The product of BMP-directed differentiation protocols for human primed pluripotent stem cells is placental trophoblast and not amnion

Abstract

The observation that trophoblast (TB) can be generated from primed pluripotent stem cells (PSCs) by exposure to bone morphogenetic protein-4 (BMP4) when FGF2 and ACTIVIN signaling is minimized has recently been challenged with the suggestion that the procedure instead produces amnion. Here, by analyzing transcriptome data from multiple sources, including bulk and single-cell data, we show that the BMP4 procedure generates bona fide TB with similarities to both placental villous TB and TB generated from TB stem cells. The analyses also suggest that the transcriptomic signatures between embryonic amnion and different forms of TB have commonalities. Our data provide justification for the continued use of TB derived from PSCs as a model for investigating placental development.

Keywords: BMP4, syncytiotrophoblast; amnion; cytotrophoblast; extravillous trophoblast; human embryonic stem cells; human primed pluripotent stem cells; placenta; scRNA-seq; snRNA-seq; trophoblast; trophoblast differentiation.

Figure 1

Comparison of procedures for BMP4 or BAP treatment among Yabe et al., Krendl et al., Io et al., and Guo et al. (A) For the generation of BAP_D8_Yabe (Yabe et al., 2016), primed H1 ESCs were seeded on a Matrigel-coated plate and cultured with mTeSR1. On the day following passaging, the medium was replaced with hESC medium (DMEM/F12 containing 20% KSR, 1 mM L-glutamine, 0.1 mM 2-ME, 1% NEAA), which had been conditioned by irradiated mouse embryonic fibroblasts (iMEFs), called MEF-conditioned medium (CM), and supplemented with 4 ng/mL FGF2. After an additional day, the medium was changed to hESC medium containing 10 ng/mL BMP4, 1 μM A83-01, and 0.1 μM PD173074 (BAP) for 8 days. (B) For the generation of BMP4_D3_Krendl (Krendl et al., 2017), primed H9 ESCs were dissociated using Accutase and seeded as single-cell monolayers on Matrigel-coated plate and cultured with mTeSR1 medium supplemented with 10 μM Y-27632. After 16–18 h, the medium was changed to RPMI1640, containing Glutamax and B27 without insulin, and supplemented with 50 ng/mL BMP4 for 72 h. (C) For the generation of pBAP_D3_Io (Io et al., 2021), primed H9 ESCs were dissociated into single cells with trypsin/EDTA and seeded on a Laminin-511 E8-coated plate and cultured with MEF-CM supplemented with BAP and 10 μM Y27632 for 3 days. (D) For the differentiation of primed hPSCs in Guo et al. (Guo et al., 2021), primed H9 ESCs were seeded on Geltrex-coated plate and cultured with AFX medium, which contains N2B27 basal medium with 5 ng/mL Activin A, 5 ng/mL FGF2, and 2 μM XAV. The next day, the medium was replaced with N2B27 basal supplemented with 50 ng/mL BMP2, 1 μM A83-01, and 1 μM PD0325901 for 5 days. In this study, RNA-seq data generated from H1_BAP_D8_Yabe, H9_BMP4_D3_Krendl, and H9_pBAP_D3_Io were used for the comparative analyses among primed ESC-derived TB models by different protocols. There is no available RNA-seq data for H9_pBAP_D5_Guo.

Figure 2

Upregulated genes and cell enrichment analyses of undifferentiated and differentiated datasets (A–C) The datasets were obtained by using BMP4-based protocols of (A) Io (pH9 versus H9_pBAP_D3), (B) Krendl (H9_BMP4 versus H9_BMP4_D3), and (C) Yabe (H1_Yabe versus H1_BAP_D8_>70). In the volcano plot, the darker shade is undifferentiated, showing upregulated genes of pH9 (purple), H9_BMP4 (darker pink), and H1_Yabe (darker green). The lighter shade is their differentiated counterpart, H9_pBAP_D3 (lighter blue), H9_BMP4_D3 (lighter pink), and H1_BAP_D8_>70 (lighter green). Only genes with |log2fold| ≥ 1 and adjusted p value (padj) ≤ 0.05 are colored. The cell enrichment results of the upregulated genes are depicted as bar plots (color coordinated), with the top chart using the Vento-Tormo et al. (2018) dataset for enrichment analyses, the middle using the Xiang et al. (2020) dataset, and the bottom using the combined Zhou et al. (2019) and Petropoulos et al. (2016) datasets (curated by Castel et al., 2020). Only the three most-enriched fetal cell types are shown here (see Figure S1 for the full cell enrichment results).

