Reconstructing human early embryogenesis in vitro with pluripotent stem cells
Preprint posted on 12 March 2021 https://www.biorxiv.org/content/10.1101/2021.03.12.435175v1
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-25853-4
Witnessing the creation of human life under the microscope. Reconstructing human early embryogenesis in vitro with pluripotent stem cells
Selected by Martin Estermann
Categories: cell biology, developmental biology, genetics, molecular biology
Background:
Human life starts at fertilization with the union of both the sperm and the ovum to form the zygote. This unique cell, through cleavage, undergoes continuous divisions without any increase in size, resulting in a solid sphere known as morula. Further division and cell differentiation results in a hollowed structure called blastocyst. Two main structures are defined, the inner cell mass, which will form the proper embryo and the trophoblast, which gives rise to the placenta and other extra embryonic tissues. Later in development, the inner cell mass differentiates into the epiblast and hypoblast, which will contribute to embryonic cells and extra embryonic tissue, respectively.
In humans, this early developmental process occurs naturally within the body of the mother, making it hard to study. In addition, human embryonic samples are difficult to obtain and are regulated by ethical and legal restrictions. Due to these reasons, there is a lack of knowledge regarding this critical developmental step. The generation of in vitro models that recapitulate this early process is therefore crucial to gain insight into this process.
With this in mind, the authors of the preprint combined human human expanded pluripotent stem cells (hEPSCs) with a cell aggregation culture method to allow the formation of 3D structures similar to human blastocysts. This research was able to recapitulate key features equivalent to those occurring between days 3 and 9/10 of human development, providing a key resource to continue exploring this process in vitro.
Key findings
1) Generation of human iPSC derived blastocyst-like structures
Human pluripotent stem cells were reprogrammed to a molecular state similar to earlier blastomere (hEPSCs) (Fig. 1A). These cells were isolated and cultured in a multi-inverted-pyramid microwell to allow cell aggregation and self-organization (Fig. 1B). The combination of ROCK and ALK5 inhibitors, WNT antagonists, BMP and FGF2 during the first 48 hours of culture induced the formation of compact cellular aggregates (Fig. 1C). Around days 3 and 4 of culture, cystic structures were visible (Fig. 1C). By day 6, the morphology of these structures resembled blastocysts, showing a similar average cell size and number to human blastocysts (Fig. 1D).
Fig. 1. Human iPSC derived blastocyst-like structures. (A) Phase-contrast hEPSCs image in 2D culture. (B) Schematic protocol of blastocyst formation from hEPSCs. (C) Representative phase-contrast images of hEPSC aggregates in 3D culture during different days (D) of culture. (D) Phase-contrast image of human blastocyst-like structures at day six of culture (D6). (Preprint Fig. 1B-E).
2) Molecular characterization of the hEPSCs derived blastocyst
To characterize the different cell lineages present in the blastocyst-like structures, the expression levels of core genes for trophectoderm, epiblast and hypoblast were evaluated by qRT-PCR (Fig. 2A). Genes involved in trophectoderm (TE) formation were strongly upregulated during blastocyst formation, compared with the 2D cultures of hEPSCs. In addition, hypoblast marker genes PDGFRA and GATA6 also showed a strong induction, except for SOX17. As expected, NANOG and POU5F1 pluripotent epiblast genes showed similar levels to the hEPSCs cells, whereas the marker KRT17 was upregulated.
To confirm the proper spatial expression of those markers, immunofluorescence was performed. GATA3 expression was evaluated on days 4 (Fig. 2B) and 5 (Fig. 2C) of incubation, being restricted to the trophectoderm. Surprisingly, GATA3 expression on day 5 was cytosolic rather than nuclear (Fig. 2C). This, together with a poor expression of E-CADHERIN (Fig. 2C), suggests a compromised trophectoderm differentiation in later stages. In contrast, KRT18, another TE marker, displayed a specific expression in the trophectodermal cells (Fig. 2D). Hypoblast/primitive endoderm marker FOX2A and epiblast/pluripotent marker SOX2 were detected in the inner mass-like cells (Fig. 2E).
Fig. 2. Characterization of the hEPSCs derived blastocyst. (A) Heatmap of trophectoderm (TE), epiblast (Epi) and hypoblast (Hypo) marker genes mRNA expression in blastocysts cultured for 4, 5 and 6 days, compared with hEPSCs cells grown in 2D. Immunofluorescence staining of blastocyst-like structures at day 4 (B), day 5 (D and E) and day 6 (C). (Preprint Fig. 3A-E).
