single-cell RNA sequencing analysis of two mice uteri
In order to find more clues about cells in mice uteri, we analyzed the scRNA-seq data of two 6-to-10-week-old female mice from the single-cell mouse cell atlas (scMCA) [7]. Using the 15 most significant principal components of the PCA, we divided the cells into 16 clusters (Figure 1A-B, Table S1). Then we clarified the identities of each cluster (Figure 1C, Table S2). Cluster 0, cluster 1, cluster 2, cluster 3, cluster 10 all highly expressed Col3a1 and Fn1, so we clarified them into stromal cells. Cluster 4, in the meantime, specifically expressed Acta2, which made us clarify them into myofibroblasts. Cluster 11, however, expressed Acta2 and Myh11 without Col3a1, so cells in cluster 11 belonged to muscle cells. Cluster 5, cluster 9, and cluster 12 all highly expressed Cd68 and Adgre1, so they were macrophages/monocytes. Cd7 and Nkg7 could be found highly expressed in cluster 8, which made cluster 8 natural killer (NK) cells. Ly6d and Cd79a were highly expressed in cluster 15, so we identified cluster 15 as B cells. Cluster 13 and cluster 7 both had high expression levels of Krt8, Krt18, and Epcam, making them epithelial cells. Cluster 6 and cluster 14 had high expression levels of Cd34 and Pecam, making them endothelial cells. In this way, we clarified the identities of 16 cell clusters, building a solid foundation for further analysis.
We identified 5 sub-clusters of stromal cells (cluster 0, cluster 1, cluster 2, cluster 3, cluster 10) and 2 sub-clusters of epithelial cells (cluster 13 and cluster 7). According to their distances in the t-SNE, we divided the 5 sub-clusters of stromal cells into 3 groups: stromal cells 1, stromal cells 2, and stromal cells 3. We compared the proportions of each group in two mice (Figure 1D). Surprisingly, we found that stromal cells 1 were exclusively in mouse 2, and stromal cells 3 were exclusively in mouse1. Stromal cells 2 could be found both in mouse 1 and mouse 2. This phenomenon was also seen in epithelial cells. Epithelial cells 1 were exclusively in mouse 2, while epithelial cells 2 were exclusively in mouse 1.
Comparison of epithelial cells revealed two uteri of distinct estrus cycles
We believed the different cell contents of two mice had further biological significance. We found that proliferating cell nuclear antigen (Pcna) was highly expressed in epithelial cells 1 (mouse 2) (Figure 2A). The protein encoded by this gene is a cofactor of DNA polymerase delta, which helps increase the processivity of leading strand synthesis during DNA replication and is regarded as the marker of proliferation [18]. Therefore, we speculated that the uterus of mouse 2 was in the regenerative phase, and the uterus of mouse1 was in the maturational phase. To further investigate this speculation, we performed GO analysis of differentially expressed genes in each epithelial cell cluster (Figure 2B-C, Table S3). The GO terms of epithelial cells 2 (mouse 1) include “positive regulation of cell motility”, “response to steroid hormone”, “skin development”, “cellular response to epidermal growth factor stimulus”, “placenta development”, and “response to wounding”. These terms showed the active rebuilt of the uterus epithelium to prepare for subsequent fertilization in the maturational status. In order to fully show the differences between two epithelia, we extracted all the epithelial cells from two uteri and clustered the cells exclusively. We gained 3 clusters (Figure 2D, Table S4). According to their Pcna expression levels, cluster 1 and cluster 2 showed proliferating characteristics (Figure 2E). We concluded that mouse 2 was in the regenerative phase, for it was entirely composed of proliferating epithelial cells. As for mouse 1, only part of the epithelial cells showed proliferating characteristics (Figure 2F).
The molecular trajectory of stromal cells in the estrus cycle
Endometrial stromal cells (ESC) perform a multitude of functions including hormonal regulation, decidualization, maternal-fetal communications, and embryo receptivity [19]. We further explored the characteristics of stromal cells in each estrus period given their significance in the uterus.
Because stromal cells 1 belonged to mouse 2, so it would be defined as regenerative_stromal, and stromal cells 3 would be defined as maturational_stromal likewise. Then we performed GO analysis of the differentially expressed genes of these two cell groups to see their functional traits.
The GO terms of regenerative_stromal included aorta morphogenesis (Figure 3A, Table S3). As we know, the key feature of the regenerative phase is angiogenesis. Besides, protein maturation, collagen catabolic process, and retinoid metabolic process were actively involved in this period.
The maturational_stromal presented more dynamic cell communication (Figure 3B, Table S3). In this period, the GO term “response to progesterone and other steroid hormones” further validated the estrus states of two mice. We also saw the term “response to transforming growth factor beta”. Transforming growth factor-beta receptor 1 mediated signaling has been reported to be required for female reproductive tract integrity and function [20]. In the maturational phase, cell movement was accelerated, which was in accord with our results. GO analysis suggested “positive regulation of cell migration” in this period. In the meantime, the elevated levels of cell communication and material transport were found in maturational_stromal, which constituted a prosperous metabolism network in stromal cells.
