Abstract
The ability to study human post-implantation development remains limited due to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (i.e., embryonic disk, bilaminar disk, yolk- and chorionic sacs, surrounding trophoblasts) remain lacking2. Mouse naïve embryonic stem cells (ESCs) have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation mouse Structured Stem cell-based Embryo Models with spatially organized morphogenesis (SEMs)3. Here, we extend these findings to humans, while using only genetically unmodified human naïve ESCs (in HENSM conditions)4. Such human fully integrated SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos including epiblast, hypoblast, extra-embryonic mesoderm, and trophoblast surrounding the latter layers. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days post-fertilization (dpf) (Carnegie stage 6a). This includes embryonic disk and bilaminar disk formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, PGC specification, polarized yolk sac with visceral and parietal endoderm, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, a trophoblast surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform may enable the experimental interrogation of previously inaccessible windows of human early post-implantation up to peri-gastrulation development.
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Supplementary Information
This file contains Supplementary Figures 1-17 and Legends, Supplementary Introduction, Supplementary Discussion, and Supplementary References.
Supplementary Table 1
Human SEM scRNA-seq analysis related gene expression list. Top markers (average log2(Fold-change)>0.25 and one-sided Wilcoxon text p-value < 0.01) of each of the 13 cell clusters in human SEMs, as identified by Seurat package using one-sided Wilcoxon test.
Supplementary Table 2
Cell annotation of human SEM cells. a. Cell annotation of human SEM. b. List of CTb-like and STb-like annotated cells. c.Normalized gene expression of top 50 differentially expressed genes that are upregulated in CTb-like compared to STb-like cells (Extended Data Fig. 12e).
Supplementary Table 3
PCR primers used in this study. Primer names, DNA sequences from 5’ to 3’ end, and the cited reference (when applicable).
Supplementary Table 4
Summary of the microscopy parameters used for imaging in this study. The spreadsheet provides information of the type of the microscope, used detection objectives, laser lines, and the voxel size for acquisition of the images published herein.
Supplementary Video 1
3D reconstruction of the day 8 human SEM (WIBR3 cell line). Immunofluorescence for epiblast- (OCT4, cyan), hypoblast- (SOX17, yellow), trophoblast-like (CK7, magenta) compartments, and nuclei (DAPI, white). 0 – 8 sec, 3D view of the outer trophoblast-like layer with enlarged multinuclear cells. 8 – 22 sec and 39 – 42 sec, 3D segmentation of the epiblast- (cyan) and hypoblas-like (yellow) structures with DAPI. 22 sec – 38 sec, inner SEM structure comprised of bilaminar disk-like structure with amnion-like and yolk sac-like compartments, surrounded by the connective tissue and the trophoblast-like cells. Immunofluorescence signal and tissue segmentation are outlined. Acquisition with Z7 microscope and processing with Imaris v10.0.1.
Supplementary Video 2
3D reconstruction of the day 8 human SEM (WIBR3 cell line). Immunofluorescence for epiblast- (OCT4, cyan), yolk sac- (SOX17, yellow), trophoblast-like compartment (CK7, magenta). 0 – 8 sec and 19 – 21 sec, 3D view of the outer trophoblast-like layer. 8 sec – 18 sec, slicing through the 3D volume showing the inner cellular structure of the SEM comprised of bilaminar disk-like structure, amnion-, and yolk sac-like compartment with the inner cavity. 3D rendering of the thresholded immunofluorescence signal. Acquisition with Z7 light-sheet microscope and processing with Imaris v10.0.1.
Supplementary Video 3
3D reconstruction of the day 6 human SEM (WIBR1 cell line). Immunofluorescence for epiblast- (OCT4, cyan), yolk sac- (SOX17, yellow), trophoblast-like compartment (CK7, magenta), and nuclei (DAPI, white). 0 – 10 sec, 3D view of the outer trophoblast-like layer. 10 – 50 sec, 3D segmentation of the epiblast- (cyan) and hypoblast-like cells (yellow) with DAPI maximum projection. 21 sec – 41 sec, slicing through the 3D volume showing the inner structure of the SEM comprised of bilaminar disk-like structure with early amnion-like compartment, connected to the outer trophoblast-like layer. Immunofluorescence signal and tissue segmentation are outlined. Acquisition with Z7 light-sheet microscope and processing with Imaris v10.0.1.
Supplementary Video 4
3D reconstruction of the human SEM at day 6 demonstrating pro-amniotic-like cavity formation within the epiblast-like compartment. 3D reconstruction of the human SEM at day 6 shows its 3D morphology (0 – 12 sec) and beginning of the proamniotic-like cavity formation within the epiblast-like compartment (12 – 18 sec). Immunofluorescence for OCT4 (epiblast, cyan), F-ACTIN (red), and nuclei (DAPI, white). The image was acquired with Zeiss LSM 800 microscope and processed with Imaris v10.0.0.
Supplementary Video 5
3D reconstruction of the human SEM at day 8 shows formation of embryonic disk-like structure and amnion-like compartment. 3D reconstruction of the embryonic disk-like structure (SOX2, cyan) and amnion-like compartment (TFAP2A, magenta) in the day 8 human SEM. 3D segmentation of epiblast-like tissue (cyan) and amnion-like tissue (pink) is denoted as the semi-transparent outline together with the corresponding immunofluorescence signal. 17 – 21 sec, human SEM epiblast-like compartment has a disk shape. The image was acquired with Z7 light-sheet microscope and processed with Imaris v10.0.0.
Supplementary Video 6
3D reconstruction of the human SEM at day 6 shows yolk sac-like morphology and polarity. 3D reconstruction of human day 6 SEM showing yolk sac-like structure (marked by SOX17, yellow). 5 – 18 sec, zoom into the visceral and parietal yolk sac-like cells having columnar and squamous cell shape, respectively. The cells exhibit apical polarization (aPKC, green). F-ACTIN (red), nuclei (DAPI, white). The image was acquired with Zeiss LSM 700 microscope and processed with Imaris v10.0.0.
Supplementary Video 7
3D reconstruction of the human SEM at day 8 shows ExEM-like cell integration underneath the yolk sac-like structure. 3D reconstruction of the human SEM at day 8 showing ExEM-like cells marked by expression of VIM (red) and located predominantly underneath the yolk sac-like structure (yellow); OCT4 (epiblast, cyan), nuclei (DAPI, grey). 3D rendering of the thresholded immunofluorescence signal. The image was acquired with Zeiss LSM 700 microscope and processed with Imaris v10.0.0.
Supplementary Video 8
3D reconstruction of human SEM at day 8 shows development of the syncytiaotrophoblast-like layer with lacunae-like structures. 3D reconstruction of the human SEM at day 8 showing development of the syncytiotrophoblast-like layer, expressing both SDC1 (magenta) and HCGB (green). The syncytiotrophoblast-like compartment forms multiple lacunae-like structures. F-ACTIN (red), nuclei (DAPI, grey). 11 – 19 sec, slicing through the 3D volume showing the inner structure of the multiple trophoblast lacunae-like structures. The image was acquired with Zeiss LSM 700 microscope and processed with Imaris v10.0.0.
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Oldak, B., Wildschutz, E., Bondarenko, V. et al. Complete human day 14 post-implantation embryo models from naïve ES cells. Nature (2023). https://doi.org/10.1038/s41586-023-06604-5
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DOI: https://doi.org/10.1038/s41586-023-06604-5
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