{"paper_id":"372920fd-3453-4724-80f3-417e4fbc6084","body_text":"Proper implantation of the embryo in the maternal endometrium is critical for a normal pregnancy, and the sophisticated transformation of the endometrium in the three different menstrual states is regulated by the collaboration of cell populations under the influence of hormones ( Kumar et al., 2011 ).\nThe attempt to use gene expression to define the menstrual cycle has been seen in previous studies, which reported a strong relationship existing between histopathology and transcriptional profiles of the samples and validated the importance of using molecular profiles to evaluate the endometrial status ( Haber et al., 2017 ). Other than humans, the endometria of mice, rats, and cows have also been analyzed to a certain extent ( Reese et al., 2001 ;  Naciff et al., 2002 ). However, there was little consistency between these microarray-based studies, for the differentially expressed genes reported in each study showed large variability. These molecular profiles remained at the tissue level. The fact that the proportions of the epithelium, stroma, immune cells, and blood vessels in individual specimens were different may cause the variability. However, single-cell analysis can solve this obstacle, as it allows us to detect cell-to-cell variability, possible subpopulations, and rare cell types.\nThe estrous cycle in mice averages 4–5 days and is a repetitive but dynamic process, reflecting changes in the levels of estradiol and progesterone secreted by the ovarian follicles ( Cora et al., 2015 ). There are different criteria for defining the estrus cycle of mice ( Vidal and Filgo, 2017 ), but no matter how the cycle is defined, mice uteri undergo the same hormone change patterns and the recurrences of regeneration and maturation as humans. In order to match with the recurring physiologic changes in humans, we divided the estrous cycle of mice into two stages: the regeneration stage and the maturation stage. The regenerative stage amounted to the proliferative phase in humans and proestrus and estrus phases in mice, the maturational stage amounted to the secretory phase in humans and the metestrus and the diestrus phases in mice. In doing so, we could seek insights and inspirations from mice single-cell RNA sequencing (scRNA-seq) data to shed light on human endometrium research.\nThe cognition of the immune conditions in the menstrual cycle has been updated rapidly, yet the amounts or the functional traits of multiple immune cell types are still under debate. The immune cells in decidua have been analyzed thoroughly ( Jiang et al., 2018 ). In sharp contrast, the features of immune cells in the regenerative stage and the maturational stage have not gained enough attention. Up to today, the complicated hormone-induced immune regulation has left a huge riddle for us to solve ( Kumar et al., 2014 ).\nBased on the research gaps mentioned above, we aimed to construct a cell atlas of mice uteri including multiple cell types at the single-cell level. We used published scRNA-seq data of two C57BL/6J mice uteri to build an integrated cell atlas from the regenerative endometrium to the maturational endometrium, and elucidated the transitions that happened in epithelial cells, stromal cells, and immune cells during the estrous cycle, hoping to provide new insights into cell dynamics in the uterus and provide a normal reference for future studies under pathologic conditions.\n\nTwo datasets from public databases were enrolled in this study.  GSE108097  from Gene Expression Omnibus ( https://www.ncbi.nlm.nih.gov/geo/ , RRID: SCR_005012), containing scRNA-seq data of 3,756 cells from two 6-to-10-week-old female C57BL/6J mice uteri, was selected to build the digital mouse cell atlas of mouse uterus ( Han et al., 2018 ). Mouse 1 contained 2041 cells, and mouse 2 contained 1715 cells. Based on the expression levels of proliferating cell nuclear antigen, differentially expressed genes of two samples, and gene ontology terms, we concluded that mouse 1 was in the maturational stage and mouse 2 was in the regeneration stage. Another scRNA-seq data of endometrial tissue was obtained from a previous study to compare the cultured endometrial stromal cells (human endometrial cells in the secretory stage were collected and cultured for 8 days in the cell culture medium consisting of a 1:1 mixture of DMEM (Cat No E15-892, GE Healthcare, United States)/Ham’s F12 (Cat No E15-890, GE Healthcare), supplemented with 10% FBS and Antibiotic-Antimycotic solution (GE Healthcare) and uncultured ones (endometrial biopsies collected from women in the secretory stage) ( Krjutškov et al., 2016 ). We used R software (version 3.6.3;  http://www.Rproject.org , RRID: SCR_001905) and Seurat package of R (version 2.3,  https://satijalab.org/seurat/ , RRID: SCR_016341) ( Satija et al., 2015 ) to process the data. Seurat is a computational strategy to process and explore scRNA-seq data. The filter criteria of cells were determined as default. It is not necessary to obtain ethical approval because we used published online datasets.\nPrincipal component analysis (PCA), uniform manifold approximation and projection (UMAP), t-distributed stochastic neighbor embedding (tSNE), and visualization were performed by the Seurat package of R ( Satija et al., 2015 ). Seurat is a computational strategy to process and explore scRNA-seq data. The fate decisions and pseudotime trajectories of endometrium stromal cells and the mononuclear phagocyte system were reconstructed by the Monocle package of R (version 2.10.1,  http://cole-trapnell-lab.github.io/monocle-release/docs/ , RRID: SCR_016339) ( Cole et al., 2014 ;  Qiu et al., 2017 ;  Wu et al., 2017 ). In short, Monocle performs differential expression and time-series analysis for single-cell expression experiments, which orders individual cells according to progress through a biological process, without knowing ahead of time which genes define progress through that process. Possible stem cells identification was performed by RaceID3 ( https://github.com/dgrun/RaceID , RRID: SCR_017045) and StemID2 ( https://github.com/dgrun/StemID , RRID: SCR_017242) ( Grün et al., 2015 ). RaceID3 is an algorithm for rare cell type identification in complex populations of single cells, while StemID2 is an algorithm based on RaceID3 for the inference of differentiation trajectories and the prediction of the stem cell identity.\nGene Ontology (GO) analyses were conducted through clusterProfiler (clusterProfiler, RRID: SCR_016884) ( Yu et al., 2012 ;  Walter et al., 2015 ) and online GO resource website ( Ashburner et al., 2000 ;  2019 ). Statistically significant GO terms ( p  < 0.05) were identified.\nHistology and immunofluorescence were conducted according to our previous procedures ( Wu et al., 2019 ). Briefly, vagina smears were firstly used to determine the mouse estrous cycle ( Supplementary Figure S1 ) ( Bertolin and Murphy, 2014 ). Then mouse uterine tissues were collected and fixed in 4% (w/v) paraformaldehyde, and then dehydrated in an ethanol gradient. Then the paraffin sections of 10 μm thickness were stained with hematoxylin and eosin. Immunostaining was carried out as follows: The series of 10 μm-thick sections were rehydrated, fixed with 4% (w/v) paraformaldehyde for 30 min, antigen retrieval was conducted by incubating in citrate antigen retrieval solution at 65°C overnight, rinsed three times with PBS, and treated with blocking solution (1% BSA) for 30 min, prior to incubation with primary antibodies at 4°C overnight. The primary antibodies rabbit anti-mouse antibodies against KRT7 (Abcam, ab181598), the primary antibodies mouse anti-mouse antibodies against PCNA (Abcam, ab29), the primary antibodies rat anti-mouse antibodies against CD34 (Biolegend, 119307) were used to detect the expression of selected proteins within the uterine tissues. The goat anti-rat-cy3 secondary antibody (Beyotime Biotechnology, A0507), goat anti-rabbit-488 secondary antibody (Invitrogen, A11008), donkey anti-mouse 546 secondary antibody (Invitrogen, A10036), and DAPI (Beyotime Biotechnology, C1002) were used to visualize the respective primary antibodies and the cell nuclei. All procedures were carried out according to the manufacturer’s instructions.\nAll statistical analyses were performed by Graphpad Prism 8.0 software ( https://www.graphpad.com/ , RRID: SCR_002798) and R software (version 3.6.3;  http://www.Rproject.org , RRID: SCR_001905). A two-sided probability value of  p  < 0.05 was considered being statistically significant.