helps achieving a deeper understanding of DNA methylation in transcriptional regulation in germ cell development.
ART-IN VITRO FERTILIZATION P-515 Wednesday, October 27, 2010 PERINATAL OUTCOME OF 2140 SINGLETONS BORN FROM TRANSFER OF FROZEN-THAWED EMBRYOS (FET) CONCEIVED BYASSISTED REPRODUCTIVE TECHNOLOGY (ART): A FRENCH CONTROL STUDY 1998-2008. S. Epelboin, E. Devouche, H. Pejoan, G. Viot, G. Apter-Danon, Scientific Committee AMP-Vigilance Reseau Follow-Up. Gynecology Obstetrics & ART, Hoˆpital Bichat-Claude Bernard, Paris, Ile de France, France; Universite Paris-Descartes, Paris, Ile de France, France; Gynecology Obstetrics & Paediatrics, Hoˆpital Beaujon, Clichy, Ile de France, France; Obstetrics Paediatrics & Genetics, Hoˆpital Cochin-Saint Vincent de Paul, Paris, Ile de France, France; Child Psychiatry, Hoˆpital Jean Rostand, Se`vres, Ile de France, France; Reseau Follow-Up, Poissy, Ile de France, France. OBJECTIVE: To assess outcome of cryo singletons born after FET and compare with singleton controls conceived with IVF or ICSI and fresh transfer. DESIGN: French population based controlled prospective study from 1998 to 2008. MATERIALS AND METHODS: Data were obtained from the French Follow-up cohort of 16002 singletons born after ART. They came from 30 different ART centers. Children were recruited from birth and medical data were collected from parental questionnaires until 72 months. We report here birth questionnaire data. The cryo population was 2140 (13%) (cryoIVF¼6%; cryo-ICSI¼7%). The control group was 13862 singletons (87%) born after IVF (34%) or ICSI (53%) and fresh transfer. IVF/ICSI ratio was higher in cryo cohort than in the fresh one (84%>65%, p< .0001). RESULTS: Maternal and paternal ages were similar in fresh and cryo cohorts (33.3 4 & 33.2 4/36 5.7 & 36 5.6 years). Primiparous/multiparous maternal status ratio appeared 1.6 higher (p< .0001) in fresh sample. The boys/girls ratio was 1.05 higher in the cryo population (p¼ .31). The caesarean/vaginal delivery ratio was the same in both populations (28%). The preterm birth rates <37 weeks were 8.6% in fresh versus 8.3% in cryo (p¼ .64).The low birth weight (LBW) <2500 g rates were higher in fresh cohort. When considering term-born children (>37WA), LBW/normal BW ratio (3.6%/1.8%) was twice higher in fresh population than in cryo (OR¼2, p< .0001). Babies born of fresh or cryo transfer measured respectively 49.7 4 and 50.1 2 cm (p< .0001). The mortality rate was 7.6& in fresh and 7.9& in cryo (NS). CONCLUSION: From this large multicentric survey of ART-born children, we observed that cryo and fresh singletons birth data were comparable, except for a lower LBW rate in cryo group. Follow-up survey at older ages will provide further evaluations of the congenital malformations fully available from 4 months questionnaire, health and development of cryo children among the ART cohort, including the imputability of the initial technics.
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P-513 Wednesday, October 27, 2010 ENDOCRINE AND EPIGENETIC CONTROL OF TROPHECTODERM (TE) DIFFERENTIATION FROM HUMAN EMBRYONIC STEM CELLS (hESCs): THE ROLE OF BONE MORPHOGENIC PROTEIN (BMP4) AND HISTONE DEACETYLASES (HDACs). T. M. Erb, S. E. Mucko, C. Schneider, P. J. Sammak. Obstetrics, Gynecology, and Reproductive Sciences, University of Pittsburgh Medical Center, Pittsburgh, PA; Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA. OBJECTIVE: Compared to spontaneous embryos, E3.5 ART embryos exhibit epigenetic variability, delayed development, and dysregulated differentiation in part due to in vitro media composition. hESCs provide a platform for evaluating endocrine and epigenetic effects on early human development to trophectoderm. State of the art media for TE-directed differentiation of hESCs are undefined, complicating mechanistic studies of early human placental development. We describe a novel, chemically defined essential media for TE derivation and evaluate whether HDACs regulate human TE differentiation. DESIGN: TE-directed hESC differentiation in defined media, phase contrast and confocal immunofluorescence microscopy, HDAC inhibition. MATERIALS AND METHODS: Pluripotent hESCs were maintained on mouse embryo fibroblasts (epiblast-ESCs) or in defined StemPro (ICMESCs). TE-directed differentiation media contained DMEM/F12 with insulin, heparin, BMP4 and the Activin A inhibitor SB431542. RESULTS: TE was produced from preimplantation ICM-ESCs, not post implantation epi-ESCs, both derived from the same source hESCs. TE forms within 48hrs in Erb’s Minimal Pluripotency Media (EMPM) with 100ng/mL of BMP4 treatment. BMP4 is sufficient, has a dose response effect, and is synergistic when combined with SB431542 for TE-directed differentiation of hESCs. Terminally differentiated syncytiotrophoblasts appeared by day 8. Neurectoderm and extraembryonic endoderm were suppressed. Tricostatin A, an HDAC inhibitor, slows differentiation in a dose dependent manner. CONCLUSION: Our in vitro model enables mechanistic studies of endocrine and environmental effects on human embryo development. We show that early placental development depends partly on highly pluripotent precursor hESCs, BMP4 and on HDAC activation. Since blastocyst development is synchronized to uterine receptivity, kinetic effects of epigenetic regulators on TE differentiation might produce delays that adversely affect implantation rates. Supported by: FAMRI 6-3401-2150, TME; R01 EB006161, PJS.
