At birth, the human placenta is the newborn's largest organ (Pryce et al., 2014), its weight being approximately double that of the heaviest organ of the body, the brain (Boyd and Hamilton, 1967). During the last six months of pregnancy, placental weight increases over sevenfold (Boyd and Hamilton, 1967) while simultaneously supporting over a thirtyfold increase in that of the fetus (Armitage et al., 1967).

The placenta's ability to transport the very substantial resources that are needed for its own growth and that of the embryo/fetus is enabled by its unique structure. Its surface area is increased by many orders of macroscopically visible branches – the chorionic villi. At term, the total area in direct contact with maternal blood is estimated to be ∼12 m2 (Boyd, 1984). At an electron microscopic level, the syncytiotrophoblasts (STBs) that form the outer surface of the villi are covered with branched microvilli, which substantially add to the surface area, by our estimate producing at least a tenfold increase (Wislocki and Dempsey, 1955). Furthermore, the human placenta is hemochorial, i.e., in direct contact with uterine blood, minimizing the cellular barriers between the maternal source of nutrients and oxygen, and the placental vasculature that carries these substances to the embryo/fetus.

At a cellular level, human placental structure is established by differentiation of its progenitor population, villous cytotrophoblasts (CTBs; Fig. 1A,B; reviewed by Maltepe and Fisher, 2015). In one pathway, the cells fuse to produce multinucleated STBs. In the other pathway, the cells leave the placenta, forming bridges (termed cell columns) that connect to the uterus and are the conduit for CTBs that invade its wall. The process is accompanied by a dramatic phenotypic switch in which the formerly epithelial cells adopt many vascular properties (Damsky and Fisher, 1998). Remarkably, these cells also breach uterine vessels that lie in their path. They penetrate the end of veins and migrate in a retrograde fashion up spiral arteries, the walls of which they occupy throughout much of their intrauterine segments. The latter process establishes the funnel-like structure of spiral arteries that reduces the flow rate and increases the volume of maternal blood perfusing the placenta.

Fig. 1.

Human placenta trophoblast subtypes targeted for laser microdissection. (A) Diagram of the human placenta in the second trimester of pregnancy. The boxed area (B) indicates the region biopsied for these studies. (B) View of the maternal-fetal interface at the cellular level. Shown are the two classes of chorionic villi. Floating villi (FV), which are covered by multinucleated syncytiotrophoblasts (STB), transport substances between the mother and the embryo/fetus while producing growth factors and hormones essential to the unique symbiosis of pregnancy. Anchoring villi (AV), contain mononuclear cytotrophoblast progenitors that either fuse to form STBs or exit the placenta proper through cell columns (CCs) that attach the embryo/fetus to the uterus. Endovascular cytotrophoblasts mediate an increase in the terminal diameter of the maternal spiral arteries, enabling increased blood flow to the placenta. The arrow indicates the direction of trophoblast invasion (modified from Damsky et al., 1994). (C-G) Photomicrographs taken before and after STB, CTB and ENDO were isolated using LMD. The sections were stained with Toluidine Blue, which enabled visualization of the cellular architecture. The dotted lines indicate the three trophoblast-containing regions that were targeted. SA, spiral artery.

Fig. 1.

Human placenta trophoblast subtypes targeted for laser microdissection. (A) Diagram of the human placenta in the second trimester of pregnancy. The boxed area (B) indicates the region biopsied for these studies. (B) View of the maternal-fetal interface at the cellular level. Shown are the two classes of chorionic villi. Floating villi (FV), which are covered by multinucleated syncytiotrophoblasts (STB), transport substances between the mother and the embryo/fetus while producing growth factors and hormones essential to the unique symbiosis of pregnancy. Anchoring villi (AV), contain mononuclear cytotrophoblast progenitors that either fuse to form STBs or exit the placenta proper through cell columns (CCs) that attach the embryo/fetus to the uterus. Endovascular cytotrophoblasts mediate an increase in the terminal diameter of the maternal spiral arteries, enabling increased blood flow to the placenta. The arrow indicates the direction of trophoblast invasion (modified from Damsky et al., 1994). (C-G) Photomicrographs taken before and after STB, CTB and ENDO were isolated using LMD. The sections were stained with Toluidine Blue, which enabled visualization of the cellular architecture. The dotted lines indicate the three trophoblast-containing regions that were targeted. SA, spiral artery.

