Generation of human blastocyst-like structures from pluripotent stem cells

AbstractHuman blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast, and primitive endoderm, all of which are essential for early development and organ formation1,2. However, due to ethical concerns and restricted access to human blastocysts, we lack a comprehensive understanding of early human embryogenesis. To bridge this knowledge gap, we need a reliable model system that recapitulates early stages of human embryogenesis. Here we report a ∼three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids also exhibited the developmental potential to undergo post-implantation morphogenesis in vitro to form structures with a cellular composition and transcriptome signature similar to human embryos that had been cultured in vitro for 8 or 10 days. In conclusion, human EPS-blastoids provide a new experimental platform for studying early developmental stages of the human embryo.HighlightsA method for generating human blastoids from EPS cells.Human blastoids resemble blastocysts in terms of morphology and cell lineage composition.Single-cell transcriptomic analyses reveal EPI, PE, and TE cell lineages in human blastoids.Human blastoids mimic in vitro the morphogenetic events of pre- and early post-implantation stages.

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Generation of human blastocyst-like structures from pluripotent stem cells

Cell Discovery ◽

10.1038/s41421-021-00316-8 ◽

2021 ◽

Vol 7 (1) ◽

Cited By ~ 1

Author(s):

Yong Fan ◽

Zheying Min ◽

Samhan Alsolami ◽

Zhenglai Ma ◽

E. Zhang ◽

...

Keyword(s):

Early Stage ◽

Three Dimensional ◽

Human Embryos ◽

Blastocyst Development ◽

Developmental Defects ◽

Cell Lineages ◽

Human Blastocyst ◽

Reliable Model ◽

Human Embryogenesis

AbstractHuman blastocysts are comprised of the first three cell lineages of the embryo: trophectoderm, epiblast and primitive endoderm, all of which are essential for early development and organ formation. However, due to ethical concerns and restricted access to human blastocysts, a comprehensive understanding of early human embryogenesis is still lacking. To bridge this knowledge gap, a reliable model system that recapitulates early stages of human embryogenesis is needed. Here we developed a three-dimensional (3D), two-step induction protocol for generating blastocyst-like structures (EPS-blastoids) from human extended pluripotent stem (EPS) cells. Morphological and single-cell transcriptomic analyses revealed that EPS-blastoids contain key cell lineages and are transcriptionally similar to human blastocysts. Furthermore, EPS-blastoids are similar with human embryos that were cultured for 8 or 10 days in vitro, in terms of embryonic structures, cell lineages and transcriptomic profiles. In conclusion, we developed a scalable system to mimic human blastocyst development, which can potentially facilitate the study of early implantation failure that induced by developmental defects at early stage.

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Comparative analysis of cell lineage differentiation during hepatogenesis in humans and mice at the single-cell transcriptome level

Cell Research ◽

10.1038/s41422-020-0378-6 ◽

2020 ◽

Vol 30 (12) ◽

pp. 1109-1126 ◽

Cited By ~ 1

Author(s):

Xin Wang ◽

Li Yang ◽

Yan-Chun Wang ◽

Zi-Ran Xu ◽

Ye Feng ◽

...

Keyword(s):

Single Cell ◽

Developmental Process ◽

Fetal Liver ◽

Cell Lineage ◽

Liver Development ◽

Erythroid Progenitor ◽

Cell Lineages ◽

Lineage Differentiation ◽

Human And Mouse

AbstractDuring embryogenesis, the liver is the site of hepatogenesis and hematopoiesis and contains many cell lineages derived from the endoderm and mesoderm. However, the characteristics and developmental programs of many of these cell lineages remain unclear, especially in humans. Here, we performed single-cell RNA sequencing of whole human and mouse fetal livers throughout development. We identified four cell lineage families of endoderm-derived, erythroid, non-erythroid hematopoietic, and mesoderm-derived non-hematopoietic cells, and defined the developmental pathways of the major cell lineage families. In both humans and mice, we identified novel markers of hepatic lineages and an ID3+ subpopulation of hepatoblasts as well as verified that hepatoblast differentiation follows the “default-directed” model. Additionally, we found that human but not mouse fetal hepatocytes display heterogeneity associated with expression of metabolism-related genes. We described the developmental process of erythroid progenitor cells during human and mouse hematopoiesis. Moreover, despite the general conservation of cell differentiation programs between species, we observed different cell lineage compositions during hematopoiesis in the human and mouse fetal livers. Taken together, these results reveal the dynamic cell landscape of fetal liver development and illustrate the similarities and differences in liver development between species, providing an extensive resource for inducing various liver cell lineages in vitro.