\n\nThe scRNA-seq data of two 6-to-10-week-old female C57BL/6J mice were processed using published Seurat pipelines ( Satija et al., 2015 ;  Han et al., 2018 ). In total, we analyzed 3,756 single cells and identified 16 cell clusters, which were grouped into eight major cell types ( Figures 1A,B ,  Supplementary Table S1 ). Then we clarified the identities of each cluster ( Figure 1C ,  Supplementary 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  Actg2  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  Cldn5  and  Pecam , making them endothelial cells. In this way, we clarified the identities of 16 cell clusters, building a solid foundation for further analysis.\nsingle-cell RNA sequencing analysis of two mice uteri.  (A)  t-distributed stochastic neighbor embedding (t-SNE) diagram of two mice uteri.  (B)  Heatmap of single cells from the two uteri revealed 16 populations.  (C)  Violin plots indicating the expression of marker genes of each cell cluster.  (D)  The cell composition of the two uteri was significantly different. T-SNE: t-distributed stochastic neighbor embedding.\nWe 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 three groups: stromal cells 1, stromal cells 2, and stromal cells 3. The proportions of each cell type in two mice were shown ( Figure 1D ). Surprisingly, we found that stromal cells 1 were exclusively in mouse 2, and stromal cells 3 were exclusively in mouse 1. 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.\nWe 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 is regarded as the marker of proliferation ( Celis and Celis, 1985 ). Previous studies have reported that for humans experiencing estrous cycles, the divergence of the expression level of  Pcna  was mainly due to the states of the estrous cycle rather than age. It has been reported that the expression of  Pcna  showed a high peak in the proliferative phase, and then decreased sharply in the secretory phase ( Li et al., 1993 ;  Noci et al., 1995 ;  Hamid et al., 2002 ). In mice, the treatment with estrogen to ovariectomized mice upregulates PCNA expression, while the co-treatment with estrogen and progesterone downregulates PCNA expression ( Annie et al., 2019 ). In addition, we found that the expression levels of metalloproteinase (MMPs) were significantly suppressed in epithelial cells 1 (mouse 2) ( Supplementary Table S2 ). So we preliminarily speculated that the uterus of mouse 2 was in the regenerative stage, and the uterus of mouse1 was in the maturational stage.\nComparison of epithelial cells and stromal cells revealed two uteri of distinct estrus stages.  (A) \n Pcna  was highly expressed in epithelial cells 1 compared to epithelial cells 2. Gene ontology (GO) analysis of differentially upregulated genes in  (B)  epithelial cells 1 and  (C)  epithelial cells 2.  (D)  t-distributed stochastic neighbor embedding (t-SNE) diagram of epithelial cells in two mice uteri.  (E) \n Pcna  was highly expressed in sub-cluster 1 and sub-cluster 2.  (F)  The epithelial cell composition of the two uteri was significantly different. Gene ontology (GO) analysis of differentially upregulated genes of stromal cells in  (G)  the regenerative stage and  (H)  the maturational stage.  (I)  Representative H&E and IF staining of PCNA expression levels in mice endometrium. (DAPI, blue; PCNA, red; KRT7, green). Scale bars, 200 μm (H&E) and 50 μm (IF). GO: Gene ontology; t-SNE: t-distributed stochastic neighbor embedding; PCNA: proliferating cell nuclear antigen; IF: immunofluorescence; H&E: hematoxylin and eosin.\nTo further investigate this speculation, functional enriched terms of the differentially expressed genes in each epithelial cell cluster were shown ( Figures 2B,C ,  Supplementary Table S3 ). Terms including “response to steroid hormone,” “skin development,” “cellular response to epidermal growth factor stimulus,” “placenta development,” and “response to wounding” were enriched in epithelial cells 2 (mouse 1). These terms showed the active rebuilt of the uterus epithelium to prepare for subsequent fertilization in the maturational status. In this period, we also found terms that indicated “intrinsic apoptotic signaling pathway,” “increased cell mobility,” and “suppressed cell adhesion,” which were all consistent with the changes that happened in the maturational status as previously reported ( Yip et al., 2013 ).