{"paper_id":"335e8a7d-f1e4-431d-be08-ef50e66f97a4","body_text":"Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379  \nhttps://doi.org/10.1186/s13287-023-03616-w\nREVIEW\nExploring distinct properties of endometrial \nstem cells through advanced single-cell analysis \nplatforms\nJin Woo Lee1,2 and Hwa‑Yong Lee3*   \nAbstract \nThe endometrium is a dynamic tissue that undergoes cyclic changes in response to ovarian hormones dur‑\ning the menstrual cycle. These changes are crucial for pregnancy establishment and maintenance. Endometrial \nstem cells play a pivotal role in endometrial regeneration and repair by differentiating into various cell types \nwithin the endometrium. However, their involvement in endometrial disorders such as endometriosis, infertil‑\nity, and endometrial cancer is still not fully understood yet. Traditional bulk sequencing methods have limitations \nin capturing heterogeneity and complexity of endometrial stem cell populations. To overcome these limitations, \nrecent single‑cell analysis techniques, including single‑cell RNA sequencing (scRNA‑Seq), single‑cell ATAC sequenc‑\ning (scATAC‑Seq), and spatial transcriptomics, have emerged as valuable tools for studying endometrial stem cells. In \nthis review, although there are still many technical limitations that require improvement, we will summarize the cur‑\nrent state‑of‑the‑art single‑cell analysis techniques for endometrial stem cells and explore their relevance to related \ndiseases. We will discuss studies utilizing various single‑cell analysis platforms to identify and characterize distinct \nendometrial stem cell populations and investigate their dynamic changes in gene expression and epigenetic patterns \nduring menstrual cycle and differentiation processes. These techniques enable the identification of rare cell popula‑\ntions, capture heterogeneity of cell populations within the endometrium, and provide potential targets for more \neffective therapies.\nKeywords Endometrial stem cells, Single‑cell analysis, ScRNA‑seq, ScATAC‑seq, Spatial transcriptomics\nIntroduction\nThe endometrium is a dynamic tissue that undergoes \ncyclic changes in response to ovarian hormones dur -\ning the menstrual cycle. These changes are critical for \nthe establishment and maintenance of pregnancy [1]. \nEndometrial stem cells are known to play a key role in \nendometrial regeneration and repair [2]. It has been \nshown that endometrial stem cells contribute to this pro-\ncess by differentiating into endometrial stromal cells [3], \nglandular epithelial cells [4], and vascular smooth mus -\ncle cells [5]. These differentiated cells then form a new \nfunctional layer of the endometrium. These cells have \nalso been implicated in the pathogenesis of endometrial \ndisorders such as endometriosis [2] and endometrial can-\ncer [6]. However, traditional bulk sequencing methods, \nwhich analyze the average gene expression or epigenetic \npattern across a population of cells, are limited in their \nability to capture the heterogeneity and complexity of \nendometrial stem cell populations within the dynamic \nOpen Access\n© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which \npermits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the \noriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or \nother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line \nto the material. 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The Creative Commons Public Domain Dedication waiver (http://creativecom‑\nmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.\nStem Cell Research & Therapy\n*Correspondence:\nHwa‑Yong Lee\nleehy@kangwon.ac.kr\n1 Department of Health Sciences and Technology, GAIHST, Gachon \nUniversity, Incheon 21999, Republic of Korea\n2 Department of Molecular Medicine, School of Medicine, Gachon \nUniversity, Incheon 406‑840, Republic of Korea\n3 Division of Science Education, Kangwon National University, \nChuncheon 24341, Republic of Korea\n\nPage 2 of 15Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \nendometrial tissue during menstrual cycle and differen -\ntiation process [7, 8].\nIn this context, various single-cell analysis techniques \nsuch as single-cell RNA sequencing (scRNA-Seq) [9, 10], \nsingle-cell ATAC sequencing (scATAC-seq) [10], and \nspatial transcriptomics [11] have been recently devel -\noped and become increasingly important for studying \nendometrial stem cells. Although there are still many \ntechnical limitations that require improvement, the uti -\nlization of single-cell analysis allows for addressing cel -\nlular heterogeneity by grouping similar cells together \nwhile separating dissimilar cells. The advantages and \ndisadvantages of the single-cell analysis platform are \nsummarized in Table  1 for a comprehensive overview. \nThis approach enables computational construction of a \npseudotime trajectory for a biological process based on \ntranscriptomic capture of unsynchronized cells from \nthe tissue. Further analysis can be