Figure 3

Upregulated genes and cell enrichment analyses of differentiated cells from BMP4-based protocols of Io et al., Krendl et al., and Yabe et al. (A–C) The comparisons include (A) H9_BMP4_D3 (light pink) versus H1_BAP_D8_>70 (light green), (B) H1_BAP_D8_>70 (light green) versus H9_pBAP_D3 (light blue), and (C) H9_pBAP_D3 (light blue) versus H9_BMP4_D3 (light pink). In the volcano plots, only genes with |log2fold| ≥ 1 and padj ≤ 0.05 are colored. The cell enrichment results of the upregulated genes are depicted as bar plots (color coordinated), with the top chart using the Vento-Tormo et al. (2018) dataset for enrichment analyses, the middle using the Xiang et al. (2020) dataset, and bottom using the combined Zhou et al. (2019), and Petropoulos et al. (2016) datasets (curated by Castel et al., 2020). Only the three most-enriched fetal cell types are shown here (see Figure S2 for the full cell enrichment results and Figure S3 for enrichment results that include H1_BAP_D8_<40).

Figure 4

Principal-component analysis (PCA) plots (A) PCA plot showing the clustering of various datasets used in this study. (B) PCA plot showing the clustering of datasets (without fully differentiated amnion sets) used in this study. Each color represents the treatment (cell type and/or media/growth condition), and the shape represents the study from which the samples were derived (see Table S1 for more details). PCA plots for additional components and variance explained by each component (scree plots) are shown in Figure S4.

Figure 5

Comparing expression of selected genes in various datasets (A) Violin-box plot showing the expression of genes upregulated in 9 week amnion samples compared with the 9 week placenta (gene list obtained from Figure 6B of Io et al.), in H9_pBAP_D3, H9_BMP4_D3, and H1_BAP_D8_>70 samples. (B) Violin plot showing the expression of amnion marker genes identified using TissueEnrich (fold change >10) from all amnion datasets available in Roost et al. for the same datasets in (A). The expression differences were compared using the non-parametric Dunn’s (Dunn, 1964) test of multiple comparisons following a significant Kruskal-Wallis test (Kruskal and Wallis, 1952), with p values adjusted with the Benjamini-Hochberg method (highly significant, ^∗∗∗p < 0.001; non-significant, NS). For (A), associated padj values for amnion versus CT, amnion-H9_pBAP_D3, amnion-H9_BMP4_D3, and amnion-H1_BAP_D8_>70 are 0.11 × 10^-1, 9.78 × 10^-9, 4.34 × 10^-13, and 1.61 × 10^-4, respectively. Similarly, for (B), padj values for amnion versus CT, amnion-H9_pBAP_D3, amnion-H9_BMP4_D3, and amnion-H1_BAP_D8_>70 are 6.82 × 10^-69, 3.16 × 10^-37, 2.39 × 10^-31, and 5.41 × 10^-20, respectively. See also Figure S5.

Figure 6

Visualization of clusters generated from single-nucleus and single-cell RNA sequencing analyses (A) Visualization of clusters generated from single-cell and single-nucleus data colored according to assignment by clustering analysis. (B) Nuclei/cells visualized according to cell type and growth conditions. H1_BAP_D8_20pcO2_Khan (pink), H1_BAP_D8_5pcO2_Khan (purple), nCT (H9_nCT_D5_Io and H9_nCT_D10_Io) (yellow), and nTE (H9_nTE_D2_Io and H9_sc.nTE_D3_Io) (green) are shown. Each dot represents a nucleus/cell. (C and D) The cell enrichment bar plots for the marker genes of clusters 6 and 8 (genes with |fold change| ≥ 1.5 and padj ≤ 0.05) along with the composition of nuclei/cells for different treatments (as percentages, relative to the total number of nuclei/cells in the condition) (see Figure S6 for cell composition of other clusters). The cell enrichment results were done using the Vento-Tormo et al. dataset, the Xiang et al. dataset, and the combined Zhou et al. and Petropoulos et al. datasets (curated by Castel et al.). Only the three most-enriched fetal cell types are shown here (see Figure S6 for the full cell enrichment results).