As for the GO analysis of myofibroblasts (Table S3), terms like “muscle structure development” and “regulation of smooth muscle cell proliferation” confirmed our previous clarification of cell identity.
These two stromal subsets in different physiological states had a huge difference in their functional traits, then we further explored how these two cell groups gained their unique traits by differentially gene expression. We performed pseudotime analysis to reconstruct the trajectory of stromal cells in two physiological states (Figure 3C). This time we also included myofibroblasts to construct a more comprehensive atlas of stromal cells in the uterus. The results showed that there were two differentiation branches: the regenerative stromal cells would transform into myofibroblast or maturational stromal cells at the first decisional point, and a small part of the maturational cells still had the ability to differentiate into myofibroblasts at the second branch. In this way, an estrus cycle was completed. The heatmap visualized changes of all the significantly branch 1-dependent genes and clustered them into 3 categories by unsupervised clustering (Figure 3D). Among these genes, the myofibroblasts-branch had high expression levels of Acta2 and Adamts1 (Figure 3E). In the meantime, the maturational_stromal-branch had high expression levels of Col6a4, Fbln2, Tcf4, and Wnt5a (Figure 3F). Col6a2 and Fbln2 encode extracellular matrix protein, and Tcf4 and Wnt5a are genes involved in Wnt signaling. Wnt signaling is important in the maturational phase, for Wnt signaling is involved in the progesterone-induced regulation of uterine stromal cells [21]. These divergent expression patterns of representative genes further confirmed our previous classification. We listed other significant branch-dependent genes, which may also play key roles in the stromal cell differentiation and need further validation (Table 1).
The immune landscape of uterus during the estrus cycle
Embryo implantation and tumor progression are similar to some extent [22]. The maternal immune system needs to find the balance and provide an appropriate environment for the fetus to grow. The current findings of NK, monocytes, and dendritic cells (DCs) during the menstrual cycle are limited. For example, findings concerning NK cell number and cytotoxic activity have been conflicting [23]. In order to gain reliable results with regard to uterine immune cells at the single-cell level, we used the most 10 significant principal components to divide all immune cells into 4 clusters (Figure 4A, Table S5). The heatmap of the top 50 markers for each cluster showed that we gained a convincing clustering result (Figure 4B).
Using known markers, we clarified the identities of each cluster (Figure 4C). Cluster 0 expressed Adgre1 and Mrc1 so we clarified them into macrophages. Cluster 1 expressed Nkg7 and Cd7, which made us clarify them into NK cells. Cluster 2 expressed Cd83 and Cd209a, so cells in cluster 2 belonged to DC cells. For their high expression of Ly6c2, we clarified cluster 3 into monocytes.
Previous researchers have proved the plausibility of using expression levels of transcripts to predict the volume of cell populations in tissues. Levels of transcripts that encode immune-related factors changed during the time-course paralleled the changes observed in the volume fractions of the immune cells [24]. We used the expression levels of marker genes to predict the numbers of the immune cells, and we found the clear difference of immune cell composition in these two estrus states. The proportions of macrophages and NK cells in two states vary considerably (Figure 4D). Macrophages dominated in the regenerative phase, while NK cells dominated in the maturational phase, which could be proved in previous studies. Macrophages in the regenerative phase have been reported to have a potential role in regeneration and proliferation of the functional layer of the endometrium [25]. The possible mechanism may lie in the fact that estrogen can recruit macrophages and neutrophils into the mouse uterus, while progesterone via its receptor antagonizes the pro-inflammatory activity of estrogen in the mouse uterus [26]. NK cells were reported to be dominant in the maturational phase [27], which played a pivotal role in the tissue homeostasis and endometrial vasculature remodeling that were necessary for embryo implantation and successful pregnancy [28].
How monocytes differentiate into DCs or macrophages are poorly understood. We performed pseudotime analysis of monocytes, macrophages, and DCs, trying to reconstruct the developmental trajectory of the mononuclear phagocyte system (Figure 4E). The branch point led to two differentiation paths: macrophages or DCs. Heatmap clustered branch-dependent genes into three categories according to their expression patterns (Figure 4F). The expression levels of marker genes of each branch over pseudotime verified the identities of two differentiation paths (Figure 4G). We listed other significant branch-dependent genes (Table 2). Among them, we detected three transcription factors: Mafb, Irf7, and Nr4a1. Mafb was detected in the macrophage branch. Mafb was reported to be essential for monocyte-macrophage differentiation in the previous study [29]. Irf7 expression was seen in the monocyte branch and Nr4a1 was expressed highly in the DC branch (Figure 4G), yet the relations between these two transcription factors and their branches have not been reported yet.
