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. 2022 Jun 15:10:835445.
doi: 10.3389/fcell.2022.835445. eCollection 2022.

A Single-Cell Characterization of Human Post-implantation Embryos Cultured In Vitro Delineates Morphogenesis in Primary Syncytialization

Yiming Wang  1   2   3   4 , Xiangxiang Jiang  1   5 , Lei Jia  6 , Xulun Wu  1   2   3   4 , Hao Wu  1   2   3   4 , Yue Wang  1   2   3   4 , Qian Li  1   3   4 , Ruoxuan Yu  1   2   3   4 , Hongmei Wang  1   2   3   4 , Zhenyu Xiao  1   3   4   7 , Xiaoyan Liang  6
Affiliations

A Single-Cell Characterization of Human Post-implantation Embryos Cultured In Vitro Delineates Morphogenesis in Primary Syncytialization

Yiming Wang et al. Front Cell Dev Biol. .

Abstract

Implantation of the human blastocyst is a milestone event in embryonic development. The trophoblast is the first cell lineage to differentiate during implantation. Failures in trophoblast differentiation during implantation are correlated to the defects of pregnancy and embryonic growth. However, many gaps remain in the knowledge of human embryonic development, especially regarding trophoblast morphogenesis and function. Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on human post-implantation embryos cultured in vitro. A hierarchical model was established, which was characterized by the sequential development of two primitive cytotrophoblast cell (pCTB) subtypes, two primitive syncytiotrophoblast subtypes, and migrative trophoblast cells (MTB) after the trophectoderm . Further analysis characterized cytoskeleton transition of trophoblast cells and morphogenesis, such as irregular nuclei, cell cycle arrest, and cellular aging during implantation. Moreover, we found syncytialization of hTSCs could mimic the morphogenesis, serving as a powerful tool for further understanding of the mechanism during the implantation stage of pregnancy. Our work allows for the reconstruction of trophoblast cell transcriptional transition and morphogenesis during implantation and provides a valuable resource to study pathologies in early pregnancy, such as recurrent implantation failure.