\nThe epithelial cells from two uteri were then clustered exclusively to further verify our hypothesis. We gained 3 clusters ( Figure 2D ,  Supplementary 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 stage, for it was entirely composed of proliferating epithelial cells. As for mouse 1, only part of the epithelial cells showed proliferating characteristics ( Figure 2F ). In addition, the expression level of  Esr1  was suppressed in the epithelial cells of cluster 0 in the mouse 1 ( Supplementary Table S4 ).\nEndometrial stromal cells (ESCs) perform a multitude of functions and undertake different functions in different estrous stages ( Cottrell et al., 2017 ). We further explored the characteristics of stromal cells of two mice to further confirm the stages of two mice. The expression levels of MMPs were significantly upregulated in epithelial cells 2 (mouse 1) ( Supplementary Table S2 ), which possibly linked with the active matrix degradation in the maturational stage.\nWe performed functional enrichment analyses of the differentially expressed genes in each stromal cell cluster to see their functional traits. The term “artery morphogenesis” was enriched in stromal cells in mouse 2 ( Figure 2G ,  Supplementary Table S3 ). One of the key features of the regeneration stage is angiogenesis. Besides, “protein maturation” and “collagen catabolic process” were activated in this period.\nThe stromal cells in mouse 1 presented more dynamic cell communications ( Figure 2H ,  Supplementary Table S3 ). The term “response to progesterone and other steroid hormones” further indicated that mouse 1 was in the maturational stage. The term “response to transforming growth factor beta” was enriched too. Also in the maturational stage, the accelerated cell movement was again seen in this stage, as enrichment analysis suggested the term “positive regulation of cell migration” was significantly enriched. We believed the elevated levels of cell communications and material transportation found in mouse 1 constituted a prosperous metabolism network in stromal cells.\nIn order to validate the data analysis results, vagina smear and hematoxylin and eosin (H&E) staining were used to determine the mouse estrous cycle ( Figure 2I ,  Supplementary Figure S1 ) ( Bertolin and Murphy, 2014 ). The expression levels of PCNA in the regenerative mice regenerative endometrium and maturational endometrium were presented ( Figure 2I ). We found that, like humans, expression level of PCNA in the regenerative endometrium was significantly higher than in the maturational endometrium, indicating the practicability of using PCNA expression level to define the estrus stage.\nTaking into account the sheer differences in proliferation and apoptosis, cell adhesion and movement, angiogenesis, and extracellular matrix remodeling, we concluded that mouse 1 was in the maturational stage and mouse 2 was in the regeneration stage.\nESCs have been reported to perform a multitude of functions including hormonal regulation, decidualization, maternal-fetal communications, and embryo receptivity ( Cottrell et al., 2017 ). After we clarified the estrous stages of two mice, we further explored the characteristics of stromal cells in each estrus period given their significances in the uterus.\nFirstly, functional enrichment analysis of upregulated genes of myofibroblasts was performed ( Figure 3A ,  Supplementary Table S3 ). Terms like “muscle structure development” and “regulation of smooth muscle cell proliferation” confirmed our previous classification of its identity.\nThe molecular trajectory of stromal cells in the two estrus stages.  (A)  Gene ontology analysis of differentially upregulated genes in myofibroblasts.  (B)  Monocle generated the pseudotemporal trajectory of stromal cells in different physiological stages.  (C)  Heat map for clustering the significantly branch-1 dependent genes that affected cell fate decisions into three clusters. q < 1E-06. The expression levels of representative genes of  (D)  myofibroblasts and  (E)  maturational stromal cells were shown in the line plots.