What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast

Abstract

Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast.

Figure 1

Features of Primary Trophoblast Cells (A) Expression of KRT7, TFAP2C, and GATA3 are good markers for mononuclear trophoblast (n = 6 donors) (gestational age 8–12 weeks). ST, syncytiotrophoblast; VCT, villous cytotrophoblast; EVT, extravillous cytotrophoblast. Scale bar, 100 μm. (B) Methylation status of individual CpG sites at the ELF5 promoter in VCT and EVT isolated by flow cytometric sorting (Figure S1A), compared with placental mesenchymal cells (PMC). Percentages show the proportion of methylated (closed circles) to non-methylated (open circles) CpG sites (n = 8 data points for each CpG per donor, samples from two donors) (results from one donor shown; both showed similar results). (C) Expression of four C19MC miRNAs in choriocarcinoma cell lines (JAR, JEG-3), primary trophoblast (M25T, M26T, M27T) (Figure S1B), embryonal carcinoma (EC) lines (2102Ep, NCCIT), hESC (H9 hESC), seminoma (TCam2), yolk sac tumor (GCT44, 1411H), and gonads (ovary, testes) (n = 3 independent experiments). Results are normalized to levels of miR-103a and plotted against the expression level for JAR cells. Normalized results are multiplied 10,000–100,000× to ensure all logged values are positive. Red solid line: 2102Ep levels; red dotted line: hESC levels. Error bars represent SE. ND, not detectable.

Figure 2

Characteristics of First Trimester Mononuclear Trophoblast Cells Flowchart depicting the characteristics of mononuclear trophoblast cells from first-trimester placentas. +ve, positive; -ve, negative. VCT, villous cytotrophoblast; EVT, extravillous cytotrophoblast.

Figure 3

2102Ep Cells Are Unlike Primary Trophoblast Cells (A) 2102Ep cells were stained by immunocytochemistry for GATA3, TFAP2C, and KRT7 with JEG-3 cells as a positive control. Flow cytometry confirmed that the few cells staining positive for KRT7 (arrows) were dead (data not shown) (n = 3 independent experiments). Scale bar, 200 μm. (B) Methylation status of the ELF5 promoter in 2102Ep EC (closed circles, methylated CpG; open circles, non-methylated CpG). (C) HLA profile of 2102Ep (n = 3 independent experiments). (D) Positive controls for HLA-G and HLA-B7 staining were JEG-3 and 721.221-HLA-B7, respectively.

Figure 4

BAP- and FGF-Treated hESC Are Unlike Primary Trophoblast Cells (A) Co-immunofluorescence of KRT7, TFAP2C, and GATA3 in H1 and CA1 hESC (n = 3 independent experiments). Scale bar, 100 μm. (B) Expression levels of POU5F1, NANOG, TFAP2C, GATA3, and ELF5 transcripts in BAP- and FGF2-treated hESC and JEG-3 cells (n = 3 independent experiments). Error bars represent SE. ND, not detectable. Assessed using paired two-tailed Student's t test. ^∗p ≤ 0.05, ^∗∗p ≤ 0.01, ^∗∗∗p ≤ 0.001, ^∗∗∗∗p ≤ 0.0001. (C) Methylation status of the ELF5 promoter in BAP or FGF2-treated CA1 hESC (closed circles, methylated CpG; open circles, non-methylated CpG). (D) Pyrosequencing shows that the ELF5 promoter in BAP-treated H1 hESC cells is also hypomethylated, compared with FGF-treated H1 hESC. See also Figure S2.

Figure 5

Expression of C19MC miRNAs and HLA Class I Molecules on BAP- and FGF-Treated hESC (A) The expression of four C19MC miRNAs is compared between BAP- and FGF-treated hESC controls. The positive control, JEG-3 cells, show C19MC levels characteristic of normal trophoblast. Mammary gland is included as negative control (n = 3 independent experiments). Error bars represent SE. ND, not detectable. (B–E) HLA class I expression by 2102Ep EC and BAP-treated or control FGF-treated hESC. mAb W6/32 detects all HLA class I molecules (B), HLA-G (C), HLA-A (D), and HLA-B (E) on BAP-treated and FGF2-treated cells (n = 3 independent experiments).