Keywords: cytoskeleton; human embryos; human trophoblast stem cells; single-cell RNA sequencing; trophoblast differentiation.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Global expression profiling of human embryo cells using single-cell RNA-seq and cell type identification. (A) Bright-field images of in vitro cultured human embryos from day 6 to day 13. Scale bars, 50 μm. (B) Cartoons of human embryo implantation morphogenesis based on Carnegie series. (C) Immunostaining of day 6 and day 8 human embryos (n = 3 and n = 3) for F-actin and CGB. DAPI, grey, DNA. Scale bars, 20 μm. (D) Immunofluorescence of day 9 and day 10 human embryos (n = 3 and n = 1) for F-actin, GATA6, and OCT4. Images showing the pro-amniotic cavity and prospective yolk sac marked by white arrows. DAPI, grey, DNA. Scale bars, 20 μm. (E) Workflow depicting the strategy of derivation of single cell from post-implantation human embryos. Number of cells every embryonic day (day 8-day 13) retained after quality filtering were shown. (F) UMAP plots showing single-cell transcriptomes of human embryos from day 8–day 13. (G) UMAP plots showing the expression patterns of the lineage markers in human embryos. Color key from grey to red indicates relative expression levels from low to high, respectively.
FIGURE 2
FIGURE 2
Distinct subtypes in early trophoblast cells during implantation. (A) Joint visualization of our datasets together with published datasets. Tang represents published datasets; Wang represents our datasets. (B) UMAP plots showing the expression patterns of early trophoblast cells in different clusters. TE, trophectoderm; pCTB, primitive cytotrophoblast; pFCC, primitive fusion-competent trophoblast cells; MTB, migrative trophoblast cells, pSTB1, primitive syncytiotrophoblast 1; pSTB2, primitive syncytiotrophoblast 2. (C) Violin plots showing the markers expression of different clusters. (D) Heat map showing representative DEGs in different clusters. Color key from purple to yellow indicates relative expression levels from low to high, respectively. (E,F) Pseudotime analysis was assigned to each cell showing embryonic day and lineage assignment, with cells colored by embryonic day (upper) and cell clusters (lower). Three-dimensional diffusion map representation of all cells at different culture time points, showing lineage assignment on each embryonic day (F).
FIGURE 3
FIGURE 3
Cytoskeleton-associated genes decreased in primitive syncytiotrophoblast. (A) Dot plots showing enriched GO terms and p-value of distinct clusters of trophoblast cells. (B) UMAP plots showing the expression levels of representative genes for trophoblast cells. Color key from grey to red indicates relative expression levels from low to high, respectively.
FIGURE 4
FIGURE 4
Nuclear enlargement and deformation in primitive syncytiotrophoblast. (A) Immunostaining of day 6 and day 8 human embryos (n = 3 and n = 3) cultured in vitro for CGB, Lamin A, and F-actin. 3D reconstruction of nuclei basing on the immunostaining results (shown in red). Dotted rectangle represented regions in the embryos that are shown with higher magnification. Scale bars, 20 μm. (B) Quantification of nuclear volume in TE, pCTB, and pSTB of human embryos. n = 3 human embryos per group. Data are shown as mean ± SEM. Unpaired two-tailed Student’s t-test, *p < 0.05; ***p < 0.001. (C) Bar graph showing the proportions of nuclear volume in TE, pCTB, and pSTB of human embryos. (D) Immunostaining of day 6 and day 8 human embryos (n = 3 and n = 3) cultured in vitro for CGB, F-actin, and Lamin A. Dotted rectangle represented regions in the embryos that are shown with higher magnification. DAPI, grey, DNA. Scale bars, 20 μm. (E) Cartoons of nuclear morphology based on immunostaining results. Protrusions are the projecting point after Elliptical Fourier Analysis. (F) Quantification of protrusion numbers in (D). Data are shown as mean ± SEM. n = 3 human embryos per group. Unpaired two-tailed Student’s t-test, **p < 0.01; ***p < 0.001. (G) Quantification of circularity in TE, pCTB, and pSTB of human embryos. n = 3 human embryos per group. Data are shown as mean ± SEM. Unpaired two-tailed Student’s t-test, *p < 0.05; ***p < 0.001. (H) Bar graph showing the proportions of nuclear circularity. (I) Immunostaining of hTSCs and STB for Lamin A, CGB, and F-actin. Dotted rectangle represented regions that are shown with higher magnification. DAPI, grey, DNA. Scale bars, 50 μm.
FIGURE 5
FIGURE 5
Loss of “cytoplasmic bridge” in primitive syncytiotrophoblast. (A) Bar graph showing the ratios of cells in different phases to total cells in each cluster according to the expression of S- and G2/M-phase genes. G1/G0-phases are the cells not in the S or G2/M phases. (B) UMAP plots showing the indicated genes in trophoblast cells. Color key from grey to red indicates relative expression levels from low to high, respectively. (C) Immunostaining of day 6 and day 10 human embryos (n = 3 and n = 3) cultured in vitro for F-actin and Aurora B. Dotted rectangle represented regions that are shown with higher magnification. Yellow arrows indicate “cytoplasmic bridges”. Scale bars, 20 μm. (D) Quantification of “cytoplasmic bridge” number in (C). Data are shown as mean ± SEM. n = 3 human embryos per group. Unpaired two-tailed Student’s t-test, *p < 0.05. (E) Immunostaining of hTSCs and STB for Aurora B, CGB, and F-actin. Dotted rectangle represented regions that are shown with higher magnification. DAPI, grey, DNA. Scale bars, 50 μm.
FIGURE 6
FIGURE 6
Primitive syncytiotrophoblast is related to cellular aging. (A) Dot plots showing enriched GO terms and p-value of distinct clusters of trophoblast cells. (B) Heat map of indicated genes in distinct clusters of trophoblast cells. The color key from purple to yellow indicates low to high gene expression levels, respectively. (C) Immunostaining of day 6 and day 8 human embryos (n = 3 and n = 3) cultured in vitro for F-actin, γH2AX, and CGB. DAPI, grey, DNA. Scale bars, 20 μm. (D) Immunostaining of hTSCs and STB for γH2AX, CGB, and F-actin. Dotted rectangle represented regions that are shown with higher magnification. DAPI, grey, DNA. Scale bars, 50 μm. (E) Quantification of the numbers of γH2AX positive nuclei in hTSCs and STB. Data are shown as mean ± SEM. n = 10 fields of view per group. Unpaired two-tailed Student’s t-test, **p < 0.01.
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