The cellular structure of the human placenta makes isolation of the various trophoblast subtypes difficult to impossible. Upon enzyme dissociation, the syncytium disintegrates and extravillous CTBs are only a small fraction of the mononuclear cells that can be isolated. To solve this problem, we have used laser microdissection (LMD) to capture STBs and column, endovascular or smooth CTBs as well as decidual cells for RNA profiling in the context of severe preeclampsia (Garrido-Gomez et al., 2017; Gormley et al., 2017). Here, we used this approach to study gene expression during the 2nd trimester of normal pregnancy. The results revealed numerous molecules that were not previously known to be expressed by STBs and/or CTBs. Among them was the cannabinoid receptor 1 (CNR1; also known as CB1), which was highly expressed by the endovascular CTB subpopulation. We went on to show that the downstream signals can influence invasion.

Blocks were prepared from biopsies of the 2nd trimester (15-20 weeks gestation) maternal-fetal interface. The focus was on areas that by macroscopic inspection included spiral arteries with dilated termini, a sign of CTB invasion. LMD enabled the capture of syncytium, cell columns and the endovascular compartment. The experimental strategy, which we used previously in an analysis of the same cell types from severe preeclampsia (Gormley et al., 2017), is depicted in Fig. 1. The dashed lines drawn on the photomicrographs to the left show the trophoblast subpopulations that were targeted for removal as shown to the right: STBs (Fig. 1C,D), cell column CTBs (CC; Fig. 1E,F), and the endovascular compartment (ENDO), containing CTBs and other cell types in spiral arteries (SA; Fig. 1G,H).

Global profiling of RNA isolated from the samples and pair-wise comparisons of the results revealed 2986 genes that were differentially expressed (DE; adjP-value <0.05) by one of the trophoblast subtypes. Principal component analysis (PCA; Fig. S1) showed that data from the same cell type clustered together separate from the other samples. Thus, consistent with their different functions, the various trophoblast subtypes analyzed had distinct transcriptomes.

Compared with the two CTB subpopulations, 2708 genes were differentially expressed in STBs (Fig. 2A; see Fig. S2 for entire heatmap and fold changes). As expected, the upregulated category included genes encoding growth-promoting molecules (e.g. GH2, AREG, CSHL1, INSL4), numerous transporters (e.g. SLC27A2, SLC26A7), ion channels (e.g. TRPV6, SCN7A) and syncytin 2 (ERVFRD-1). The transcript for MEOX2 was also differentially expressed in STBs, suggesting the possibility that this homeobox gene plays a role in fate specification or maintenance of this cell type. Unexpectedly, the expression of LGR5, which encodes a cell surface molecule that marks stem cells (e.g. intestinal and colonic; Leung et al., 2018), was unique to STBs. Of interest was detection of the mRNA encoding neurotensin (NTS), raising the possibility that this small neuropeptide is released from the surface of the placenta into maternal blood during pregnancy, following which it seems likely that enteric and other effects are possible (Bugni and Pothoulakis, 2013).

Fig. 2.

Heatmaps of differentially expressed STB, CTB and ENDO RNAs. (A) The 25 most highly upregulated and 25 most highly downregulated transcripts in syncytiotrophoblasts (STB) compared with cytotrophoblasts (CTB) and endovascular cytotrophoblasts (ENDO) (total=2708, Table S2). (B) Heatmap of the most highly differentially expressed transcripts in CTB (total=2070, Table S2) relative to STB and ENDO. (C) Heatmap of the most highly differentially expressed transcripts in ENDO (total=2753, Table S2) relative to STB and CTB. The data are displayed as a heatmap showing the relative RNA fold change as a variation in color from blue (decreased expression) to white (average expression) to red (increased expression). Cells captured from four placentas were analyzed.

Fig. 2.

Heatmaps of differentially expressed STB, CTB and ENDO RNAs. (A) The 25 most highly upregulated and 25 most highly downregulated transcripts in syncytiotrophoblasts (STB) compared with cytotrophoblasts (CTB) and endovascular cytotrophoblasts (ENDO) (total=2708, Table S2). (B) Heatmap of the most highly differentially expressed transcripts in CTB (total=2070, Table S2) relative to STB and ENDO. (C) Heatmap of the most highly differentially expressed transcripts in ENDO (total=2753, Table S2) relative to STB and CTB. The data are displayed as a heatmap showing the relative RNA fold change as a variation in color from blue (decreased expression) to white (average expression) to red (increased expression). Cells captured from four placentas were analyzed.