\nThe analyses of two stromal subsets in the last section showed that stromal cells in different physiological stages had huge differences in their functions. Therefore, we wanted to further explore how these two cell groups gained their unique traits by differentially gene expression. The pseudotime trajectory of stromal cells in two physiological stages was constructed ( Figure 3B ). The results showed that there were two differentiation branches: the regenerative stromal cells would transform into myofibroblasts 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 decisional point. In this way, an estrus cycle was completed. All the significantly expressed branch 1-dependent genes were clustered into three categories by unsupervised clustering ( Figure 3C ). Among these genes, the myofibroblasts-branch genes had high expression levels of  Acta2  and  Adamts1  ( Figure 3D ). In the meantime, the maturational_stromal-branch genes had high expression levels of  Col6a4 ,  Fbln2 ,  Tcf4 , and  Wnt5a  ( Figure 3E ). 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 ).\nSignificant branch-dependent genes of mice stromal cells in different physiological stages.\nEmbryo implantation and tumor progression are similar to some extent ( Holtan et al., 2009 ). The maternal immune system needs to find the balance and provides 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 are conflicting ( Oertelt-Prigione, 2012 ). In order to gain reliable results with regard to uterine immune cells at the single-cell level, all immune cells were clustered exclusively into 4 clusters ( Figure 4A ,  Supplementary Table S5 ). The heatmap of the top 50 markers for each cluster showed that we gained a convincing clustering result ( Figure 4B ).\nThe immune landscape of the uterus during the two estrus stages.  (A)  t-distributed stochastic neighbor embedding (t-SNE) diagram of immune cells in two mice uteri.  (B)  Heatmap of immune cells from the two uteri revealed four populations.  (C)  Violin plots indicating the expression of marker genes of each cell cluster.  (D)  The immune cell composition of the two uteri was different, macrophages dominated in the regenerative stage, while natural killer (NK) cells dominated in the maturational stage.  (E)  Monocle generated the pseudotemporal trajectory of the phagocyte system.  (F)  Heat map for clustering the significantly branch-1 dependent genes that affected cell fate decisions into three clusters. q < 1E-06.  (G)  The expression levels of representative genes of macrophages/dendritic cells (DCs)/monocytes and three transcription factors were shown in the line plots. t-SNE: t-distributed stochastic neighbor embedding; NK: natural killer; DC: dendritic cells.\nUsing known markers, the identities of each cluster were clarified ( 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 levels of  Ly6c2 , we clarified cluster 3 into monocytes. We found a clear difference in immune cell composition in these two estrus stages. The proportions of macrophages and NK cells in the two stages varied considerably ( Figure 4D ). Macrophages dominated in the regenerative stage, while NK cells dominated in the maturational stage.\nThe mechanism of monocytes differentiating into DCs or macrophages is poorly understood. The developmental trajectory of the mononuclear phagocyte system was constructed ( Figure 4E ). The branch point led to two differentiation paths: macrophages or DCs. Branch-dependent genes were clustered 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,  Irf7  expression was seen in the monocyte branch, and  Nr4a1  expressed highly in the DC branch ( Figure 4G ).\nSignificant branch-dependent genes of the mononuclear phagocyte system in two mice uteri.\nAs for NK cells ( Table 3 ), NK cells in the regenerative stage showed intense inflammatory responses with high expression levels of  Tnf ,  Il1b , and  S100a8 . It also expressed  Cxcl2  and  Ccrl2 , which respectively promoted the recruitment of neutrophils and themselves ( Regan-Komito et al., 2017 ). In contrast, upregulated genes of NK cells in the maturational stage mostly were immune-suppressive genes like  Serpinb9 ,  Stat3 ,  Cd96 , and  Cd55 . They also secreted substances that were advantageous for follow-up pregnancy like  Ccl2 .