P-514 Wednesday, October 27, 2010 PRIMORDIAL GERM CELL DERIVATION FROM MOUSE iPS CELLS AND ASSOCIATED EPIGENETIC CHANGES. X. Li, J. Deng, L. Sun, D. Diep, K. Zhang, B. Beutler. Department of Genetics, The Scripps Research Institute, La Jolla, CA; Obstetric & Gynecology Department, Wayne State University, School of Medicine, Detroit, MI; Bioengineering Department, University of California, San Diego, La Jolla, CA. OBJECTIVE: Induced pluripotent stem cells (iPS cells) have the pluripotency that is similar to that of ES cells, and provide an accessible tool to study the germ cell development. However, in vitro germ line differentiation from iPS cells has not been reported. In this study, we aimed to derive the primordial germ cells (PGC) and more mature germ cells in vitro from iPS cells, and use this differentiation system as a platform for studying the poorly understood process of epigenetic reprogramming experienced by PGCs, which is
FERTILITY & STERILITYÒ
important for the erasure of imprinting and epimutations, and the return to pluripotency. DESIGN: Develop the putative PGCs and more mature germ cells from mouse iPS cells carrying an Oct4-GFP reporter, and use the targeted bisulfite resequencing technology to study the DNA methylation changes involved in PGCs and germ cell line formation. MATERIALS AND METHODS: We differentiated the mouse iPS cells into EBs, subsequently isolated PGCs and continuously grew germ cell lines. The isolated Oct4-GFP+ cells mixed in tissue from embryonic gonads, as well as more mature germ cells at various stages of differentiation were transplanted into the testes of germ cell-depleted mice to study the efficiency of gametogenesis from iPS cells. We utilized bisulfite padlock sequencing to determine the DNA methylation status of 33,000-39,000 CpG sites in mouse imprinted genes, miRNA genes and genes on X and Y chromosomes, and compared the global methylation patterns in iPS cells and PGCs. RESULTS: iPS cells support the earliest stages of germ lineage formation in cell culture, as demonstrated by the formation of PGCs and subsequent more mature germ cells. Differentially methylated region and associated genes involved in early reprogramming in PGCs were identified. CONCLUSION: iPS cells sustain the early gametogenesis and can be employed as a potential source of gametes. This in vitro differentiation system helps achieving a deeper understanding of DNA methylation in transcriptional regulation in germ cell development.
ART-IN VITRO FERTILIZATION P-515 Wednesday, October 27, 2010 PERINATAL OUTCOME OF 2140 SINGLETONS BORN FROM TRANSFER OF FROZEN-THAWED EMBRYOS (FET) CONCEIVED BYASSISTED REPRODUCTIVE TECHNOLOGY (ART): A FRENCH CONTROL STUDY 1998-2008. S. Epelboin, E. Devouche, H. Pejoan, G. Viot, G. Apter-Danon, Scientific Committee AMP-Vigilance Reseau Follow-Up. Gynecology Obstetrics & ART, Hoˆpital Bichat-Claude Bernard, Paris, Ile de France, France; Universite Paris-Descartes, Paris, Ile de France, France; Gynecology Obstetrics & Paediatrics, Hoˆpital Beaujon, Clichy, Ile de France, France; Obstetrics Paediatrics & Genetics, Hoˆpital Cochin-Saint Vincent de Paul, Paris, Ile de France, France; Child Psychiatry, Hoˆpital Jean Rostand, Se`vres, Ile de France, France; Reseau Follow-Up, Poissy, Ile de France, France. OBJECTIVE: To assess outcome of cryo singletons born after FET and compare with singleton controls conceived with IVF or ICSI and fresh transfer. DESIGN: French population based controlled prospective study from 1998 to 2008. MATERIALS AND METHODS: Data were obtained from the French Follow-up cohort of 16002 singletons born after ART. They came from 30 different ART centers. Children were recruited from birth and medical data were collected from parental questionnaires until 72 months. We report here birth questionnaire data. The cryo population was 2140 (13%) (cryoIVF¼6%; cryo-ICSI¼7%). The control group was 13862 singletons (87%) born after IVF (34%) or ICSI (53%) and fresh transfer. IVF/ICSI ratio was higher in cryo cohort than in the fresh one (84%>65%, p< .0001). RESULTS: Maternal and paternal ages were similar in fresh and cryo cohorts (33.3 4 & 33.2 4/36 5.7 & 36 5.6 years). Primiparous/multiparous maternal status ratio appeared 1.6 higher (p< .0001) in fresh sample. The boys/girls ratio was 1.05 higher in the cryo population (p¼ .31). The caesarean/vaginal delivery ratio was the same in both populations (28%). The preterm birth rates <37 weeks were 8.6% in fresh versus 8.3% in cryo (p¼ .64).The low birth weight (LBW) <2500 g rates were higher in fresh cohort. When considering term-born children (>37WA), LBW/normal BW ratio (3.6%/1.8%) was twice higher in fresh population than in cryo (OR¼2, p< .0001). Babies born of fresh or cryo transfer measured respectively 49.7 4 and 50.1 2 cm (p< .0001). The mortality rate was 7.6& in fresh and 7.9& in cryo (NS). CONCLUSION: From this large multicentric survey of ART-born children, we observed that cryo and fresh singletons birth data were comparable, except for a lower LBW rate in cryo group. Follow-up survey at older ages will provide further evaluations of the congenital malformations fully available from 4 months questionnaire, health and development of cryo children among the ART cohort, including the imputability of the initial technics.
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