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Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010005161x ◽

1974 ◽

Vol 32 ◽

pp. 320-321

Author(s):

J. P. Revel

Keyword(s):

Developmental Stages ◽

Three Dimensional ◽

Cell Movement ◽

Cellular Interactions ◽

Serial Sections ◽

Cell Sheets ◽

Freeze Etching ◽

Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

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Generation of three-dimensional pannus-like tissues in vitro from single cell suspensions of synovial fluid cells from arthritis patients

Rheumatology International ◽

10.1007/s00296-003-0333-0 ◽

2004 ◽

Vol 24 (2) ◽

pp. 71-76 ◽

Cited By ~ 1

Author(s):

Samuel Solomon ◽

Madhan Masilamani ◽

Subhasis Mohanty ◽

J�rg E. Schwab ◽

Eva-Maria Boneberg ◽

...

Keyword(s):

Synovial Fluid ◽

Single Cell ◽

Three Dimensional ◽

Cell Suspensions ◽

Synovial Fluid Cells

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Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽

10.3390/cells10113111 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3111

Author(s):

Po-Yu Lin ◽

Denny Yang ◽

Chi-Hsuan Chuang ◽

Hsuan Lin ◽

Wei-Ju Chen ◽

...

Keyword(s):

Stem Cells ◽

Single Cell ◽

Embryonic Cells ◽

Cell Stage ◽

Developmental Potential ◽

Functional Features ◽

Early Mouse ◽

Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

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Protocol for controlled modeling of human epiblast and amnion development using stem cells

10.21203/rs.2.11520/v1 ◽

2019 ◽

Cited By ~ 2

Author(s):

Yi Zheng ◽

Jianping Fu

Keyword(s):

Stem Cells ◽

Primordial Germ Cells ◽

Primitive Streak ◽

Human Embryos ◽

Vitro Fertilization ◽

Human Embryology ◽

Post Implantation ◽

Advance Knowledge ◽

Early Human

Abstract Due to the inaccessibility of post-implantation human embryos and the restriction on in-vitro fertilization (IVF) embryos cultured beyond 14 days, the knowledge of early post-implantation human embryogenesis remains extremely limited. Recently, we have developed a microfluidic in-vitro platform, based on human pluripotent stem cells (hPSCs), which is capable of recapitulating several key developmental landmarks of early human post-implantation embryonic development, including lumenogenesis of the epiblast (EPI), amniogenesis, and specification of primordial germ cells (PGCs) and of primitive streak (PS) cells. Given its controllability and reproducibility, the microfluidic platform provides a powerful experimental platform to advance knowledge of human embryology and reproduction. This protocol describes the preparation of the microfluidic device and its implementation for modeling human post-implantation epiblast and amnion development using hPSCs.

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Mitochondria and human preimplantation embryo development

Reproduction ◽

10.1530/rep-08-0444 ◽

2009 ◽

Vol 137 (4) ◽

pp. 619-624 ◽

Cited By ~ 44

Author(s):

Martin Wilding ◽

Gianfranco Coppola ◽

Brian Dale ◽

Loredana Di Matteo

Keyword(s):

