Establishment and comparison of human term placenta-derived trophoblast cells†

Abstract

Research on the biology of fetal-maternal barriers has been limited by access to physiologically relevant cells, including trophoblast cells. In this study, we describe the development of a human term placenta-derived cytotrophoblast immortalized cell line (hPTCCTB) derived from the basal plate. Human-term placenta-derived cytotrophoblast immortalized cell line cells are comparable to their primary cells of origin in terms of morphology, marker expression, and functional responses. We demonstrate that these can transform into syncytiotrophoblast and extravillous trophoblasts. We also compared the hPTCCTB cells to immortalized chorionic trophoblasts (hFM-CTC), trophoblasts of the chorionic plate, and BeWo cells, choriocarcinoma cell lines of conventional use. Human-term placenta-derived cytotrophoblast immortalized cell line and hFM-CTCs displayed more similarity to each other than to BeWos, but these differ in syncytialization ability. Overall, this study (1) demonstrates that the immortalized hPTCCTB generated are cells of higher physiological relevance and (2) provides a look into the distinction between the spatially distinct placental and fetal barrier trophoblasts cells, hPTCCTB and hFM-CTC, respectively.

Keywords: BeWo; immortalization; pregnancy; term placenta; trophoblast.

Graphical Abstract

Figure 1

Human placenta-derived immortalized trophoblast and stromal cell line and their characterization. (A) Schematic diagram of generation of immortalized human placental trophoblast cell lines (hPTC) and immortalized human placental stromal cell lines (hPSC). (B) The representative phase contrast images of primary human placental trophoblast cells (pPTC at Passage 1), immortalized human placental trophoblast cells (hPTC at Passage 10), pPTC stained with β-galactosidase at Passage 2, hPTC stained with β-galactosidase at Passage 13, human placental stromal cells (pPSC at Passage 1), immortalized human placental stromal cells (hPSC at Passage 10). hPTCs show similar epithelial cell morphology as their primary cell origin, and both cells grow in colonies. Red arrow pointing to P2 pPTC cells showing senescent cells (blue cells); other stained cells can be appreciated in the background also. No similar stained senescent cells can be appreciated in P13 hPTC . Immortalized hPTC cells didn’t show senescent cells. hPSCs show similar mesenchymal cell morphology as their primary cell origin. Scale bar, 50μm. (C) Representative immunocytochemical staining images showing epithelial and stromal cell-specific marker protein expressions in primary and immortalized cells. Epithelial cell-specific marker cytokeratin-7 (red) expression was detected in both primary and immortalized trophoblast cell populations (yellow arrows). Stromal cell-specific marker vimentin (green) was detected only in the primary isolate of trophoblast cells due tostromal cells contamination coming with the isolation process. Regardless of whether primary or immortalized, the populations for trophoblast cells were negative for vimentin expression (yellow arrows). Both primary and immortalized stromal cells show vimentin expression (green); interestingly they were both expressed low level of cytokeratin-7 (red). Scale bar, 50μm. (D) Cell signaling pathways activation in primary and immortalized cells. Western blot analysis showing p38 MAPK pathway activation in primary and immortalized placental trophoblast cells. PTCs were treated with LPS for 30 min to mimic infection and CSE for 6 hr to mimic oxidative stress, while PSCs were treated with CSE for 6 hr and 12 hr. Controls are non-treated samples. Both primary and immortalized cells show similar patterns of p-p38MAPK, indicating the activation of these pathways. Immortalized cells show the presence of p53 protein expression proving SV40T-based immortalization. Actin was detected for loading control.

Figure 2

Characterization of hPTCCTB-derived differentiated hPTCSTB. (A) Schematic diagram of forskolin- induced cytotrophoblast cells differentiation to syncytiotrophoblast cells. (B) Representative immunofluorescent images showing cell-cell adhesion marker, E-cadherin, and pan-trophoblast marker, cytokeratin-7, for their reduction in hPTCSTB cells in comparison to hPTCCTB cells. Magnification is at 10X. Unsupervised heatmap (C) and the volcano plot (D) showing differentially expressed genes (DEGs) in comparison of hPTCCTB vs. hPTCSTB. Significant genes (adjusted P < 0.05) with log2-fold change are represented in blue dots for upregulated and in reddots for downregulated genes. Statistically non-significant differential genes (P > 0.05) are represented by gray dots. (E) GO enrichment analysis for the three main ontologies of the biological process (blue) terms, the cellular component (green) terms, and the molecular function (orange) terms inDEGs of hPTCCTB vs. hPTCSTB comparison. x-axis represent the GO terms, y-axis shows the gene number enriched in the GO terms. Bubble charts of top 20 biological processes (F) and pathwayenrichment analyses (KEGG) (G) in DEGs of hPTCCTB vs. hPTCSTB comparison. hPTCCTB, immortalized human placental cytotrophoblast cells. hPTCSTB, primary placental syncytiotrophoblast cells. DAPI, 4',6-diamidino-2-phenylindole. GO, gene ontology. KEGG, Kyoto encyclopedia of genes and genomes.