With regard to cell column CTBs, 2070 genes were differentially expressed in this subpopulation (Fig. 2B; see Fig. S2 for entire heatmap and fold changes). Receptors for metabolites were the most highly upregulated. They included FABP7a. We have previously described CTB expression of this molecule at the maternal-fetal interface (Winn et al., 2007). The alpha-ketoglutarate receptor (OXGR1) was highly expressed. This result suggested that CTBs might be using metabolic products in the basal plate as an energy source, which could couple the initial stages of invasion to maternal metabolism. As shown by our previous work (reviewed by Maltepe and Fisher, 2015), molecules that are involved in adhesion/de-adhesion were upregulated in columns (ITGB6, COL17A1, ACAN, VIT). An mRNA encoding troponin I (TNNI2), a protein involved in muscle contraction, was DE, possible evidence of its involvement in movement of these cells through the columns and into the uterus. In accord with the dramatic phenotypic changes the cells undergo, the DNA demethylase TET1 was upregulated, suggesting that alterations at the level of the epigenome might play a role in specifying the fate of this CTB population.

Overall, 2753 genes were differentially expressed in the endovascular compartment (Fig. 2C; see Fig. S2 for entire heatmap and fold changes). The most highly upregulated genes included the BMP4 antagonist CHRDL1, which mitigates migration and invasion of breast cancer cells (Cyr-Depauw et al., 2016), the secretory leukocyte peptidase inhibitor SLPI, which plays a role in host defense (Majchrzak-Gorecka et al., 2016) and a calcium channel involved in cell signaling (TRPC4), which promotes vessel relaxation and permeability (Freichel et al., 2004). We detected expression of molecules that could control leakage of blood from modified spiral arteries (FGG, CP) and the complement cascade (CFH, C3). To the best of our knowledge, this list included previously unseen identifications: OMD, RORB and MEDAG. CNR1 was also in this category, which raised the possibility that endogenous and exogenous cannabinoids could impact CTB invasion and remodeling of the uterine vasculature.

Finally, we detected expression of mRNAs that were indicative of contamination with other cell types, not unexpected because endovascular CTBs lie adjacent to the decidua, which expresses relaxin (RLN1) (Goldsmith and Weiss, 2009) and PRL (Jones et al., 1998), which we detected. NK cells infiltrate the walls of spiral arteries (Moffett and Colucci, 2015), which we found evidence for in the expression of molecules these immune cells produce, including GZMA, KLRC1 and KLRC3. We also detected molecules expressed by T cells (TRDJ2, TRDC).

In parallel, we have been developing a mass spectrometry (MS)-based proteomic method for analyzing the same trophoblast subpopulations, captured by LMD, which were the focus of the transcriptomic analysis. Given that typical estimates of correlations between mRNA and protein expression are below 50% (Maier et al., 2009; Vogel and Marcotte, 2012), we were interested in those that were significant at both levels, making them more likely to have functional effects. For these experiments, we employed a shotgun proteomic approach in which the number of times a peptide is identified enables relative quantification of the parent protein (i.e. spectral counting; Zhang et al., 2013). In all, we showed that 108 differentially expressed mRNAs had similar relative protein abundances within STB, CTB or ENDO (Fig. S3).

Examples of these data are shown in Fig. 3. The upper left hand panel illustrates the labeling scheme, which was subsequently omitted for the sake of simplification. Data are organized in columns. Molecules that were upregulated in STB are shown to the left, CTB in the middle and ENDO to the right. Each cell type is designated by a different bisected symbol (square, circle or diamond). Left or right shading denotes RNA or protein values, respectively, which is colored (green, blue or red) to aid interpretation.

Fig. 3.

Plots of relative RNA and protein abundances in trophoblast subtypes. The upper left hand panel illustrates the labeling scheme. Data are organized in columns. Molecules that were upregulated in STBs are shown to the left, cell column CTBs in the middle and endovascular CTBs to the right. Each cell type is designated by a different bisected symbol (square, circle or diamond). Left or right shading denotes RNA or protein values, respectively, which is colored (green, blue or red) to aid interpretation. RNA data were from four biological replicates, protein data were from three biological replicates.

