CD90 Marks a Mesenchymal Program in Human Thymic Epithelial Cells In Vitro and In Vivo

Abstract

Thymic epithelium is critical for the structural integrity of the thymus and for T cell development. Within the fully formed thymus, large numbers of hematopoietic cells shape the thymic epithelium into a scaffold-like structure which bears little similarity to classical epithelial layers, such as those observed in the skin, intestine or pancreas. Here, we show that human thymic epithelial cells (TECs) possess an epithelial identity that also incorporates the expression of mesenchymal cell associated genes, whose expression levels vary between medullary and cortical TECs (m/cTECs). Using pluripotent stem cell (PSC) differentiation systems, we identified a unique population of cells that co-expressed the master TEC transcription factor FOXN1, as well as the epithelial associated marker EPCAM and the mesenchymal associated gene CD90. Using the same serum free culture conditions, we also observed co-expression of EPCAM and CD90 on cultured TECs derived from neonatal human thymus in vitro. Single cell RNA-sequencing revealed these cultured TECs possessed an immature mTEC phenotype and expressed epithelial and mesenchymal associated genes, such as EPCAM, CLDN4, CD90 and COL1A1. Importantly, flow cytometry and single cell RNA-sequencing analysis further confirmed the presence of an EPCAM+CD90+ population in the CD45- fraction of neonatal human thymic stromal cells in vivo. Using the human thymus cell atlas, we found that cTECs displayed more pronounced mesenchymal characteristics than mTECs during embryonic development. Collectively, these results suggest human TECs possess a hybrid gene expression program comprising both epithelial and mesenchymal elements, and provide a basis for the further exploration of thymus development from primary tissues and from the in vitro differentiation of PSCs.

Keywords: CD90/Thy1; cell identity; epithelial and mesenchymal components; human thymic epithelial cells; pluripotent stem cell differentiation; primary cells culture.

Figure 1

Human pluripotent stem cell differentiation identifies a FOXN+EPCAM+CD90+ population. (A) Flow cytometry analysis of PSC-derived endodermal cultures for the expression of CD90, EPCAM, FOXN1:GFP and CD104. Data shows representative results of two independent PSC lines: HES3 and MEL1. (B) RNA-sequencing analysis of GFP+EPCAM+CD90+ (pos) and GFP+EPCAM+CD90- (neg) fractions showing the relationships between individual samples representing each population in a multi-dimensional scaling (MDS) plot. (C) Histogram representation of the expression of thymic associated genes, NOTCH ligand genes and collagens. The Y axis shows expression in counts per million (CPM) for GFP+EPCAM+CD90+ (CD90+) and GFP+EPCAM+CD90- (CD90-) populations. p values are of the comparison between CD90 positive and CD90 negative populations. Data is shown as the mean+/- SEM for biological replicates n = 3. Statistical significance was calculated using an unpaired t test, p values are indicated for each individual graph.

Figure 2

Derivation and characterization of neonatal human thymus-derived monolayer cell cultures. (A) Bright field images showing freshly derived and passaged monolayer adherent cells from neonatal human thymus. Sale bar, 100 mm. (B) Flow cytometry analysis for CD104 and EPCAM expression on neonatal thymus-derived monolayer cell cultures. (C) Histogram representation of flow cytometry analysis for CD90 expression of neonatal thymus-derived monolayer cell culture (left) and of indicated fractions in (B) identified based on CD104 and EPCAM expression. (D) Histogram representation of gene expression levels of TEC associated genes including keratins in each of the four indicated populations measured in counts per million (from RNA-sequencing analysis). The indicated p values relate to the comparison of the EPCAM+ CD104+ double positive (DP) with EPCAM- CD104- double negative (DN) populations. Data shown in +/- with biological replicates n=3. Statistical significance was calculated with an unpaired t test (E). Heatmap representation of the log fold change in the expression levels of genes found to be statistically significant (p value < 0.05) related to EMT, epithelial polarity, Rho and TGFβ signaling in the four indicated fractions. DP, EPCAM+ CD104+ double positive; DN, EPCAM- CD104- double negative; SP, EPCAM-CD104+ double positive; UN, unsorted sample.

Figure 3

Singe cell RNA-sequencing of neonatal human thymus-derived monolayer cell cultures. (A) Uniform Manifold Approximation and Projection (UMAP) analysis of single cell RNA-sequencing analysis showing cells from the four donors (Donor 18-21) integrated with primary neonatal human thymic epithelial cells from the Human Thymus Cell Atlas (26). Samples were separated by their original sample identity and grouped by cell type clustering. (B) Feature plots of key genes expressed in monolayer cultured neonatal human thymic cells. (C) Dot plot representation of cluster specific genes in the monolayer cultured neonatal human thymic cells. Distinct classes of integrins, laminins, cell cycling, chemokines and their ligands (CXCLs and CCLs), mTEC associated keratins, collagens, and serum amyloids (SAAs) are identified specific to respective clusters. Color intensity in each dot represents the average expression. Dot size represents the percentage of cells expressing that gene in its respective cluster.

Figure 4

Single cell RNA-sequencing analysis of epithelial and mesenchymal gene expression in primary human cTECs and mTECs. (A) Validation of MAGIC program for gene-gene association analysis with established cTEC and mTEC markers and functional genes. Scatter plots show the distribution of cells associated with CLDN4 expression representing mTECs (X axis) and LY74 representing cTECs (Y axis). Color intensity represents the level of gene expression as indicated by color key. (B) Scatter plots showing the MAGIC imputed values calculated for epithelial and mesenchymal gene expression associated with cTECs and mTEC genes in neonate human TECs. (C) Dot plot representation of the expression epithelial, mesenchymal and TEC genes in primary human TECs reported in the human thymic cell atlas (26). Color intensity in each dot represents the average expression. Dot size represents the percentage of cells expressing that gene in its respective cluster.

Figure 5

Identification of EPCAM+CD90+ cells in primary neonatal human TECs. (A) Feature plot representation of single cell RNA-sequencing analysis of human neonatal human thymic epithelial cells expressing EPCAM, CD90, FOXN1 and PDPN separately and triple positive populations expressing these genes. (B) Representative flow cytometry plots (Donor 19) showing the expression of EPCAM and CD90 on primary neonatal human TECs. Non-thymocyte cells (middle) are enriched by an FCS-A gate (left). Thymic stromal cells (right) are enriched from the CD45- population. (C) Quantification summary of the frequency of CD90+ EPCAM+ double positive cells within the CD45- thymic stromal cell population of six human thymus donors. Data is shown as the mean +/- SEM. Flow cytometry results contributing to the data pots in (B) are shown in Figure S5 .