\nSignificantly differentially expressed genes of immune cells in different physiological stages.\nThe number of macrophages was higher in the regenerative stage. In this stage, macrophages were pro-inflammatory by secreting resistin, which was a systemic pro-inflammatory cytokine targeting both leukocytes and adipocytes ( Table 3 ) ( Nagaev et al., 2006 ). However, it also showed high expression levels of selenoprotein  Msrb1  and  Hes1  to inhibit inflammatory responses ( Lee et al., 2017 ;  Zhang et al., 2019 ). As for macrophages in the maturational stage, it mainly showed the M2 phenotype with high expression levels of  Il10 ,  Cd206 , and  Ccl7 , which were consisted of the characteristics of decidual macrophages ( Xuan et al., 2015 ).\nIn the regenerative stage, 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 ( Lee et al., 2016 ;  Yashiro et al., 2019 ). Its pro-inflammatory role was also embodied in the high expression of  Ccrl2 , which played a significant role in inflammation ( Regan-Komito et al., 2017 ). DCs in the maturational stage were immune-suppressive ( Table 3 ). DCs expressed  PD-L1 ,  Jchain , and  Anxa1 , which were correlated with immune tolerance and refrained the secretion of pro-inflammatory cytokines ( Källberg and Leanderson, 2008 ;  Sena et al., 2016 ).\nMonocytes in the maturational stage secreted multiple chemokines like  Thbs1 ,  Ccl2 ,  Ccl7 ,  Ccl8 ,  Lyn , and  Anxa1  to facilitate the migration of themselves ( Table 3 ) ( Asano et al., 2015 ;  Liu et al., 2015 ). Monocytes also expressed M1 markers like  Cd86  and  Lgals8  to enhance inflammatory responses ( Patel et al., 2017 ). By contrast, monocytes in the regenerative stage 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 responses ( Buck et al., 2013 ;  Ruiz-Rosado et al., 2016 ).\nTaken together, we found that the immune responses were suppressed in the maturational stage compared to the regenerative stage by reducing inflammatory cytokines secretion and facilitating differentiation into immune-suppressive cells.\nThe mesenchymal-to-epithelial transition has been proposed as a possible reason to explain the periodically endometrial epithelial tissue regeneration ( Huang et al., 2012 ). 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 ( Patterson et al., 2013 ). We tried to identify multipotent ESCs in the scRNA-seq data using published pipelines ( Grün et al., 2016 ). We failed to find the cell groups that expressed all the proposed stem cell surface molecules like  Cd73 ,  Cd90 ,  and Cd105 . We hypothesized that expressions of  Cd73 ,  Cd90 , and  Cd105  may be induced during  in-vitro  culture. Another scRNA-seq dataset containing cultured ESCs and uncultured ones was used to show the diversity of two culture environments ( Krjutškov et al., 2016 ). 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 ) and  Cd44  had significant higher expressions in the cultured group ( Figure 5B ). We found that cell group had a higher entropy score and more connections with other cell types ( Figures 5C,D,F ,  Supplementary Table S6 ), had the potential to differentiate into stromal cells, epithelial cells, and immune cells ( Figure 5E ), which make it the potential multipotent ESCs  in vivo . This cell subcluster expressed higher expression of  Cd34 ,  Pdgfrb , and  Aldh1a2  ( Figures 5F–I ). The representative immunofluorescence staining of CD34 in mice endometrium was shown, indicating the existence of this endometrial mesenchymal stem cells ( Figure 5J ).\nIdentify  Pdgfrb \n \n + \n \n Aldh1a2 \n \n + \n \n Cd34 \n \n + \n  endometrial mesenchymal stem cells  in vivo .  (A)  Cells directly harvested from biopsy clustered separately with cells after  in-vitro  culture.  (B)  The heat map presented the differentially expressed  Cd73  ( Nt5e ),  Cd90  ( Thy1 ),  Cd44 ,  Pdgfrb , and  Eng  in cells directly harvested from biopsy and after  in-vitro  culture.  (C)  Lineage tree generated by StemID2.  (D)  Histograms for the number of links, the delta-entropy, and the StemID2 score generated by StemID2. A high score indicates a higher likelihood that the cluster is an actual stem cell cluster. The t-SNE map with color code representation of log-transformed expression across marker genes of  (E)  epithelial cells, stromal cells, immune cells, and  (F)  stem cells.  (G–I)  The expression level of Pdgfrb, Aldh1a2, and Cd34 in cluster 5 and the rest of the uterine stromal cells.  (J)  Representative IF staining of CD34 expression levels in mice endometrium. (DAPI, blue; CD34, red). Scale bars, 50 μm. IF: immunofluorescence;  p -value<0.0001.\nTo conclude, we found the transcriptional signature of endometrial mesenchymal stem cells  in vivo . And the cell group 5 that we presented here showed a great possibility to be the cells responsible for re-epithelialization.\n\nThere is still a huge void in uterus-related research at the single-cell level. In humans, the sequence data of  Cd13 \n +  stromal and  Cd9 \n \n + \n  epithelial cells from endometrial tissues have been generated ( Krjutškov et al., 2016 ). Researchers also used scRNA-seq to map the temporal transcriptomic changes in cultured primary ESCs along a decidual time-course and in response to the withdrawal of differentiation signals ( Lucas et al., 2020 ). In mice, the transcriptional profiles of uterine epithelial cells at five developmental stages, ranging from neonatal to mature stages were analyzed ( Wu et al., 2017 ). These studies contained a limited number of cell types. A scMCA covering major cell types was completed ( Han et al., 2018 ). It included the uterus and mice other major organs as well, yet without an in-depth analysis of the changes that happened in the endometrium at different estrus stages and left opportunities for later researchers to delineate the dynamic endometrial cell transformation of all cell types in the estrus cycle at the single-cell level. Recently, mesenchymal cells in the adult mouse endometrium have been characterized and five subpopulations have been identified ( Kirkwood et al., 2021 ). Our research is a systematical single-cell level study with a special focus on constructing a mice cell atlas at different estrus stages and showed that different estrus stages had sheer different cell composition.\nWe identified three transcription factors in the differentiation path of the mononuclear phagocyte system . Mafb  was reported to be essential for monocyte-macrophage differentiation in the previous study ( Goudot et al., 2017 ).  C1qb , as the complement component of C1q complex, was one of the  Mafb  target genes and conformably upregulated in the macrophage branch ( Table 2 ) ( Hamada et al., 2020 ).\nIrf7  has been reported to be involved in the regulatory pathway initiated in DCs during their response to microbial stimuli but dispensable in DC development ( Owens et al., 2012 ).  Nr4a1  has been reported to be the target for modulating the inflammatory phenotypes of monocytes and macrophages ( Hanna et al., 2012 ). In our analyses,  Irf7  expression was seen in the monocyte branch, and  Nr4a1  expressed highly in the DC branch, so the relations between these two transcription factors and their branches needed further experiments. Likewise, understanding the decisional mechanism of fibroblast-to-myofibroblast differentiation may be the key to tackle endometriosis and adenomyosis. Among the highly expressed genes that determined the myofibroblast differentiation ( Table 1 ), there were a lot of them have not been reported, which shed light on future research.\nGenerally, the immune cells in the uterus endometrium showed a pro-inflammatory tendency in the regenerative stage and an anti-inflammatory tendency in the maturational stage. Yet the macrophages in the regenerative stage also expressed certain immune-suppressive genes, indicating the possibility that tissue-resident macrophages existed and contributed to endometrium repair. The findings concerning uterus tissue-resident macrophages are rare. Putative tissue-resident macrophages have been reported to be spatially restricted and in association with areas of repaired, re-epithelialized endometrium ( Cousins et al., 2016 ). In our pseudotime analysis ( Figure 4E ), we found a branch in the path of macrophage differentiation, which indicated the existence of a subtype of macrophages and might be the uterus tissue-resident macrophages. The molecular profiles of mouse uterus tissue-resident macrophages remained unknown, which needed further research. Our results may provide insights into mechanisms regarding tissue remodeling and aid in tackling endometrium abnormalities like endometritis.