3) hEPSCs derived blastocyst can organize into post-implantation structures
To address if the in vitro generated blastocysts have the capacity to develop beyond implantation, they were transferred to a 96-well ultra-low attachment U-shaped plate and cultured in vitro with modified IVC1 media (Fig 3A). The structures reorganized into disk-shaped structures with SOX2 positive inner compartment surrounded by GATA3 positive extra embryonic compartment (Fig. 3B). The structure generated was similar to human post implantation embryos.
Fig. 3. Blastocyst can organize into post-implantation structures. (A) Illustration of the in vitro post implantation differentiation protocol (top) and corresponding phase-contrast images of a representative structure at each step (bottom). (B) Immunofluorescence staining of a post implantation-like structure at endpoint. GATA3 (cyan) indicates extra-embryonic structures and SOX2 (yellow) the embryonic compartment. (Preprint Fig. 4A and B).
Why we choose this paper:
There is a lack of knowledge in human early embryogenesis. Mouse model organisms, despite giving a great approximation of this process do not recapitulate this process accurately. There is a current demand on in vitro methodologies to study this process in humans, and this research provides the first step into this unknown field. This research showed a great approximation of the early embryogenesis process equivalent to days 3 to 10 of human development. This protocol is not perfect and as the authors noted, there is a lot of room for improvement, but it is the first step towards unraveling the mysteries of human early embryogenesis and to generate new and improved in vitro models. This research marks a before and after in the field of human early development and differentiation.
Questions to the authors:
- Is there any limitation (ethical or technical) to continue the development of blastocyst-like structures beyond post-implantation stages?
- Are the epiblast cells generated with this methodology capable of differentiating in vitro into ectoderm, endoderm and mesoderm?
- Repeated implantation failure is one of the most common causes of pregnancy failure in in vitro embryonic transference, despite the good quality of the embryos. How feasible would it be to evaluate the implantation success in vitro (in endometrial cultured cells) or in vivo in humanized mice?
Posted on: 6 May 2021
doi: https://doi.org/10.1242/prelights.28869
Read preprint
(1 votes) Sign up to customise the site to your preferences and to receive alerts
Register hereAlso in the cell biology category:
Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments
Structural basis of respiratory complexes adaptation to cold temperatures
RIPK3 coordinates RHIM domain-dependent inflammatory transcription in neurons
Also in the developmental biology category:
Temporal constraints on enhancer usage shape the regulation of limb gene transcription
OGT prevents DNA demethylation and suppresses the expression of transposable elements in heterochromatin by restraining TET activity genome-wide
Actin polymerization drives lumen formation in a human epiblast model
Also in the genetics category:
Temporal constraints on enhancer usage shape the regulation of limb gene transcription
A long non-coding RNA at the cortex locus controls adaptive colouration in butterflies
AND
The ivory lncRNA regulates seasonal color patterns in buckeye butterflies
AND
A micro-RNA drives a 100-million-year adaptive evolution of melanic patterns in butterflies and moths
A revised single-cell transcriptomic atlas of Xenopus embryo reveals new differentiation dynamics
Also in the molecular biology category:
Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments
Nanos2+ cells give rise to germline and somatic lineages in the sea anemone Nematostella vectensis
Plant plasmodesmata bridges form through ER-driven incomplete cytokinesis
AND
Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants
preLists in the cell biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
| List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
| List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
| List by | Dey Lab, Amanda Haage |
Also in the developmental biology category:
GfE/ DSDB meeting 2024
This preList highlights the preprints discussed at the 2024 joint German and Dutch developmental biology societies meeting that took place in March 2024 in Osnabrück, Germany.
| List by | Joyce Yu |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
Single Cell Biology 2020
A list of preprints mentioned at the Wellcome Genome Campus Single Cell Biology 2020 meeting.
| List by | Alex Eve |
Society for Developmental Biology 79th Annual Meeting
Preprints at SDB 2020
| List by | Irepan Salvador-Martinez, Martin Estermann |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EDBC Alicante 2019
Preprints presented at the European Developmental Biology Congress (EDBC) in Alicante, October 23-26 2019.
| List by | Sergio Menchero et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
SDB 78th Annual Meeting 2019
A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.
| List by | Alex Eve |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Young Embryologist Network Conference 2019
Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London
| List by | Alex Eve |
Pattern formation during development
The aim of this preList is to integrate results about the mechanisms that govern patterning during development, from genes implicated in the processes to theoritical models of pattern formation in nature.
| List by | Alexa Sadier |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
| List by | Shikha Nayar |
Also in the genetics category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
| List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
| List by | Nándor Lipták |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
| List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
| List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Also in the molecular biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |