The trophoblast cell-specific proteome
The main cellular components of the highly vascularized placenta are trophoblasts, decidual cells, endothelial cells and primitive mesenchymal cells. Together they mediate fetal nutrient uptake, waste elimination, and gas exchange via the maternal blood supply. Trophoblasts are the cells that surround the fertilized egg, also known as the blastocyst. There are three types of trophoblasts: cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts, each responsible for different parts of the placental development during pregnancy. Transcriptome analysis shows that 62% (n=12111) of all human proteins (n=19670) are detected in trophoblast cells and 1342 of these genes show an elevated expression in any trophoblast cells compared to other cell type groups.
The trophoblast cell transcriptome
The scRNA-seq-based trophoblast cell transcriptome can be analyzed with regard to specificity, illustrating the number of genes with elevated expression in each specific trophoblast cell type compared to other cell types (Table 1). Genes with an elevated expression are divided into three subcategories:
Protein expression of genes elevated in trophoblast cells
In-depth analysis of the elevated genes in trophoblast cells using scRNA-seq and antibody-based protein profiling allowed us to visualize the expression patterns of these proteins in different types of trophoblast cells: cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts.
As shown in Table 1, 451 genes are elevated in cytotrophoblasts compared to other cell types. Cytotrophoblasts are considered to be trophoblastic stem cells since the layer surrounding the blastocyst remains the same while the daughter cells differentiate and proliferate to function in several different roles. When the placenta begins to develop the blastocyst (fertilized egg) is implanted in the endometrium. This process is enabled by cytotrophoblast cells that secrete enzymes enabling the embryo to get fully covered by the endometrial epithelium. Cytotrophoblasts make up the external layer of the blastocyst as well as the internal layer of the placental trophoblast cell layer. Examples of proteins with elevated expression in cytotrophoblasts include the highly conserved paternally expressed 10 (PEG10), which is involved in cell proliferation, differentiation and apoptosis, and PAGE family member 4 (PAGE4), with function in transcription regulation.
As shown in Table 1, 760 genes are elevated in Syncytiotrophoblasts compared to other cell types. Syncytiotrophoblasts form when undifferentiated and highly proliferative cytotrophoblasts fuse together. Syncytiotrophoblasts are specialized epithelial cells that cover the floating placental villi and are involved in maintaining pregnancy through the production of growth factors and hormones. Since the syncytiotrophoblasts are in direct contact with the maternal blood, these cells are closely involved in the exchange of gas, nutrients and waste between the mother and the fetus. Examples of proteins elevated in syncytiotrophoblasts include chorionic somatomammotropin hormone 2 (CSH2), a hormone only produced during pregnancy, involved in stimulating lactation, fetal growth and metabolism, and the protein called glycoprotein hormones, alpha polypeptide (CGA), a subunit in various hormones affecting receptor binding on target cells as well as activation of downstream signaling pathways.
As shown in Table 1, 648 genes are elevated in Extravillous trophoblasts compared to other cell types. Extravillous trophoblasts are formed when cytotrophoblasts proliferate to form anchoring villi that attach to the uterine wall. From the anchoring villi, extravillous trophoblasts form by detaching from the placental villi and migrating before penetrating the decidualized uterus. This is essential for both altering vascularisation, allowing steady, adequate blood supply to the growing fetus, as well as physically attaching the placenta to the mother's uterine wall. Examples of proteins with elevated expression in extravillous trophoblasts include major histocompatibility complex, class I, G (HLA-G), which plays a central role in the establishment of immune tolerance during pregnancy and pappalysin 2 (PAPPA2), a metalloproteinase thought to be a local regulator of insulin-like growth factor.
Trophoblast cell function
The placenta is made up of various kinds of cells, mainly stemming from trophoblasts. Upon proliferation and differentiation around the embedded blastocyst, trophoblasts start developing into what ultimately forms the different parts of the placenta. There are three types of trophoblasts: cytotrophoblasts, syncytiotrophoblasts and extravillous trophoblasts, each responsible for different parts of the placental development during pregnancy.
Cytotrophoblasts are considered to be trophoblastic stem cells since the layer surrounding the blastocyst remains the same while the daughter cells differentiate and proliferate to function in several different roles. When the placenta begins to develop the blastocyst (fertilized egg) is implanted in the endometrial wall, whereafter the trophoblasts start developing rapidly, eventually forming two distinct layers of cells known as cytotrophoblasts and syncytiotrophoblasts. Cytotrophoblasts secrete enzymes enabling the embryo to get fully covered by the endometrial epithelium. If the cytotrophoblast function is inadequate, implantation of the blastocyst will most likely fail.
The syncytiotrophoblast layer extends over the placental surfaces of all villous trees, completely lining the intervillous space. It is easy to identify during the first half of pregnancy, whereafter the nuclei start grouping up and the layer becomes thinner to reduce the diffusion distance between fetal and maternal blood. The syncytiotrophoblast layer is in direct contact with the maternal blood and constitutes the site with the highest metabolic and endocrine activity. The main functions of the syncytiotrophoblast layer are transfer and control of nutrients and gases, resynthesis of proteins and lipids, and transport of electrolytes and amino acids between mother and child.
When the cytotrophoblasts start proliferating they eventually form so-called anchoring villi that attach to the uterine wall. From the anchoring villi, extravillous trophoblasts form by migrating trophoblasts detaching from the placental villi before penetrating the decidualized uterus. This is essential for both altering vascularisation, allowing steady, adequate blood supply to the growing fetus, as well as physically attaching the placenta to the mother's uterine wall during pregnancy.
The histology of organs that contain trophoblast cells, including interactive images, is described in the Protein Atlas Histology Dictionary.
Here, the protein-coding genes expressed in trophoblast cells are described and characterized, together with examples of immunohistochemically stained tissue sections that visualize corresponding protein expression patterns of genes with elevated expression in different trophoblast cell types.
The transcript profiling was based on publicly available genome-wide expression data from scRNA-seq experiments covering 13 different normal tissues, as well as analysis of human peripheral blood mononuclear cells (PBMCs). All datasets (unfiltered read counts of cells) were clustered separately using louvain clustering and the clusters obtained were gathered at the end, resulting in a total of 192 different cell type clusters. The clusters were then manually annotated based on a survey of known tissue and cell type-specific markers. The scRNA-seq data from each cluster of cells was aggregated to average normalized protein-coding transcripts per million (pTPM) and the normalized expression value (nTPM) across all protein-coding genes. A specificity and distribution classification was performed to determine the number of genes elevated in these single cell types, and the number of genes detected in one, several or all cell types, respectively.
It should be noted that since the analysis was limited to datasets from 13 organs only, not all human cell types are represented. Furthermore, some cell types are present only in low amounts, or identified only in mixed cell clusters, which may affect the results and bias the cell type specificity.
Relevant links and publications
Uhlén M et al., Tissue-based map of the human proteome. Science (2015)