Figure 3

Comparison of trophoblast cell lines for extravillous trophoblast (EVT) differentiation. (A) Schematic diagram of differentiation of extravillious trophoblast cells with 50ng/mL of NRG1 and 2.5mM-10mM PD0325901 for 6 days. (B) Immunocytochemistry analysis was performed to validate differentiated EVT marker human leukocyte antigen-G (HLA-G) protein expression. Elongated shapes of EVT cells were pointed with yellow arrows in phase contrast images. hFM-CTC cells showedHLA-G expression however didn’t show morphological change. BeWo cells show least HLA-G expression and did not show morphological changes. (C) Representative immunofluorescent images of cytokeratin-7 protein expression in immortalized human placental trophoblast cells (hPTCEVT), human chorion trophoblast cells (CTC) and BeWo cells. NC, negative control. Scale bar, 50μm.

Figure 4

Global transcriptomic profiling of human trophoblast cell lines based onRNA seq analysis. (A) PCA plot for trophoblast cells: human placental cytotrophoblast cells (hPTCCTB), their derivative syncytiotrophoblast cells (hPTCSTB), two commercial trophoblast cell lines, CTBPRO2® (CTB_com) and BeWo; and human fetal membrane-derived chorion trophoblast cell line (hFM-CTC)1. Unsupervised heatmaps and volcano plots showing differentially expressed genes from 3 distinct PCA plot clusters’ comparisons: hPTCCTB vs. hFM-CTC (B-C) and hPTCCTBvs. BeWo (D-E). hPTCCTB, immortalized human placental cytotrophoblast cells. hPTCSTB, primary placental syncytiotrophoblast cells. CTB_com, commercially available cytotrophoblast cells. hFM-CTC, human fetal membrane-derived chorion trophoblast cells.

Figure 5

Gene ontology (GO) enrichment analysis and KEGG pathway enrichment analysis based on RNA seq-DEGs. (A-D) hPTCCTB vs. hFM-CTC transcriptomics comparison.(E-H) hPTCCTB vs. BeWo transcriptomics comparison. (I-M) hFM-CTC vs. BeWo transcriptomics comparison. GO enrichment analysis for the three main ontologies of the biological process (blue) terms, the cellular component (green) terms, and the molecular function (orange) terms in DEGscomparison. x-axis represent the GO terms, y-axis shows the gene number enriched in the GO terms (A, D, G). Bubble charts of top 20 biological processes (B, F, K) and pathway enrichment analyses (KEGG) (C, G, L) in DEGs comparison. (D, H, M) Gene set enrichment analysis (GSEA) of transcriptomics between hPTCCTB and hFM-CTC (D), hPTCCTB and BeWo (H) and hFM-CTC and BeWo (M) cells. GO, gene ontology. KEGG, Kyoto encyclopedia of genes and genomes. GSEA, gene set enrichment analysis. hPTC, immortalized human placental cytotrophoblast cells. hFM-CTC, human fetal membrane- derived chorion trophoblast cells.

Figure 6

Comparison of trophoblast cell lines for trophoblast cell-specific markers in the normal culture conditions. Representative immunofluorescence images showing proliferating cytotrophoblast cells f marker GATA3 expression (A) and typical trophoblast marker cytokeratin-7 expression (B) in normal culture condition. Scale bar, 50μm. hPTCCTB, human placental cytotrophoblast cells. hFM-CTC, human fetal membrane-derived chorion trophoblast cells. DAPI, 4',6-diamidino-2-phenylindole. NC, negative control.

Figure 7

Chorion trophoblast cellscannot differentiate into syncytiotrophoblasts, unlike placental trophoblast cells. hPTCCTB, hFMCTCand BeWo cells were treated with forskolin (20μM for BeWo, 50μM for hPTCCTB and hFMCTC)for 3 days of syncytialization. Immunocytochemistry analysis was performed to validate syncytialization with STB markers, glial cells missing transcription factor 1 (GCM1) (A), human chorionic gonadotrophin hormone (CGB) (B), and mononucleated, proliferating cell marker GATA3 protein expressions (C). GCM1 and CGB were detected abundantly in the differentiated hPTCSTB andBeWoSTB cells (orange arrows in A and B, respectively); however, barely in the hFM-CTC cells (red arrows in A and B). (C) GATA3 protein expression was decreased in differentiated hPTCSTB and BeWoSTB cells (orange arrows); however, hFM-CTC cells show unchanged expression of GATA3 indicating prevention of syncytialization in these cells. Multinucleated syncytialized cells were pointed with yellow arrows in phase contrast images (A-C). Unsupervised heatmap (D) and the volcano plot (E) showing differentially expressed genes (DEGs) in hFM-CTC cells compared to BeWoSTB cells. Significant genes (adjusted P < 0.05) with log2-fold change are represented in blue dots for upregulated and in red dots for downregulated genes. Statistically non-significant differential genes (P > 0.05) are represented by gray dots. Scale bar, 50μm. hPTCSTB, human placental syncytiotrophoblast cells. hFM-CTC, human fetal membrane- derived chorion trophoblast cells. DAPI, 4 Œ,6-diamidino-2-phenylindole. NC, negative control.