performed to resolve \nfunctional processes through evaluation of differential \ngene expression across identified clusters or trajectories \n[9]. Therefore, single-cell analysis platforms enable us to \nidentify rare cell populations and capture heterogene -\nity of cell populations within the endometrium, which \nmight be targeted for more effective therapies for endo -\nmetrial diseases [12]. For example, scRNA-Seq has been \nused to identify distinct subpopulations of endometrial \nstromal cells including proliferative and secretory stro -\nmal cells during menstrual cycle and to characterize \ngene expression changes that occur during endometrial \ndifferentiation [13, 14]. The inclusion of epigenetic data \nsuch as chromatin accessibility is crucial for providing \na comprehensive understanding of the regulatory land -\nscape during development. In recent studies, researchers \nhave expanded transcriptomic analyses to incorporate \nsingle-cell assay for transposase-accessible chromatin \nsequencing (scATAC-Seq). This powerful method allows \nfor characterization of potential gene regulatory net -\nworks through identification of changes in accessible \nchromatin that occur with cell-state changes [15]. By \ncombining scATAC-Seq and scRNA-Seq, an unparalleled \nresolution can be achieved for a developing human endo-\nmetrium. Indeed, scATAC-seq has been used to identify \ncell type-specific epigenetic changes during menstrual \ncycle and to identify putative regulatory elements associ -\nated with endometrial differentiation [12, 16].\nIn this review, we will summarize current state-of-the-\nart techniques for various single-cell analyses of endome-\ntrial stem cells and their related diseases. We will discuss \nrecent studies that have used single-cell analysis to iden -\ntify and characterize distinct endometrial stem cell pop -\nulations and to investigate dynamic changes in gene \nexpression and epigenetic patterns of endometrial stem \ncells during menstrual cycle and differentiation into spe -\ncific cell lineages. We will also highlight potential roles \nof endometrial stem cells in the development and pro -\ngression of various uterine diseases through their ability \nto accumulate genetic mutations, express genes associ -\nated with uterine diseases, and interact with other cells \nwithin tissue microenvironment. Overall, this review \ndemonstrates the power of various single-cell analyses for \nadvancing our understanding of the molecular mecha -\nnisms underlying endometrial development and function \nas well as for developing new therapies for endometrial \nstem cell-related disorders.\nWhy single‑cell analysis can be an effective \nstrategy for studying endometrial stem cells?\nSingle-cell analysis has emerged as a powerful tool for \ninvestigating properties of endometrial stem cells. Single-\ncell analysis provides their gene expression, epigenetic \nTable 1 The advantages and disadvantages of the single‑cell analysis platform\nAdvantages of single-cell analysis Disadvantages of single-cell analysis\nHigh Resolution: Provides detailed insights into individual cell character‑\nistics\nTechnical Challenges: Complex and resource‑intensive procedures may \npose technical difficulties\nHeterogeneity Exploration: Allows for the study of cellular diversity \nwithin tissues\nCost: Can be expensive, particularly when analyzing large numbers of sin‑\ngle cells\nPrecision Medicine: Facilitates personalized treatment strategies based \non individual cell profiles\nData Analysis Complexity: Handling and interpreting large datasets require \nadvanced bioinformatics expertise\nRare Cell Detection: Enables the identification of rare cell populations \nwithin a sample\nCell Isolation Issues: Challenges in isolating and capturing individual cells \nwithout bias\nEarly Disease Detection: May detect cellular changes at early stages of dis‑\nease development\nLimited Sample Size: Some techniques may require a significant number \nof cells, limiting applications in samples with low cell numbers\nCell Fate Mapping: Helps in understanding cellular developmental trajec‑\ntories\nCell Stress Response: The process of isolating single cells can induce stress‑\nrelated changes in gene expression\nIdentification of Cell Types: Allows for the identification and characteriza‑\ntion of specific cell types\nStandardization Challenges: Lack of standardized protocols may lead \nto variability in results between studies\n\nPage 3 of 15\nLee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n \nmodifications, protein expression, and other molecular \nfeatures. One advantage of single-cell analysis is its abil -\nity to identify and characterize rare or heterogeneous cell \npopulations that might be missed by bulk tissue analysis \n[7]. For example, endometrial stem cells are a relatively \nrare population within the endometrium. It can be diffi -\ncult to isolate and study them using traditional methods. \nSingle-cell analysis allows researchers to identify and \nstudy these cells with greater precision. Another advan -\ntage of single-cell analysis is its ability to reveal molecular \nmechanisms underlying stem cell differentiation and self-\nrenewal [17]. By comparing gene expression profiles of \nstem cells at different stages of differentiation, research -\ners can gain insights into regulatory networks and signal -\ning pathways involved in these processes. Several reasons \noutlining the usefulness of single-cell analysis in endome-\ntrial stem cell research will be discussed.