In order to study the different functional traits of immune cells in two states, we drew scatter plots to find the genes that were visual outliers of the average expression levels of two estrus states, which could highlight genes that exhibited dramatic responses to estrus phase change.
As for NK cells (Figure 4H), NK cells in the regenerative phase showed intense inflammatory responses with high expression levels of Tnf, Il1b, and S100a8 [30]. It also expressed Cxcl2 and Ccrl2, which respectively promoted the recruitment of neutrophils and themselves [31]. In contrast, genes that were up-regulated in the maturational state mostly were immune-suppressive genes like Serpinb9, Stat3, Cd96, and Cd55. NK cells also secreted substances that were advantageous for follow-up pregnancy like Ccl2.
The number of macrophages was higher in the regenerative phase. In this phase, macrophages were pro-inflammatory by secreting resistin, which is a systemic pro-inflammatory cytokine targeting both leukocytes and adipocytes (Figure 4I) [32]. However, it also showed high expression levels of selenoprotein Msrb1 and Hes1 to inhibit inflammatory responses [33, 34]. As for macrophages in the maturational phase, it mainly showed the M2 phenotype with high expression levels of Il10, Cd206, and Ccl7, which consisted of the characteristics of decidual macrophages [35].
In the regenerative phase, the functions of DC were mainly embodied in influencing other cells. It secreted Ccl22 to recruit regulatory T (Treg) cells and Ccl5 to recruit CCR5-positive cytokine-induced killer cells [36, 37]. Its pro-inflammatory role was also embodied in the high expression of Ccrl2, which played a significant role in inflammation [31]. DCs in the maturational phase were immune-suppressive (Figure 4J). DCs expressed PD-L1, Jchain, and Anxa1, which were correlated with immune tolerance and refrained the secretions of pro-inflammatory cytokines [38, 39].
Monocytes in the maturational phase secreted multiple chemokines like Thbs1, Ccl2, Ccl7, Ccl8, Lyn, and Anxa1, to facilitate the migration of themselves (Figure 4K) [40, 41]. Monocytes also expressed M1 marker Cd86 and Lgals8 to enhance inflammatory responses [42]. By contrast, monocytes in the regenerative phase showed more intense cytolytic ability by producing S100a8, Tnf, and Il1b. It also secreted Cxcl16 and Ccl3 to facilitate the recruitment of monocytes and Mmp14, Trem2, and Mif to induce inflammatory cytokines [43, 44].
Taken together, we found that the immune responses were suppressed in the maturational phase compared to the regenerative phase by reducing inflammatory cytokines secretion and facilitating differentiation into immune-suppressive cells.
Identify Pdgfrb+ Aldh1a2+ Cd34+ endometrial mesenchymal stem cells in vivo
The mesenchymal-to-epithelial transition has been proposed as a possible reason to explain the periodically endometrial epithelial tissue regeneration and postpartum endometrial recovery [45]. A previous study has reported a group of cells expressed both the epithelial cell marker, pan-cytokeratin, and the stromal cell marker, vimentin as well [46]. We tried to identify multipotent ESCs in the scRNA-seq data, so we performed StemID2. We failed to find the cell cluster that expressed all the proposed stem cell surface molecules like Cd73, Cd90, and Cd105 [47]. We hypothesized that expressions of Cd73, Cd90, and Cd105 may be induced during in-vitro culture. To verify this hypothesis, we used another scRNA-seq data of endometrial tissues [8]. The authors generated expression data of cultured ESCs and uncultured ones. The diversity between these two sets of samples was greater than we expected. The cells from two different culture environments clustered separately (Figure 5A). The stem cell surface markers mentioned above were expressed differently in the two cell groups. We found that stem cell makers like Cd90 (Thy1), Cd44 had significant higher expressions in the cultured group (Figure 5B), so we decided to use Pdgfrb as the stem cell marker, which was known to be enriched in endometrial mesenchymal stem/stromal cells [48]. StemID showed that cluster 5 may be the possible multipotent ESCs, which had a higher expression of Pdgfrb, a higher entropy score, and more connections with other cell types (Figure 5C-D, F). It had the potential to differentiate into stromal cells, epithelial cells, and immune cells (Figure 5E). Besides, this cluster also highly expressed Cd34 and Aldh1a2 (Figure 5F-I) [49]. Cd34+Klf4+ stromal-resident stem cells have been reported to directly contribute to endometrial regeneration [50]. The cluster5 highly expressed Cd34, but without detectable expression of Klf4. To conclude, we found the transcriptional signature of endometrial mesenchymal stem cells in vivo. And the cluster 5 that we explored here showed a great possibility to be the cells responsible for re-epithelialization.