Global proteomic analyses of human cytotrophoblast differentiation/invasion

Abstract

During human pregnancy, cytotrophoblasts (CTBs) from the placenta differentiate into specialized subpopulations that play crucial roles in proper fetal growth and development. A subset of these CTBs differentiate along an invasive pathway, penetrating the decidua and anchoring the placenta to the uterus. A crucial hurdle in pregnancy is the ability of these cells to migrate, invade and remodel spiral arteries, ensuring adequate blood flow to nourish the developing fetus. Although advances continue in describing the molecular features regulating the differentiation of these cells, assessment of their global proteomic changes at mid-gestation remain undefined. Here, using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), which is a data-independent acquisition strategy, we characterized the protein repertoire of second trimester human CTBs during their differentiation towards an invasive phenotype. This mass spectrometry-based approach allowed identification of 3026 proteins across four culture time points corresponding to sequential stages of differentiation, confirming the expression dynamics of established molecules and offering new information into other pathways involved. The availability of a SWATH CTB global spectral library serves as a beneficial resource for hypothesis generation and as a foundation for further understanding CTB differentiation dynamics.

Keywords: Cytotrophoblast; Human; Placenta; Proteomics; SWATH-MS.

Fig. 1.

Global proteomic profiling of cytotrophoblast differentiation/invasion. (A) Simplified workflow schematic of cytotrophoblast proteomic analysis using SWATH-MS. (B) Distribution of relative protein abundances, spanning four orders of magnitude. (C) Tissue enrichment analysis of annotated proteins from the dataset. n=4 biological replicates.

Fig. 2.

Differentially expressed protein changes during cytotrophoblast differentiation/invasion. (A) Unsupervised hierarchical clustering of 477 differentially expressed (DE) proteins relative to 0 h. (B) Principal component analysis of DE proteins by biological replicate (n=4). (C) Distribution of DE protein expression changes at each time point relative to 0 h. (D) Gene ontology (GO) enrichment analysis of DE proteins. The most enriched categories from the three GO terms (biological process, cellular component and molecular function) were primarily matched to respiratory/metabolic processes, exosomal/vesicular components and metabolic/adhesive functions, respectively (Fisher's exact test; P<0.01, Benjamini-Hochberg corrected).

Fig. 3.

Dynamics of differentially expressed proteins during cytotrophoblast differentiation/invasion. (A) Unsupervised fuzzy clustering of 477 differentially expressed (DE) proteins identified four clusters with distinct expression profiles. n=number of proteins assigned to each cluster. (B) Heatmap of gene ontology (GO) enrichment analysis of each cluster. GO terms overrepresented in each cluster (Fisher's exact test; P<0.05, unadjusted) were visualized by fold enrichment as a qualitative comparison to functionalities in other clusters. (C) Representative GO terms enriched in clusters 1 and 2 (Fisher's exact test; P<0.05, Benjamini-Hochberg corrected) were visualized for biological processes, cellular component or molecular function (MF).

Fig. 4.

Protein expression profiles of families/complexes during cytotrophoblast differentiation/invasion. (A) Representative protein families/complexes with significant convergent expression profiles (P<0.01, distance measurement). (B) Representative protein families/complexes with significant divergent expression profiles (P<0.05, distance measurement). Asterisks indicate a protein that is significantly differentially expressed.

Fig. 5.

Selected cytotrophoblast proteins with robust expression changes. Additional cutoffs (P<0.01, absolute log 2 fold change ≥|1.5|) were applied to the differentially expressed (DE) proteins. (A) Heatmap depicting expression changes of 42 robust DE proteins relative to 0 h. (B,C) Predicted protein-protein interaction networks of enriched pathways. Robust DE proteins were visualized in Cytoscape using the STRING database, and molecules in significantly enriched pathways were separated to individual clusters (P<0.05, FDR corrected). (D-F) Representative images from second trimester tissue sections of the basal plate (BP). Sections were stained with the anti-cytotrophoblast marker cytokeratin 7 (red), with DAPI (blue) and with (D) PD-L1, (E) SQSTM1 or (F) NID1 (green). n≥4 biological replicates. Scale bars: 20 µm in D; 100 µm in E,F. AV, anchoring villi; BP, basal plate; FV, floating villi; Dec, decidua.