Embryo Development ◽

Preimplantation Embryo ◽

Anaerobic Respiration ◽

Mitochondrial Metabolism ◽

Human Reproduction ◽

Aerobic Respiration ◽

Human Embryos ◽

Developmental Potential ◽

Preimplantation Embryo Development

Human reproduction, like all biological systems, is characterised by a large level of variability. In this field, the variability is observed as a large difference in implantation potential of human embryos developing in vitro, despite similarities in observable parameters such as rate of development and morphology of these embryos. One of the underlying factors that determines developmental potential in these embryos is the availability of energy in the form of ATP for development. Here, we suggest that, despite the evidence suggesting that mitochondrial metabolism is relatively inactive during preimplantation embryo development, aerobic (mitochondrial) metabolism contributes a major role in the supply of ATP. A second pathway, anaerobic respiration, is also active and the two pathways work in synchrony to supply all the ATP necessary. We discuss the differences in the two forms of energy production and suggest that, although anaerobic respiration can supplement deficiencies in the energy supply in the short term, this is not sufficient to substitute for aerobic respiration over long periods. Therefore, we suggest that deficiencies in the levels of aerobic respiration can explain variability in the implantation potential of apparently equivalent embryos.

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Functional 3D Human Liver Bud Assembled from MSC-Derived Multiple Liver Cell Lineages

Cell Transplantation ◽

10.1177/0963689718780332 ◽

2018 ◽

Vol 28 (5) ◽

pp. 510-521 ◽

Cited By ~ 4

Author(s):

Jing Li ◽

Feiyue Xing ◽

Feng Chen ◽

Liumin He ◽

Kwok-Fai So ◽

...

Keyword(s):

Human Liver ◽

Early Stage ◽

Three Dimensional ◽

Alternative Methods ◽

Liver Sinusoidal Endothelial Cells ◽

Medicine Research ◽

Cell Lineages ◽

Self Assembled ◽

Liver Tissues

The severe shortage of donor liver organs requires the development of alternative methods to provide transplantable liver tissues such as stem cell-derived organoids. Despite several studies describing the generation of vascularized and functional liver tissues, none have succeeded in assembling human liver buds containing hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs). Here, we report a reproducible, easy-to-follow, and comprehensive self-assembly protocol to generate three-dimensional (3D) human liver buds from naïve mesenchymal stem cells (MSCs), MSC-derived hepatocytes, and HSC- and LSEC-like cells. By optimizing the ratio between these different cell lineages, the cell mixture self-assembled into 3D human liver buds within 72 h in vitro, and exhibited similar characteristics with early-stage murine liver buds. In a murine model of acute liver failure, the mesenteric transplantation of self-assembled human liver buds effectively rescued animal death, and triggered hepatic ameliorative effects that were better than the ones observed after splenic transplantation of human hepatocytes or naïve MSCs. In addition, transplanted human liver buds underwent maturation during injury alleviation, after which they exhibited a gene expression profile signature similar to the one of adult human livers. Collectively, our protocol provides a promising new approach for the in vitro construction of functional 3D human liver buds from multiple human MSC-derived hepatic cell lineages; this new technique would be useful for clinical transplantation and regenerative medicine research.

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Modelling human embryogenesis: embryo-like structures spark ethical and policy debate

Human Reproduction Update ◽

10.1093/humupd/dmaa027 ◽

2020 ◽

Vol 26 (6) ◽

pp. 779-798

Author(s):

Ana M Pereira Daoud ◽

Mina Popovic ◽

Wybo J Dondorp ◽

Marc Trani Bustos ◽

Annelien L Bredenoord ◽

...

Keyword(s):

