Download - Gist Croft, "Self-organization of the in vitro attached human embryo and its implications"
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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
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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
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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)
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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
(Langmans Medical Embryology; Human Embryology and Teratology)
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New ex vivo models of
Be Morris et al, 2012; Bedzhov et al, 2014a,b: Zernicke-Goetz Lab Kang 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
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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
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Human embryos attach and develop in vitro
DPF6 DPF8 DPF10 DPF12 DPF14
Deglincerti and Croft et al, Nature 2016
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In vitro culture of the human blastocysts DPF6
Scale bar = 50 um
ICM
Trophectoderm
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Morphology: Phalloidin ICM: OCT4, GATA6
TE: CDX2
DPF6 Number of cells: 267 37 (n=8)
Deglincerti and Croft, et al, Nature 2016
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The molecular signature of the human blastocyst is more similar to the cow than the mouse
Mouse blastocyst
Cow blastocyst
Human blastocyst
(Rossant, 2015)
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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
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Morphology: Phalloidin, DAPI ICM: NANOG; SOX17
DPF6 Number of cells: 267 37 (n=8)
- Epi and PE specified - ICM cell-sorting incomplete
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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, Nstor Saiz, Stefano Di Talia, Anna-Katerina Hadjantonakis, MSKCC
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What happens after DPF6?
DPF6 DPF7
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Attachment occurs at ~DPF7.5, always on the side of the ICM
in vitro
in vivo
Deglincerti and Croft et al, Nature 2016
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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
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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, Nstor Saiz, Stefano Di Talia, Anna-Katerina Hadjantonakis, MSKCC
DPF8 profiles
Epiblast NANOG OCT4 HI
PE
GATA6 SOX17
TE
GATA3 CDX2 LOW/VAR
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DPF10 Number of cells: 890 226 (n=4)
Morphology: Phalloidin ICM: OCT4; GATA6
TE: GATA3; CDX2 Deglincerti and Croft et al, Nature 2016
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DPF10: Epiblast cavitation amniotic cavity
Morphology: Phalloidin ICM: OCT4; GATA6
TE: CDX2
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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
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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
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DPF8: Second stage of TE lineage progression
ICM: OCT4 TE: CK7; HCGB
Deglincerti and Croft et al, Nature 2016
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DPF10: Diversification of TE lineage: CTB and SCTB
ICM: OCT4 TE: CK7; HCGB
Deglincerti and Croft et al, Nature 2016
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DPF12: further specialization of TE lineages
ICM: OCT4 TE: CK7; HCGB
DAPI GATA3 Phalloidin GATA3 Phalloidin HCGB DAPI DAPI
Deglincerti and Croft et al, Nature 2016
CS5C (DPF12)
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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
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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: nave, primed, germ layers and cells PE TE Anticipation of gastrulation molecular and geometric controls prepatterning
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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 nave human pluripotent stem cells elusive and what would they look like?
Day 0 Day 2, BMP4 treatment
OCT4 DAPI CDX2 BRA SOX2
Human ES cell colony
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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
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What happens after DPF12?
ICM: OCT4 TE: CK7; HCGB
DAPI GATA3 Phalloidin GATA3 Phalloidin HCGB DAPI DAPI
Deglincerti, Croft et al, Nature 2016
CS5C (DPF12) CS6 (DPF14-17): gastrulation
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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
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Acknowledgements Blastocyst 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
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