Fig. 3.

Plots of relative RNA and protein abundances in trophoblast subtypes. The upper left hand panel illustrates the labeling scheme. Data are organized in columns. Molecules that were upregulated in STBs are shown to the left, cell column CTBs in the middle and endovascular CTBs to the right. Each cell type is designated by a different bisected symbol (square, circle or diamond). Left or right shading denotes RNA or protein values, respectively, which is colored (green, blue or red) to aid interpretation. RNA data were from four biological replicates, protein data were from three biological replicates.

With regard to STBs (Fig. 3, left column), CSHL1 was the most highly expressed differentially expressed molecule at both mRNA and protein levels. We validated translation of an open reading frame (chromosome 4 open reading frame 36; C4ORF36). High placental levels of this mRNA (ENSG00000163633) have been reported (Fagerberg et al., 2014). Its fungal protein analog (MGG_01005) is dynein light chain Tctex-type 1 (dynlt1/3; Li et al., 2018), which transports various types of cellular cargo. CYP19A1 (aromatase) and GH2 (growth hormone 2) were also expressed at higher levels in the syncytium.

With regards cell column CTBs (Fig. 3, middle column), the highly-expressed differentially expressed molecules included several identifications that, to our knowledge, were not known to be produced by the placenta. They included pannexin 1 (a major ATP release and nucleotide eflux channel; Chekeni et al., 2010; Elliott et al., 2009), pyroglutamyl-peptidase I (PGPEP1; an exopeptidase; Cummins and O'Connor, 1998) and lymphocyte antigen 6K (LY6K; a biomarker of lung and esophageal carcinomas; Ishikawa et al., 2007). We were also interested to find that a sialyltransferase (alpha-n-acetylgalactosaminide alpha-2,6-sialyltransferase 6; Samyn-Petit et al., 2000) was upregulated in this CTB subpopulation. Its identification may be related to the observation that placental proteins carry unusual forms of glycosylation not found on the same molecules produced by other cell types (McMaster et al., 1998).

With regard to the endovascular compartment (Fig. 3, right column), the highly-expressed differentially expressed molecules included the hydrolase acylphosphatase 2, and fibulin 2, a secreted extracellular matrix glycoprotein that can stabilize the basement membranes of epithelial cells (Ibrahim et al., 2018). We also confirmed the differential expression of immune molecules: butyrophilin 3A2, which can inhibit T-cell activity, as do other members of this family (Messal et al., 2011; Rhodes et al., 2016) and galectin 3, which has numerous other functions, including a role in cell adhesion (Pugliese et al., 2015) and epithelial-stromal signaling (AbuSamra et al., 2019).

Next, we used Ingenuity Pathways Analysis (IPA) to infer functional similarities and differences among the trophoblast subtypes. Pathways were assigned a z-score based on the predicted impact of differentially expressed genes (DEGs), either activation (+) or deactivation (−). Those shown in Fig. 4 were significantly over-represented (either positively or negatively) in all of the trophoblast subpopulations. Again, there was significant overlap between the in silico predictions of the pathways that were activated in STB and ENDO versus column CTBs. It seems possible that this convergence could be driven, in part, by the fact that these two regions are in direct contact with maternal blood. In keeping with this theory, many of the pathways identified as potentially activated were involved in responding to signals from circulating molecules: corticotropin releasing hormone, relaxin, renin, angiotensin, NO, adrenomedullin, fatty acids (via PPAR, RXR), purines and pyrimidines (via P2Y receptors) or growth hormone. We also found potential positive regulation of pathways that are involved in neuronal functions, including synaptogenesis and CREB signaling. The STB and column CTB data suggested that these cells may be able to respond to cannabinoids; cell columns and endovascular compartments upregulated the expression of genes that are involved in TH2 (tolerogenic) responses. There were a large number of unique pathways that were predicted to be activated solely by the endovascular trophoblasts and other cells types of the remodeled spiral arteries (ENDO). The putative pathways were involved in immune functions (acute phase signaling, dendritic cell maturation, IL6, Tec kinase signaling or osteoarthritis), cell movement (colorectal cancer metastasis as well as G12/13, integrin and ILK signaling), MAPK signaling, vascular biology (cardiac hypertrophy) and prolactin responses.