\nWe reported a novel group of markers for  in vivo  endometrial mesenchymal stem cells:  Pdgfrb ,  Aldh1a2 , and  Cd34 , which has not been reported before.  Cd34 \n \n + \n \n Klf4 \n \n + \n  stromal-resident stem cells have been reported to directly contribute to endometrial regeneration ( Yin et al., 2019 ).  PDGFRα \n + / CD34 \n +  Celll group 5 highly expressed  Cd34 , but without detectable expression of  Klf4 . Many proposed stem cell surface molecules like  Cd73 ,  Cd90 , and  Cd105  failed to express highly in  in vivo  endometrial mesenchymal stem cells ( Kyurkchiev et al., 2010 ).\nHowever, several limitations of our study should be acknowledged. Firstly, this is an RNA-seq based bioinformatics study and caution must be taken when further extrapolating these results  in vivo.  Future study is needed to evaluate the presence of a transcript corresponds to its expression at the protein level. Experiments such as further functional verification of reported endometrial mesenchymal stem cells are crucial in the future. Secondly, the limited number of mice that participated in this study may affect the statistical power and the strength of our conclusions. Thirdly, one of our goals in this study is to seek insights from mice scRNA-seq data and shed light on human endometrium research. However, species-specificities in uterine physiology exist, which may dampen the practicability of our study.\nWe thoroughly compared the functional traits and molecular profiles of each cell type from the regenerative stage to the maturational stage ( Figure 6 ). With a template for the interactions of different cell types at the normal condition, researchers can better identify morphologically unaffected abnormalities in future studies. We also depicted the transitions and transcription factors that shaped the differentiation of ESCs and the mononuclear phagocyte system, and many of them have not been reported before. The genes that have not been reported before can inspire subsequent basic research. The cell atlas of mice uteri presented here would improve our understanding of the functional changes that occurred in the endometrium during the estrus cycle.\nDigital Cell Atlas of Mouse Uterus: from regenerative stage to maturational stage. Based on the  Pcna  expression, differentially expressed genes, and gene ontology terms of two stages, we built up the cell atlas of the mouse uterus from the regenerative to maturational endometrium. The number and function of immune cells varied sharply in two periods. In the regenerative stage, mice epithelial cells expressed Pcna highly, and the term “aorta morphogenesis” was enriched in stromal cells. Macrophages dominated in this stage. The immune system was boosted with intensely secreted cytokines and chemokines. In the maturational stage, cells presented more dynamic cell communication instead of proliferating. Terms “response to progesterone and other steroid hormones” and “response to transforming growth factor beta” were enriched. NK cells dominated in this stage. The immune responses were suppressed compared to the regenerative stage by reducing inflammatory cytokines secretion and facilitating differentiation into immune-suppressive cells.  Pcna : proliferating cell nuclear antigen; NK: natural killer; DC: dendritic cells.\n\nThis study is a systematical single-cell level study to construct a mice cell atlas from the regenerative stage to the maturational stage, including epithelial cells, stromal cells, and immune cells. The functional traits and molecular profiles for each cell type in these two stages are thoroughly compared. The mice cell atlas also delineates the transitions that shape the differentiation of endometrial stromal cells and the mononuclear phagocyte system. This study found putative uterus tissue-resident macrophages and  Pdgfrb \n \n + \n \n Aldh1a2 \n \n + \n \n Cd34 \n \n + \n  endometrial mesenchymal stem cells  in vivo .","source_license":"CC-BY-4.0","license_restricted":false}