Examining the State of the Science of Mammalian Embryo Model Systems Proceedings of a Workshop (2020) / Chapter Skim
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4 Stem CellBased Models of Human Embryos
4 Stem CellBased Models of Human Embryos
Pages 49-74
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From page 49... ...
(Brivanlou, Rivron) • Geometrically confining human embryonic stem cells and adding a single growth factor (bone morphogenetic protein 4)
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• Microfluidic systems can be used to model features of peri implantation human development using human pluripotent stem cells -- such as self-organization into multicellular struc tures, lumenogenesis, and even differentiation into amniotic ectoderm-like cells -- which enables research on cell signaling dynamics, signaling propagation, and inductive signals that drive the formation of different compartments. Microfluidic systems appear to be more controllable and reproducible than the conventional 3D culture environment.
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GASTRULOIDS: MODELING HUMAN EMBRYOS AND EMBRYONIC TISSUES Ali Brivanlou, the Robert and Harriet Heilbrunn Professor at The Rockefeller University, introduced synthetic self-organizing models of human embryos called "gastruloids" and highlighted lessons about the basic science of human development that can be gleaned from these models. From a utilitarian perspective, he also made the case for the use of synthetic models as a tool for addressing diseases whose roots can be traced to very early human embryonic development.
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Brivanlou delved further into the rationale for studying human embryo models -- "gastruloids" -- that are derived from human embryonic stem cells (hESCs) (Warmflash et al., 2014)
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Creating Model Human Gastruloids Derived from Human Embryonic Stem Cells Brivanlou described how these model human gastruloids are created using hESCs. When the hESCs are cultured under standard conditions, the variations in size and density of colonies -- some colonies are touching other colonies, some colonies are completely independent of one another -- and the presence of individual randomly dispersed cells make it impossible to replicate the system and quantify it at a subcellular level.
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The third lesson is that a wave of Wnt signaling controls primitive streak formation downstream of the BMP4 signaling, which can be functionally demonstrated using grafting in a chick embryo (Martyn et al., 2019)
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Morphologically they appear similar; their molecular signatures indicate that they are close but not identical. Single-cell RNA sequencing is in progress to compare the specimens and evaluate the similarities and differences between their respective cell types in the hope that new cell types might be identified.2 Modeling Human Disease in Gastruloids and Neuruloids In the final part of his presentation, Brivanlou commented on the application of human embryo models to cure human diseases.
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The center section of gastruloids can be used to generate another type of self-organizing structure called neuruloids, which can also be used to model Huntington's disease. Neuruloids are self-organizing ectodermal structures that display radial distribution of cell types, with brain cells at the center, surrounded by neural crest and neural placodes (sensory organs)
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He presented Figure 4-1 to illustrate the functions of the FIGURE 4-1 TGFβ signals (e.g., BMP, Nodal) and cell fates in human embryonic stem cells.
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. Wnt and Activin/Nodal signaling behave differently than BMP signaling in that they respond adaptively to ligand stimulation, Warmflash said.
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One hypothesis is that the order in which the cell sees these signals is important, he added. If they see the Wnt signaling and then the Nodal signaling, they will differentiate.
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Future Directions for Synthetic Embryology Warmflash emphasized that the goal of this work is not to build embryos from stem cells; rather, the goal is to build systems to answer specific questions about how the embryo works. The current state of knowledge and
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BLASTOIDS: MODELING THE PRE-IMPLANTATION EMBRYO Nicolas Rivron, a group leader at the Institute of Molecular Biotechnology, Austrian Academy of Sciences, described how his laboratory models early embryonic development in 3D by combining trophoblast stem cells and embryonic stem cells to form blastoids, which model some features of the pre-implantation embryo. He emphasized that the work he would describe is strictly based on mouse stem cells, not human stem cells.
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ESCs are dependent on Wnt and LIF, and trophoblast stem cells (TSCs) are dependent on fibroblast growth factor 4 (FGF4)
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Features of Blastoids Rivron provided an overview of the features of the blastoid. He explained that the blastoids make analogs of the three cell types that form the blastocyst -- epiblast, trophoblast, and primitive endoderm -- which later develop into the placenta, yolk sac, and body to form the whole organism.
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• Communication between embryonic stems cells and trophoblast stem cells drives the development of blastoids. • Blastoids recapitulate aspects of implantation and early development in utero.
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that activate the MAP kinase and TGFβ and Wnt signaling pathways, among others, in the trophoblast. Further functional analysis of these embryonic inductions demonstrated that embryonic cells regulate trophoblast proliferation and self-renewal, Rivron said.
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His laboratory uses human pluripotent stem cells (hPSCs) -- both induced pluripotent stem cells and hESCs -- to develop sensitive models for studying pre-implantation human development.
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Autonomous BMP Signaling Drives Amniotic Differentiation Fu and colleagues investigated the molecular mechanisms for driving amniotic cell differentiation from hPSCs in their 3D culture environment. Their model did not have any exogenous external signals driving the stem cells to differentiate, so they looked at BMP signaling pathways.
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. Controlled Microfluidic Modeling of Human Embryo Development Conventional 3D culture environments can be used to generate selforganizing multicellular structures that bear similarities to certain facets of human development, Fu said.
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At certain time points, molecular asymmetry was very evident in the stem cells: only the cells that were directly exposed to BMP4 stimulation expressed TFAP2A, the gastrulating cells expressed T/Brachyury and were continuously differentiating, and all of the cells expressed CDX2. (CDX2 is an extraembryonic marker, but it also expresses in the posterior primitive streak.)
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The researchers have also used comparative transcriptome analysis to compare the cell types generated by their synthetic system with cell types developed from primate monkey embryos (Nakamura et al., 2016)
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Primitive endoderm may be one of many elements required for blastoids to take the next developmental step, Rivron surmised. He noted that this type of work demonstrates certain advantages of using stem cell–based embryo models over the classical approach to embryology, which involves knocking out genes in actual embryos and looking for a phenotype.
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Human Versus Nonhuman Model Systems Speakers were asked whether the models that had been discussed might eventually obviate the need to study human embryonic material directly. A workshop participant went on to ask which elements might be essential to continue studying using human material versus those that might be replaced with models in nonhuman primates or other species.
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The gene is expressed in the fertilized egg and then expressed ubiquitously in all adult cell types. If the mutation were a dominant mutation, it follows that the earliest effect of the mutant would occur at the earliest time point of biological activity, Brivanlou said.
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This suggests that the amniotic ectoderm compartment corresponds to the amniotic compartment in the peri-implantation human embryo. However, the underlying question of whether his lab has established the equivalency of the cell types generated in their in vitro models cannot be answered until data become available that allow them to perform comparisons with in vivo human or primate embryos.
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