Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions

Abstract

Introduction: Trophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells.

Methods: After enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT.

Results: The trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR.

Conclusion: We verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

Keywords: HLA-G; culturing; differentiation; immune cell; placenta; pregnancy; stem cell; trophoblast.

Figure 1

Macroscopic and microscopic pictures of trophoblast cells. (A) Cultured trophoblasts from a representative experiment (donor QH1) are shown. CTB were evaluated by (B) light microscopy or (C) after May Grunwald-Giemsa staining. The cells form a dense cluster, are rounded in structure and adhere to the plate. (D) Cells at the EVT stage have a more spindle-shaped appearance.

Figure 2

Cell frequencies by flow cytometry after enrichment and during CTB culturing. (A) Representative gating strategy from one donor (RC2), to select singlet cells by forward-sideward scatter that were viable (7-AAD^neg) and non-hematopoietic (CD45^neg). (B) Representative flow cytometry dot blots and histogram plots of trophoblasts from one donor (RC2), after cell enrichment but before culturing (upper panel) and during culturing in CTB stem cell medium (lower panel). (C) Mean cell frequencies from seven donors showing the positivity for each of the markers of interest, both after cell enrichment but before culturing (left graph) and during culturing in CTB stem cell medium (right graph).

Figure 3

Expression of classical trophoblast markers in primary trophoblast cultures. (A) Messenger RNA expression of GATA3, TFAP2c, ELF3, and ELF5 (n = 6 primary cultures; n = 2 MSC cultures). (B) Approximately 750 microRNA were screened in primary CTB/EVT (n = 3), along with JAR, JEG-3, and two MSC lines. Out of these, 116 microRNAs were included for hierarchical clustering. Twenty-five C19 microRNA (green boxes) were typically expressed in trophoblasts. (C) Expression level of five typical C19 microRNAs (open bars) and average level of all C19 microRNAs per group (black bars). All graphs are represented by means with SD.

Figure 4

Expression of cell adhesion and epithelial markers in primary trophoblast cultures. (A) Expression of cell adhesion and trophoblast markers at the RNA level by culture days. Cultures (n = 7) were analyzed at the CTB and EVT phase for mRNA expression of ITGA1, ITGA5, ITGA6, TEAD4, EPCAM, and CGA. (B) Representative gating strategy from one donor (RC2), to select singlet cells by forward-sideward scatter that were viable (7-AAD^neg). The figure shows dot blots of the markers studied in CTB (upper two panels) and EVT after 6 days of differentiation (lower two panels). (C) Expression of cell adhesion and epithelial markers at the protein level by culture days. Cultures (n = 4) were analyzed by flow cytometry at the CTB and EVT phase for surface expression of integrins, CDH1, and three members of the ErbB family.

Figure 5

Expression of HLA-C in trophoblasts. (A) Messenger RNA expression of HLA-C according to the HLA-C alleles. RNA from CTB cultures (n = 7) was analyzed by quantitative PCR using primer sets that are specific for different HLA-C alleles. The vertical axes represent relative mRNA expression of HLA-C alleles, corrected for the average signal of two reference genes (GAPDH, ACTB). The graph shows means with SEM of two independent experiments per culture. (B) HLA-C mRNA expression was determined by quantitative PCR using generic primers that targeted all alleles (left panel). HLA-C was also studied by flow cytometry using antibodies against pan-HLA class I (right panel). (C) Messenger RNA expression of HLA-A, -B, -C, -DRB1, and -G (left panel) was studied in CTB and EVT cultures. RNA was analyzed by quantitative PCR using locus-specific primer sets. The graph shows means with SEM. Surface expression of individual HLA-A and HLA-B alleles was investigated by flow cytometry using human monoclonal antibodies on four blood cell samples and two trophoblast cultures (right panel). The graph displays medians with interquartile ranges.

Figure 6

Expression of HLA-G in trophoblasts. (A) HLA-G mRNA expression was determined by quantitative PCR using generic primers that targeted all alleles (left panel). HLA-G was also studied by flow cytometry using antibodies against HLA class I and two epitopes on HLA-G1 and -G5 isoforms (MEM-G9 and 87G), respectively (middle and right panel). (B) HLA-G1, G2, and G3 isoforms were separately studied by using isoform-specific primer sets (annealing locations indicated by black bars in the left panel). The results in the right panel are displayed as means with SD of n = 4 primary cultures. (C) HLA-G expression according to HLA-G 3’-UTR haplotype. Messenger RNA expression of HLA-G from seven CTB cultures was analyzed by quantitative PCR (corrected for the average signal of reference genes GAPDH and ACTB; left panel), whereas information of HLA-G surface expression by flow cytometry against MEMG9 was available for six CTB cultures (right panel). Expression data were grouped according to haplotype: UTR-1 (n = 4), UTR-2 (n = 2), UTR-4 (n = 2) or UTR-7 (n = 1). The graphs show floating bars (min to max with line at median). (D) Soluble HLA-G levels were determined by ELISA in supernatant of CTB (n = 3), EVT (n = 3), JEG-3 and JAR cell lines, and MSC (n = 2). The graph shows medians with the whiskers representing minimum and maximum values.

Figure 7

Cytokine secretion by trophoblasts in the culture medium. The level of 20 different cytokines and growth factors was analyzed by Luminex analysis in the medium from four cultures (QG1, QG2, RG3, and QH1 in triplicate). IL-2, IL-5, IL-6, IL-8, and TNF-α were detected above the minimum threshold value and are shown in this figure. The graph shows medians and the whiskers representing minimum and maximum values.

Figure 8

Expression of activation markers on CD4⁺ and CD8⁺ T cells following co-culturing of lymphocytes with EVT. First, EVT (culture QH1) were differentiated for six days. Then, lymphocytes from four different donors were added to the EVT for three days. HLA-DR and CD69 were studied by flow cytometry in both the CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell population, both in the absence of EVT (first two columns) or in the presence of EVT (third column). The graph shows means with SEM. *P < 0.05.