\nVarious types of endometrial stem cells\nEndometrial stem cells constitute a subset of cells \nendowed with the ability for self-renewal and multiline -\nage differentiation into various types of cells constitut -\ning the endometrial tissue [18, 19]. In fact, endometrium \nencompasses several types of tissue-resident stem cells, \nsuch as epithelial-like stem cells [4], stromal-like stem \ncells [3], and perivascular endometrial stem cells [20]. \nEach of these stem cell types possesses distinctive molec -\nular and functional properties.\nStem cells with epithelial characteristics are believed \nto reside within the basal layer of the endometrial tis -\nsue, situated proximately to the basal laminae. These \nepithelial-like stem cells exhibit the capacity for both self-\nrenewal and multilineage differentiation, enabling them \nto generate the glandular epithelium of the endome -\ntrial tissue. Epithelial-like stem cells undergo regulation \nthrough an intricate interaction with various endocrine \nand paracrine factors, such as hormones and/or growth \nfactors from the adjacent immune and stromal cells. \nFor instance, Janzen et al. revealed that the self-renewal \ncapacity and differentiation potential of EpCAM/CD44 \npositive epithelial-like stem cells, along with the Wnt/β-\ncatenin signaling and its downstream regulators such as \nAxin2, c-Myc, CD44, and ID2, can be regulated by pro -\ngesterone and estrogen [21].\nStem cells with stromal characteristics within endo -\nmetrial tissue are believed to reside in the perivascular \nregion of the stromal area, which functions as connec -\ntive tissue providing structural support for endometrial \nblood vessels and glands [22]. These stem cells pos -\nsess the capability to differentiate into various cell types \nsuch as endothelial cells, fibroblasts, and smooth mus -\ncle cells [23]. Moreover, stromal-like stem cells exhibit \nimmunomodulatory characteristics, potentially playing \na significant role in the regulation of immune responses \nwithin the endometrial tissue. According to the results of \nLeñero et  al., therapeutic potential of  CD146+ stromal-\nlike stem cells were found to be predominantly facilitated \nby secretion of a blend of enriched factors, including let-\n7e-5p, miR-182-3p, miR-320e, and miR-378 g [24]. These \nsecretory factors specifically interact with the immune \nsystem and influence angiogenesis by modulating mac -\nrophage polarization, T cell activity, and transcriptional \nregulation of various immune regulatory cytokines \nincluding IL-1β, IL-6, and TNF-α.\nPerivascular stem cells are situated within the perivas -\ncular region of the endometrial tissue, displaying the \ncapacity to differentiate into both stromal and epithe -\nlial cell types [25, 26]. It is believed that these stem cells \ncontribute to the establishment and upkeep of the vas -\nculature within the endometrium. These stem cells are \nidentified by the expression of distinct cell surface bio -\nmarkers, including CD146, PDGFRβ, and SUSD2 [2]. \nLi et  al. employed flow cytometry, utilizing antibodies \nagainst CD10, CD13, CD44, CD73, CD90, and CD105, \nto isolate perivascular endometrial stem cells in humans. \nThey illustrated the cells’ ability to undergo differentia -\ntion into adipocytes, neuron-like cells, and osteoblasts \n[27].\nHigh heterogeneity of endometrial tissue\nThe human endometrium exhibits remarkable cel -\nlular diversity, with various cell types contributing to \nits complex functions. Epithelial cells lining the lumi -\nnal surface undergo cyclic changes and participate in \nembryo implantation and glandular secretion [28]. Stro -\nmal cells that are highly plastic in nature can support \ntissue remodeling and respond to hormonal cues [29]. \nImmune cells including macrophages, natural killer cells, \nand T cells can interact with other cell types to regulate \nimmune responses and tissue remodeling [30]. Endothe -\nlial cells ensure proper vascularization and nutrient \nexchange. Supporting cells such as smooth muscle cells, \nperivascular cells, and fibroblasts provide structural sup -\nport [31]. Understanding the dynamic interplay and func-\ntional roles of these diverse cell populations within the \nendometrium is crucial for comprehending endometrial \nbiology, reproductive processes, and associated disor -\nders. Currently, understanding the generation of cellular \ndiversity from a single stem cell, regulatory mechanisms \ngoverning dynamic tissue regeneration, and utilization \nof this diversity to mount appropriate responses to exter -\nnal perturbations are central challenges in endometrial \nresearch [32].