Self-Renewing Trophoblast Organoids Recapitulate the Developmental Program of the Early Human Placenta

Abstract

Defective placentation is the underlying cause of various pregnancy complications, such as severe intrauterine growth restriction and preeclampsia. However, studies on human placental development are hampered by the lack of a self-renewing in vitro model that would recapitulate formation of trophoblast progenitors and differentiated subtypes, syncytiotrophoblast (STB) and invasive extravillous trophoblast (EVT), in a 3D orientation. Hence, we established long-term expanding organoid cultures from purified first-trimester cytotrophoblasts (CTBs). Molecular analyses revealed that the CTB organoid cultures (CTB-ORGs) express markers of trophoblast stemness and proliferation and are highly similar to primary CTBs at the level of global gene expression. Whereas CTB-ORGs spontaneously generated STBs, withdrawal of factors for self-renewal induced trophoblast outgrowth, expressing the EVT progenitor marker NOTCH1, and provoked formation of adjacent, distally located HLA-G⁺ EVTs. In summary, we established human CTB-ORGs that grow and differentiate under defined culture conditions, allowing future human placental disease modeling.

Keywords: Wnt signalling; cell fusion; cytotrophoblast organoids; differentiation; extravillous trophoblast lineage; human placenta; self-renewal.

Figure 1

Establishment of Human CTB Organoids from Isolated First-Trimester Villous CTBs (A) Representative phase contrast images of CTB-ORGs (n = 8) before (P0) and after first passaging (P1). Small spheroid-like structures are visible after 4–7 days and reach a diameter of approximately 300–800 μm within 3 weeks. (B) H&E staining and immunofluorescence of tissue sections of CTB-ORGs revealed densely packed cell clusters of epithelial origin with fused regions in the center. Representative pictures of CTB-ORGs (n = 8 different cultures between P2 and 4) and first-trimester placenta (n = 6; 6th to 7th week of gestation) showing cytokeratin 7 (KRT7) (white), hepatocyte growth factor activator inhibitor type 1 (HAI1) (white), E-cadherin (red), and nuclei (DAPI, blue) are shown. Arrows mark mononuclear CTBs in organoids and villous cytotrophoblasts (vCTBs), whereas arrowheads depict multinuclear structures and STBs in organoids and placental tissues, respectively. VS, villous stroma. In negative controls primary antibodies were replaced with mouse monoclonal isotype IgG (mAB-IgG). (C) Representative electron transmission microscopy images of a CTB-ORG at P3 out of three different cultures (P2 and P3) analyzed. Stippled line in (a) indicates boundary between CTB and syncytiotrophoblasts (STB). ^∗Glycogen deposits in CTBs; nu, nucleus; (b) illustrates nuclei and transport vesicles (marked by arrowheads) in magnified STBs. Inset picture (1), showing the micro-villous surface of STBs, is depicted at a higher magnification on the right hand side. (D) Time-dependent growth (phase contrast images) of a representative clonal organoid line, generated from a single cell of an organoid culture at P3. See also Figures S1 and S2.

Figure 2

Human Trophoblast Organoids Express Markers of CTB Identity, Stemness, and Proliferation (A–C) Representative immunofluorescence images of first-trimester placentae (n = 4) and CTB-ORGs between P2 and P5 (n = 6 different cultures) are shown. EVT, extravillous trophoblast; vCTB, villous cytotrophoblast; STB, syncytiotrophoblasts; VS, villous stroma; nuclei were counterstained with DAPI. Trophoblasts expressing markers of stemness or proliferation are marked with arrows. Stippled line in pictures of 6th-week placentae demarcates STBs from underlying vCTBs. (A) Immunofluorescence of markers of trophoblast stemness and identity in serial sections of CTB-ORGs and 6th-week placenta. In negative controls primary antibodies were replaced with rabbit isotype IgG (rAB-IgG). (B) qPCR showing mRNA expression of CTB identity and stemness markers in CTB-ORGs (P2–P4; n = 6 different cultures), primary vCTBs (n = 4), and placental fibroblasts (FIB) (n = 4). Mean values ± SD measured in duplicates (normalized to TBP) are shown ^∗p < 0.05; ns, not significant; nd, not detectable; AU, arbitrary units; TFAP2A and TFAP2C mRNAs encode AP-2α and AP-2γ, respectively. (C) Immunofluorescence of cyclin A (CCNA) and KI67 in CTB-ORGs and 6th-week placenta. (D) Methylation status of individual CpG sites in the proximal ELF5 promoter region (−379 to −28) in two different CTB-ORGs (P1 and P3), vCTB and FIB isolated from 6th-week placenta. Percentages indicate proportion of methylated (filled circles) to unmethylated (open circles) CpG residues. See also Figure S3.

Figure 3

CTB Organoids Express Markers of Syncytiotrophoblasts in the Inside Region (A) Immunofluorescence of different STB markers in serial sections of a representative CTB-ORG culture (n = 4 different cultures analyzed between P2 and P5) and in 6th-week placental tissue (n = 3). Stippled line depicts the border between CTB and STB. ENDOU, poly(U)-specific endoribonuclease; GCM1, glial cell missing 1; CGβ, chorionic gonadotrophin β. Nuclei were stained with DAPI. Note that GCM1 is predominantly expressed in STBs, but can also be detected in a small number of vCTBs (marked by arrows). (B) qPCR showing mRNA expression of STB markers in CTB-ORGs (n = 6 different cultures, P2–P4), vCTBs (n = 4), and STBs (n = 3), generated in vitro by differentiating vCTBs for 72 hr on fibronectin. Mean values ± SD, measured in duplicate, are shown. ^∗p < 0.05; AU, arbitrary units. CGB, CGβ mRNA. See also Figure S4.