Stem Cells ◽

Human Development ◽

Human Embryo ◽

Embryoid Bodies ◽

Embryo Research ◽

Human Embryos ◽

Pubmed Database ◽

Human Embryo Research ◽

Post Implantation

Abstract BACKGROUND Studying the human peri-implantation period remains hindered by the limited accessibility of the in vivo environment and scarcity of research material. As such, continuing efforts have been directed towards developing embryo-like structures (ELS) from pluripotent stem cells (PSCs) that recapitulate aspects of embryogenesis in vitro. While the creation of such models offers immense potential for studying fundamental processes in both pre- and early post-implantation development, it also proves ethically contentious due to wide-ranging views on the moral and legal reverence due to human embryos. Lack of clarity on how to qualify and regulate research with ELS thus presents a challenge in that it may either limit this new field of research without valid grounds or allow it to develop without policies that reflect justified ethical concerns. OBJECTIVE AND RATIONALE The aim of this article is to provide a comprehensive overview of the existing scientific approaches to generate ELS from mouse and human PSCs, as well as discuss future strategies towards innovation in the context of human development. Concurrently, we aim to set the agenda for the ethical and policy issues surrounding research on human ELS. SEARCH METHODS The PubMed database was used to search peer-reviewed articles and reviews using the following terms: ‘stem cells’, ‘pluripotency’, ‘implantation’, ‘preimplantation’, ‘post-implantation’, ‘blastocyst’, ‘embryoid bodies’, ‘synthetic embryos’, ‘embryo models’, ‘self-assembly’, ‘human embryo-like structures’, ‘artificial embryos’ in combination with other keywords related to the subject area. The PubMed and Web of Science databases were also used to systematically search publications on the ethics of ELS and human embryo research by using the aforementioned keywords in combination with ‘ethics’, ‘law’, ‘regulation’ and equivalent terms. All relevant publications until December 2019 were critically evaluated and discussed. OUTCOMES In vitro systems provide a promising way forward for uncovering early human development. Current platforms utilize PSCs in both two- and three-dimensional settings to mimic various early developmental stages, including epiblast, trophoblast and amniotic cavity formation, in addition to axis development and gastrulation. Nevertheless, much hinges on the term ‘embryo-like’. Extension of traditional embryo frameworks to research with ELS reveals that (i) current embryo definitions require reconsideration, (ii) cellular convertibility challenges the attribution of moral standing on the basis of ‘active potentiality’ and (iii) meaningful application of embryo protective directives will require rethinking of the 14-day culture limit and moral weight attributed to (non-)viability. Many conceptual and normative (dis)similarities between ELS and embryos thus remain to be thoroughly elucidated. WIDER IMPLICATIONS Modelling embryogenesis holds vast potential for both human developmental biology and understanding various etiologies associated with infertility. To date, ELS have been shown to recapitulate several aspects of peri-implantation development, but critically, cannot develop into a fetus. Yet, concurrent to scientific innovation, considering the extent to which the use of ELS may raise moral concerns typical of human embryo research remains paramount. This will be crucial for harnessing the potential of ELS as a valuable research tool, whilst remaining within a robust moral and legal framework of professionally acceptable practices.

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Chemically-defined induction of a primitive endoderm and epiblast-like niche supports post-implantation progression from blastoids

10.1101/510396 ◽

2019 ◽

Cited By ~ 3

Author(s):

Erik J. Vrij ◽

Yvonne S. Scholte op Reimer ◽

Javier Frias Aldeguer ◽

Isabel Misteli Guerreiro ◽

Jop Kind ◽

...

Keyword(s):

Small Molecules ◽

Three Dimensional ◽

Embryoid Bodies ◽

Primitive Endoderm ◽

Amniotic Cavity ◽

Large Numbers ◽

Body Modifications ◽

Free Serum ◽

Post Implantation

AbstractThe early mammalian conceptus (blastocyst) contains two supporting extraembryonic tissues - the trophectoderm and the primitive endoderm (PrE) - that encase and guide the epiblast (Epi) to eventually form the all body. Modifications of the conceptus exposed key genes regulating these tissues co-development. However, the combinations of signalling pathways underlying the interplay of PrE and Epi remains elusive. Stem cell-based models including embryoid bodies and blastoids can be generated in large numbers and subjected to high-content screens. Here, we use combinatorial screens of proteins, GPCR ligands and small molecules to rapidly (72 hours) and efficiently (80%) guide embryoid bodies to form a three-dimensional PrE-/Epiblast-like niche in chemically-defined conditions (gel-free, serum-free). This bipotent niche spontaneously progresses, without growth factors, to form a pro-amniotic cavity surrounded by a polarized Epi covered with parietal and visceral endoderm-like cells. In blastoids, these molecules enhance the ratio and number of Gata6+/Nanog+ cells and promote the survival, expansion and morphogenesis of a post-implantation-like Epi in vitro. Altogether, modelling early development in chemically-defined conditions delineates the pathways sufficient to form a functional PrE/Epiblast niche that fuels post-implantation development.

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