Gist Croft, "Self-organization of the in vitro attached human embryo and its implications"
•
2 likes•1,547 views
November 7, 2016 For over 35 years, the “14-Day Rule,” prohibiting in vitro experimentation on embryos beyond 14 days, stood as an ethical line in the sand for embryo research around the world. Throughout the arc of the rule’s existence it had not been questioned, as scientists had been unable to grow embryos in vitro either up to, or beyond, 14 days; a practical limitation that served as a backstop to the ethical rule. However, in May 2016, labs in the U.S. and the U.K. were the first to report being able to sustain human embryos in vitro for up to 13 days. This development and other advances in in vitro research involving organized, embryo-like cellular structures have raised a number of questions about the rule, its genesis, application, and future scope. This conference convened experts in bioethics, stem cell research, embryology, and law to discuss the ethical underpinnings and future scope of the rule. Questions discussed included: - What are the historical, ethical and scientific rationales for establishing the 14-Day Rule? - Should the 14-Day Rule be revisited in light of recent advances? - Should the 14-Day Rule even apply to research involving the in vitro culture of embryo-like cellular structures? This event was free and open to the public. This event was sponsored by the Harvard University Office of the Vice Provost for Research, the Edmond J. Safra Center for Ethics at Harvard University, and the Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics at Harvard Law School, and the Harvard Stem Cell Institute, with support from the International Society for Stem Cell Research and the Center for Bioethics at Harvard Medical School. View the full agenda and learn more on the website: http://petrieflom.law.harvard.edu/events/details/advances-in-in-vitro-research-and-the-14-day-rule.
Recommended
More Related Content
What's hot
What's hot (20)
Viewers also liked
Viewers also liked (20)
Similar to Gist Croft, "Self-organization of the in vitro attached human embryo and its implications"
Similar to Gist Croft, "Self-organization of the in vitro attached human embryo and its implications" (20)
More from The Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics
More from The Petrie-Flom Center for Health Law Policy, Biotechnology, and Bioethics (20)
Recently uploaded
Recently uploaded (20)
Gist Croft, "Self-organization of the in vitro attached human embryo and its implications"
- 1. Self-organization of the in vitro attached human embryo and its implications Gist Croft, PhD Brivanlou Laboratory of Stem Cell Biology and Molecular Embryology The Rockefeller University The Ethics of Early Embryo Research and the Future of the 14 Day Rule Petrie-Flom Center, Harvard Law School 11 / 7 / 2016
- 2. Derived from the ICM of the blastocyst Evans, Martin, Kauffman, 1981 (mouse), Thomson, 1998 (human) iPSCs: reprogram somatic cells with pluripotency genes, Yamanaka, 2006 Pluripotent Self-renewing Experimental model of human development Make cell types for cell-replacement and disease-modeling Embryonic Stem Cells: Origins and Utility NIH Kang et al, 2009 (iPS mouse) Need to understand early cell fate choices in vivo, origin and trajectory of ES cells
- 3. Human embryo development after implantation remains a black box Zygote D1 2-cell D1-D2 4-cell D2 Multi-cell D3 Morula D3-D4 Blastocyst D5-6 Stage: DPF: Post-blastocyst D7+ (B. Behr, Stanford IVF)
- 4. CS4 (DPF7) CS5C (DPF12) CS6 (DPF14-17): gastrulation Mouse Organizational landmarks of pre-gastrulation development: mouse vs. human Mouse Egg cylinder Human Germ disc Attached blastocyst CS5B (DPF9) Bilaminar disc Trilaminar disc (Langman’s Medical Embryology; Human Embryology and Teratology)
- 5. New ex vivo models of Be Morris et al, 2012; Bedzhov et al, 2014a,b: Zernicke-Goetz LabKang et al, 2013; Schrode et al, 2014: Hadjantonakis Lab New ex vivo approaches provide insights into early mouse embryo development First and second cell fate decisions tissue morphogenesis and self organization
- 6. Can we adapt approach for in vitro culture of human blastocysts? 2013 Rockefeller IRB Protocol approved culture ?? thaw zona pellucida removed cryopreserved blastocysts blastocyst 2005 National Academies of Science 2005 and 2010 Stem Cell Research Guidelines: bioethical consensus and mandate for in vitro culture up to DPF14 or primitive streak 2016 Deglincerti and Croft, et al. Nature 2104 consented donation of surplus IVF embryos for culture experiments 2015 first experiments
- 7. Human embryos attach and develop in vitro DPF6 DPF8 DPF10 DPF12 DPF14 Deglincerti and Croft et al, Nature 2016
- 8. In vitro culture of the human blastocysts – DPF6 Scale bar = 50 um ICM Trophectoderm
- 9. Morphology: Phalloidin ICM: OCT4, GATA6 TE: CDX2 DPF6 Number of cells: 267 ± 37 (n=8) Deglincerti and Croft, et al, Nature 2016
- 10. The molecular signature of the human blastocyst is more similar to the cow than the mouse Mouse blastocyst Cow blastocyst Human blastocyst (Rossant, 2015)
- 11. Morphology: Phalloidin ICM: OCT4; GATA6 TE: GATA3 DPF6 Number of cells: 267 ± 37 (n=8) - GATA3 marks TE - ICM cell-sorting incomplete Deglincerti and Croft et al, Nature 2016
- 12. Morphology: Phalloidin, DAPI ICM: NANOG; SOX17 DPF6 Number of cells: 267 ± 37 (n=8) - Epi and PE specified - ICM cell-sorting incomplete
- 13. Divergent transcriptional profile of mouse vs. human TE ICM determinants similar TE: GATA3+ OCT4LOW GATA6 LOW CDX2 LOW/VAR/CYTO Mouse Human TE: CDX2+ GATA3+ ICM: NANOG OCT4 HI GATA6 HI/LOW SOX17 ICM: NANOG OCT4 HI GATA6 HI/LOW SOX17 Cartoon adapted from Nadine Schrode, Néstor Saiz, Stefano Di Talia, Anna-Katerina Hadjantonakis, MSKCC
- 14. What happens after DPF6? DPF6 DPF7
- 15. Attachment occurs at ~DPF7.5, always on the side of the ICM in vitro in vivo Deglincerti and Croft et al, Nature 2016
- 16. DPF8 Number of cells: 268 ± 8 (n=4) ICM: OCT4; GATA6 TE: GATA3 - Compaction of the Epiblast - Physical sorting of Epi and PE Deglincerti and Croft et al, Nature 2016
- 17. Mouse Human after attachment before attachment Second cell fate decision, ICM Epiblast vs. PE: similar determinants; delay in human relative to mouse Adapted from Nadine Schrode, Néstor Saiz, Stefano Di Talia, Anna-Katerina Hadjantonakis, MSKCC DPF8 profiles Epiblast NANOG OCT4 HI PE GATA6 SOX17 TE GATA3 CDX2 LOW/VAR
- 18. DPF10 Number of cells: 890 ± 226 (n=4) Morphology: Phalloidin ICM: OCT4; GATA6 TE: GATA3; CDX2 Deglincerti and Croft et al, Nature 2016
- 19. DPF10: Epiblast cavitation amniotic cavity Morphology: Phalloidin ICM: OCT4; GATA6 TE: CDX2
- 20. Yolk sac TE Yolk sac cavity ICM: OCT4; GATA6 TE: GATA3; CDX2 DPF10: Yolk sac cavity, lined by a newly described human-specific cell type (yolk sac TE cells) OCT4LO/GATA6LO/CDX2+ Morphology: Phalloidin CS5B (DPF9) Bilaminar germ disc CS5C (DPF12) Deglincerti, Croft et al, Nature 2016
- 21. DPF10: First expression of CD24 in embryo; exclusively marks Epiblast GATA3 CD24 GATA6 OCT4 Deglincerti, Croft et al, Nature 2016 DPF10: First timepoint Epi cells match hESC
- 22. DPF8: Second stage of TE lineage progression ICM: OCT4 TE: CK7; HCGB Deglincerti and Croft et al, Nature 2016
- 23. DPF10: Diversification of TE lineage: CTB and SCTB ICM: OCT4 TE: CK7; HCGB Deglincerti and Croft et al, Nature 2016
- 24. DPF12: further specialization of TE lineages ICM: OCT4 TE: CK7; HCGB DAPI GATA3 Phalloidin GATA3 Phalloidin HCGBDAPI DAPI Deglincerti and Croft et al, Nature 2016 CS5C (DPF12)
- 25. Early and autonomous diversification of TE lineages in vitro Different blastocyst transcriptional profiles and delayed ICM cell-sorting vs. mouse ysTE, a new human-specific embryonic cell type Autonomous formation of species-specific amniotic and yolk sac cavities Embryo self-organization in the absence of maternal input after attachment Self-organization of the in vitro attached human embryo DPF10+ epiblast: new ex vivo benchmark for origin of hESCs
- 26. Future Directions Reproductive and maternal fetal medicine new measures of embryo quality placental disorders maternal fetal interface, immune tolerance comparative embryology Developmental roadmap of stem cell fate epiblast: naïve, primed, germ layers and cells PE TE Anticipation of gastrulation molecular and geometric controls prepatterning
- 27. Warmflash, et al, Nature Methods 2014; Etoc, et al, Developmental Cell 2016 Unresolved questions in embryonic stem cell biology Why do hESC apparently differentiate in forward and reverse (form TE-like cells)? Why are naïve human pluripotent stem cells elusive and what would they look like? Day 0 Day 2, BMP4 treatment OCT4DAPI CDX2 BRA SOX2 Human ES cell colony
- 28. Blastocyst Pluripotent hESC 2days BMP4 induced “gastrulation” model Comparison of cell fate regulation ex vivo and in vitro Molecular markers Signalling pathways Cell polarity Tissue architecture DPF10 DPF12
- 29. What happens after DPF12? ICM: OCT4 TE: CK7; HCGB DAPI GATA3 Phalloidin GATA3 Phalloidin HCGBDAPI DAPI Deglincerti, Croft et al, Nature 2016 CS5C (DPF12) CS6 (DPF14-17): gastrulation
- 30. Morphology: Phalloidin ICM: OCT4; GATA6 TE: GATA3 Croft et al, unpublished CS6 (DPF14-17): gastrulation DPF14 Number of cells: 1012 ± 127 (n=8) Transition to a volcano-shaped structure Horseshoe distribution of cells Centrifugal dispersion of cells
- 31. AcknowledgementsBlastocyst donors and IVF clinic Alessia Deglincerti: co-first author Ali Brivanlou and Eric Siggia Magdalena Zernicka-Goetz Kat Hadjantonakis, Lauren Pietila Stephanie Tse, Corbyn Nchako Cecilia Pelligrini: technical support RU BIRC: Alison North, Pablo Ariel, Kaye Thomas, Tao Tong Amy Wilkerson: bioethics and IRB Protocol Arlene Hurley and Donna Brassil Hospital/IRB facilitation office Supported by Starr foundation Tri-I Stem Cell Initiative grant and Rockefeller Private funds