Fig. 4.

Ingenuity Pathway Analysis of the differentially expressed genes among trophoblast subpopulations. Pathways were assigned a z-score based on the predicted impact of DEGs, either activation (+) or deactivation (−). Significant pathways had a −log(P-value)>1.3, an absolute z-score>2, and contained at least four members in a unique combination. The data are displayed as a heatmap showing the z-score as a variation in color from blue (decreased expression) to red (increased expression) for each trophoblast subtype. The results were notable for the number signaling pathways that were identified.

Fig. 4.

Ingenuity Pathway Analysis of the differentially expressed genes among trophoblast subpopulations. Pathways were assigned a z-score based on the predicted impact of DEGs, either activation (+) or deactivation (−). Significant pathways had a −log(P-value)>1.3, an absolute z-score>2, and contained at least four members in a unique combination. The data are displayed as a heatmap showing the z-score as a variation in color from blue (decreased expression) to red (increased expression) for each trophoblast subtype. The results were notable for the number signaling pathways that were identified.

Given that the gene expression data were specific to various trophoblast subtypes, we were interested in showing that the differentially expressed molecules had the expected immunolocalization patterns. With regard to STBs, we chose neurotensin, a small peptide gut hormone with brain and nervous system effects including analgesia (Cordomi et al., 2016; Pérez de Vega et al., 2018; Tschumi and Beckstead, 2019), which to our knowledge was not known to be produced by the human placenta. The immunolocalization signal for this molecule (Fig. 5A-C) was detected as a vesicular pattern in the syncytium at higher density in the apical region of the cells, suggesting possible release into the maternal circulation. In addition, we were interested in the expression of C4orf36, also in STBs, the peptide product of which was detected by mass spectrometry. Immunostaining revealed a dense punctate pattern in STBs (Fig. 5D-F). Within the cell columns, we confirmed the expression of PGPEP1 by the cytotrophoblasts in that region (Fig. 5G-I).

Fig. 5.

Immunolocalization of differentially expressed proteins: NTS, C4ORF36 and PGPEP1. Tissue sections were immunostained for the antigen of interest and cytokeratin (CK), a trophoblast marker. Nuclei were localized with DAPI. Each panel was a confocal z-stack maximum intensity projection. (A-C) Neurotensin (NTS) was detected in a vesicular pattern localized to the apical region of syncytiotrophoblasts (STB). Within any one tissue section the vesicle density varied among regions, although no areas lacked immunoreactivity. Furthermore, no differences based on gestational age were observed. (D-F) C4ORF36, which localized to STBs, had a uniformly dense punctate pattern. No variation was observed across the second trimester. (G-I) PGPEP1 was highly upregulated in cytotrophoblast columns (CC) of anchoring villi (AV). The results shown were represented of the immunostaining observed in a minimum of three biological replicates.

Fig. 5.

Immunolocalization of differentially expressed proteins: NTS, C4ORF36 and PGPEP1. Tissue sections were immunostained for the antigen of interest and cytokeratin (CK), a trophoblast marker. Nuclei were localized with DAPI. Each panel was a confocal z-stack maximum intensity projection. (A-C) Neurotensin (NTS) was detected in a vesicular pattern localized to the apical region of syncytiotrophoblasts (STB). Within any one tissue section the vesicle density varied among regions, although no areas lacked immunoreactivity. Furthermore, no differences based on gestational age were observed. (D-F) C4ORF36, which localized to STBs, had a uniformly dense punctate pattern. No variation was observed across the second trimester. (G-I) PGPEP1 was highly upregulated in cytotrophoblast columns (CC) of anchoring villi (AV). The results shown were represented of the immunostaining observed in a minimum of three biological replicates.

To begin to explore the role of CTB CNR1 within the uterine wall, we confirmed the cell expression of this receptor by using antibody-based methods. Immunolocalization with an antibody that was specific for this antigen showed binding to a subset of cytokeratin (CK)-positive CTBs within the uterine wall and in the endovascular compartment (Fig. 6A-C). Immunoblotting with the same antibody detected a band of the expected molecular weight in CTB lysates (