Figure 4

RNA-Seq and Bioinformatic Analyses of Placental Organoids and Primary Trophoblasts (A) Hierarchical clustering of genome-wide mRNA expression profiles. A total of 7,417 genes, differentially expressed between CTB-ORG cultures (P2 and P4) and FIBs (n = 2 from 6th week), and expression of these genes in vCTB (n = 3 from 6th- to 7th-week placentae) and differentiated STB (n = 3; 6th- to 7th-week placentae) are shown. Analysis is based on p < 0.05. (B) Venn diagram on the left shows that of 2,293 of trophoblast-upregulated mRNAs (>2-fold change relative to FIB; p < 0.05), 1,456 were common to CTB-ORG and vCTB. CTB-ORGs also express 1,500 STB-specific mRNAs (Venn diagram on the right-hand side) and share 1,042 common genes with both vCTB and STB. (C) Clustered heatmap depicting selected expression patterns of markers of CTB stemness and cell fusion. TFAP2A and TFAP2C mRNAs encode AP-2α and AP-2γ, respectively. CGB5, mRNA of the chorionic gonadotrophin β5 gene; ENDOU, poly(U)-specific endoribonuclease.

Figure 5

Loss of Wnt Activators Promotes Formation of EVTs and Their Progenitors in Trophoblast Organoid Cultures (A) Upper pictures show representative phase contrast images of (CTB-ORG cultures) at P2 in the absence (4 days, Wnt⁻) or presence (Wnt⁺) of R-spondin and CHIR99021. Lower immunofluorescence images depict HLA-G expression in representative CTB-ORGs (P2). In total n = 10 different CTB-ORGs (P2–P5) were analyzed. Note that EVTs, which have deeply migrated into Matrigel, are lost during the fixation procedure. Hence, HLA-G⁺ cells of Wnt⁻ CTB-ORGs, shown in (A and D), likely represent EVTs of the distal cell column. Nuclei are stained with DAPI. RNA (B) and protein (C) expression of vCTB lineage/stem cell-specific genes, and of STB and EVT markers in Wnt⁺ and Wnt⁻ CTB-ORGs. (B) qPCR analyses in different CTB-ORGs (n = 5; P2–P5). Expression was normalized to TBP. Mean values ± SD, measured in duplicate, are shown, ^∗p < 0.05; AU, arbitrary units. (C and D) Representative western blots performed with different CTB-ORGs (n = 4; P2-P5) are shown (C). GAPDH was used as loading control. PRG2, proteoglycan 2; (D) immunofluorescence showing NOTCH1 and adjacent HLA-G expression in a CTB-ORG (P3) and in 6th-week placenta. Representative images of CTB-ORGs (n = 10 different cultures; P2–P5) and first-trimester placental tissues (n = 4) are depicted. CC, cell column; EVT, extravillous trophoblast; VS, villous stroma; See also Figures S5 and S6.

Figure 6

Differential Expression Patterns of Nuclear β-Catenin and T Cell Factors in Distinct Trophoblast Subtypes of Organoids and the Effects of Wnt Inhibition on EVT Formation (A and B) Representative pictures of n = 6 (A) and n = 4 (B) different CTB-ORGs cultures (analyzed between P2 and P4) are depicted. CC, cell column; CTB, cytotrophoblast; EVT, extravillous trophoblast; STB, syncytiotrophoblast; Nuclei are visualized with DAPI. (A) Localization of β-catenin in CTB-ORGs in the absence (Wnt⁻) or presence (Wnt⁺) of R-spondin and CHIR99021. Trophoblasts displaying nuclear β-catenin are marked with arrows. (B) Expression of T cell factors (TCFs) in Wnt⁺ and Wnt⁻ CTB-ORGs, co-stained with trophoblast subtype markers. Stippled line indicates NOTCH1⁺ progenitor population. Serial sections are shown in the Wnt⁻ condition. (C) qPCR analyses showing TCF7 (encoding TCF-1), TCF7L1 (encoding TCF-3), and TCF7L2 (encoding TCF-4) mRNA expression (normalized to TBP) in different Wnt⁺ and Wnt⁻ CTB-ORGs (n = 5, P2–P4). Mean values ± SD, measured in duplicates, are shown ^∗p < 0.05; AU, arbitrary units. See also Figure S7.

Figure 7

Inhibition of Wnt Response Prevents EVT Formation (A) Representative phase contrast images of different CTB-ORG cultures (n = 4, P2–P4) grown for 4 days under Wnt⁺ or Wnt⁻ conditions in the absence (ctrl) or presence of inhibitor of Wnt response 1 (IWR-1). The latter was supplemented 48 hr after removal of R-spondin and CHIR99021 for an additional 2 days. Inset pictures 1–4 (boxed areas) are depicted at a higher magnification. Trophoblast outgrowths are marked with stippled lines. (B) Immunofluorescence images of representative Wnt⁺ or Wnt⁻ CTB-ORGs (n = 4, P2–P4) treated with IWR-1 as mentioned above. Sections were co-stained with DAPI to visualize nuclei. (C) Model system showing the role of Wnt in trophoblast organoid self-renewal and differentiation. β-cat, β-catenin; CTB, cytotrophoblast; EVT, extravillous trophoblast; STB, syncytiotrophoblasts; TCF, T cell factor.