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

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

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)

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)

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

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

Human embryos attach and develop in vitro

DPF6 DPF8 DPF10 DPF12 DPF14

Deglincerti and Croft et al, Nature 2016

In vitro culture of the human blastocysts DPF6

Scale bar = 50 um

ICM

Trophectoderm

Morphology: Phalloidin ICM: OCT4, GATA6

TE: CDX2

DPF6 Number of cells: 267 37 (n=8)

Deglincerti and Croft, et al, Nature 2016

The molecular signature of the human blastocyst is more similar to the cow than the mouse

Mouse blastocyst

Cow blastocyst

Human blastocyst

(Rossant, 2015)

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

Morphology: Phalloidin, DAPI ICM: NANOG; SOX17

DPF6 Number of cells: 267 37 (n=8)

- Epi and PE specified - ICM cell-sorting incomplete

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

What happens after DPF6?

DPF6 DPF7

Attachment occurs at ~DPF7.5, always on the side of the ICM

in vitro

in vivo

Deglincerti and Croft et al, Nature 2016

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

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

DPF10 Number of cells: 890 226 (n=4)

Morphology: Phalloidin ICM: OCT4; GATA6

TE: GATA3; CDX2 Deglincerti and Croft et al, Nature 2016

DPF10: Epiblast cavitation amniotic cavity

Morphology: Phalloidin ICM: OCT4; GATA6

TE: CDX2

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

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

DPF8: Second stage of TE lineage progression

ICM: OCT4 TE: CK7; HCGB

Deglincerti and Croft et al, Nature 2016

DPF10: Diversification of TE lineage: CTB and SCTB

ICM: OCT4 TE: CK7; HCGB

Deglincerti and Croft et al, Nature 2016

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)

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

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

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

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

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

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

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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