Examining the State of the Science of Mammalian Embryo Model Systems Proceedings of a Workshop (2020) / Chapter Skim
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3 Examining the Development of Extraembryonic Lineages
3 Examining the Development of Extraembryonic Lineages
Pages 29-48
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From page 29... ...
• There are morphological, physiological, and molecular differ ences between primates and other mammals in early develop ment, including the expression of chorionic gonadotropin in the human trophoblast lineage. These differences are impor tant to consider when selecting a model to study the earliest stages of human trophoblast development.
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From page 30... ...
Clark opened the session by commenting that the talks would focus on the difficult question of what gives an embryo organismal potential. MODELING TROPHOBLAST DIFFERENTIATION USING PLURIPOTENT STEM CELLS Mana Parast, a professor in residence of pathology at the University of California, San Diego, presented on the use of pluripotent stem cells to model trophoblast differentiation in humans.
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From page 31... ...
. In floating villi, CTB undergo cell–cell fusion to form STB cells that secrete human chorionic gonadotropin (hCG)
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From page 32... ...
The niche for TSCs is presumed to be within the CTB layer, Parast added. Markers of Human Trophoblast Differentiation Two specific markers of CTB and TSCs found in the human but not in the mouse were highlighted by Parast, and she noted that there are many other differences as well.
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From page 33... ...
According to unpublished work that Parast has done with Kathy Niakan, this supposition is supported by evidence that VGLL1 and p63 are expressed in the very early trophectoderm layer of human blastocyst-stage embryos. Modeling Human Trophoblast Differentiation Prior to the derivation of human TSCs, models of human trophoblast differentiation were poor because the cell lines available were not fully representative of the in vivo cell type, Parast explained.
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From page 34... ...
A subsequent re-plating of the cells on feeder-conditioned media derives a more differentiated cell type. The advantage of this approach is that they were able to separate trophoblast lineage specification from terminal trophoblast differentiation so that the cells could be compared with primary placentaderived CTB cells, Parast said.
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From page 35... ...
As to whether it is strictly an in vitro phenomenon of "transdifferentiation," Parast countered that there may be in vivo stress conditions whereby cells derived from ESCs or EpiSCs can contribute to trophoblast. Alternatively, hPSCs or a subpopulation thereof may not even be pluripotent stem cells -- they might be totipotent, because certain types of mouse ESCs have been shown to cycle between the totipotent and pluripotent state (Macfarlan et al., 2012)
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From page 36... ...
By around day 4, uneven surfacing is evident in the colonies, a feature that becomes more extensive as culture proceeds and that reflects the emergence of STB. The cells produce increasingly larger amounts of hCG between days 6 and 8, probably reflecting the requirement for hCG production by the embryo as it implants, the need to ensure successful maternal recognition of pregnancy, and the continued production of progesterone by the maternal ovary.
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From page 37... ...
. BOX 3-2 Data Collected on the BAP-Treated hESC Model of Trophoblast Differentiation • The BMP4/BAP-exposed cells produce a complement of hormones, including progesterone and hCG, and they express a complement of genes consistent with an identity as placental trophoblast.
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From page 38... ...
These cells are positive for HLA-G and share some markers with the extravillous trophoblast cells that later in placental development invade the endometrium from the tips of anchoring placental villi. The single-cell RNAseq analysis also revealed how proliferating cytotrophoblast closer to the embryo proper give rise to two more differentiated populations of c ytotrophoblast, one with some of the markers defining the motile cells, and the other carrying some STB markers shared by the large STB cells (West et al., 2019)
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From page 39... ...
. MOLECULAR INNOVATION IN THE HUMAN TROPHOBLAST LINEAGE Paul Robson, an associate professor and the director of single-cell biology at The Jackson Laboratory in Farmington, Connecticut, discussed molecular evolutionary innovation in the human trophoblast lineage.
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From page 40... ...
Differences Between Primates and Other Mammals in Early Development Robson explored some of the morphological, physiological, and molecular differences between primates and other mammals in early development, noting that different features of human versus mouse embryology are well established and had been discussed by other speakers. Morphological differences between primates have been demonstrated by a cladistic analysis of the ontogeny of primate fetal membranes, which positions species accurately in the phylogenetic tree based on differential features in early embryonic development (Luckett, 1976)
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From page 41... ...
Transgenic mice have been used to study the regulation of human CGA, Robson said. This work found that anterior pituitary expression was faithfully maintained in the mice, but there was no expression in the mouse trophoblast, suggesting that there are transcription factors in the human trophoblast that are not present in the mouse trophoblast to drive expression of this gene.
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From page 42... ...
Classic trophoblast transcription factors such as GATA2, GATA3, and AP2A all have SMAD1/5/8 binding sites and immediately upregulate upon BMP4 induction, which explains how trophoblast is being driven from ESCs. Markers of Primitive Syncytium Derived from Human Embryonic Stem Cells Robson highlighted several markers of hESC-derived primitive syncytium established by RNA-sequencing data comparing the human villous placenta (i.e., later-stage trophoblast)
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From page 43... ...
As with LMO2, there are species-specific differences that are indicative of active evolutionary change around the regulatory regions that bind GCM1 and drive expression of these transcriptional regulators in primitive syncytium. Early Development as a Site for Rapid Mutation and Selection Robson concluded by emphasizing the extent of the molecular and functional differences between species in the primitive syncytium and predicting that there are significant differences in other cell types at these early stages of development.
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From page 44... ...
PANEL DISCUSSION Variability in Trophoblast-Related Terminology Panelists were asked by Amander Clark, the session moderator, to explain why they refer to the trophoblast system as a syncytium and how that relates to the syncytiotrophoblast, CTB, mural trophoblast, or polar trophoblast. She also asked how the primitive syncytium relates to the cell types differentiated in their pluripotent stem cell models.
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From page 45... ...
Cells Used to Direct Trophoblast Stem Cell Derivation A workshop participant asked Parast whether the protocol she follows to direct TSC derivation used undirected pluripotent cells or BMP-derived cells. She replied the cells were already BMP-derived cells at the end of the first step.
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From page 46... ...
In the blastocyst it is not syncytial, but it becomes syncytial as it implants. Robson added that primitive syncytium probably forms at the polar end of the human blastocyst upon attachment.
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From page 47... ...
Role of Trophoblast Signaling in Inducing Gastrulation The panelists were asked whether early human trophoblast must signal to the epiblast to induce the processes of gastrulation, given that (1) in humans the first trophoblast develops as primary syncytium and (2)
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