\nCellular diversity within the endometrium plays a cru -\ncial role in maintaining tissue functionality and adapt -\ning to environmental stimuli. However, unraveling \n\nPage 4 of 15Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \nmechanisms responsible for generating and regulating \nthis diversity remains a significant scientific endeavor. To \naddress these issues, Díaz-Gimeno et al. have performed \ncomprehensive transcriptomic profiling of the endome -\ntrium during various time points encompassing the mid-\nsecretory phase. Their research unraveled certain levels of \nmolecular characteristics of the endometrium associated \nwith the crucial window of implantation (WOI), a narrow \ntimeframe during which the endometrium is receptive to \nembryo implantation [33]. However, simple comparative \nanalysis of the transcriptome with traditional approaches \nbetween patients and healthy controls has provided lim -\nited insights into the underlying molecular mechanisms \ninvolved in uterine disorders such as endometriosis, \nrecurrent implantation failure (RIF), and recurrent preg -\nnancy loss (RPL) [34]. Cells within a given tissue have tra-\nditionally been perceived as functionally identical units. \nConventional detection methods often rely on capturing \noverall response of these cells [35]. Thus, conventional \ntechnologies commonly employ bulk population-level \nmeasurements, neglecting distinctive cellular behaviors \narising from cell-to-cell variations such as cell metabo -\nlism, differentiation, and growth [36]. The collective \nfunctionality of a complex tissue is actually derived from \na heterogeneous population of cells that exhibit subtle \nvariations among them. Moreover, the endometrium \nundergoes dynamic changes during the menstrual cycle, \nwhich further adds to its complexity (Fig. 1).\nIn this context, single-cell analysis provides a powerful \napproach for uncovering the hidden diversity and com -\nplexity within tissues. By studying individual cells instead \nof averaging their characteristics in the tissue, research -\ners can discern heterogeneity and understand functional \nimplications of these individual cellular differences. The \nability to study transcription patterns and epigenetic \nregulation in single cells was limited by technologi -\ncal constraints in the past. However, recent advances \nin bioanalytical technologies have enabled the study of \ntranscription pattern [37] and epigenetic regulation [38] \nin single cells, which in turn allow researchers to profile \ngene expression and signaling pathways of individual \ncells in a heterogeneous population, thereby providing a \nmore detailed understanding of the heterogeneity of the \nendometrium at a single-cell level. By examining gene \nexpression profiles of individual cells, researchers can \nidentify cell subpopulations and their molecular charac -\nteristics, including their differentiation potential, prolif -\neration rates, and interaction with neighboring cells. For \nFig. 1 Comprehensive landscape of identified cell types in normal endometrial tissue and the current understanding of their lineage hierarchy. This \nfigure provides an overarching depiction of the diverse cell types recognized within normal endometrial tissue, along with the existing perspective \non their lineage relationships. The pivotal role of endometrial stem cells as precursors for both epithelial and stromal cell lineages is outlined, \nunderscoring their significance in the generation of various differentiated cell types, each characterized by distinctive surface markers. Specifically, \nType I epithelial progenitor cells exhibit the capacity to undergo differentiation into columnar epithelial cells, ciliated epithelial cells, and secretory \nepithelial cells. Meanwhile, Type II epithelial progenitor cells demonstrate the potential to differentiate into basal epithelial cells and squamous \nepithelial cells. Furthermore, the lineage progression extends to secretory endometrial stromal cells, which exhibit the capability to differentiate \ninto perivascular stromal cells, fibrocytes, and myofibroblasts. Additionally, fibroblastic endometrial stromal cells manifest the ability to undergo \ndifferentiation into distinct cell types, including lipid‑rich cells, glycogen‑rich cells, and decidual cells. The figures presented in this article were \ncrafted by our group\n\nPage 5 of 15\nLee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n \nexample, Garcia-Alonso et al. have integrated scRNA-Seq \nand spatial transcriptomics to investigate the cellular \ncomposition and heterogeneity within the endometrium. \nThey analyzed approximately 100,000 cells from prolif -\nerative and secretory phases of the endometrium sourced \nfrom 15 women in their reproductive age [11]. Through \na meticulous examination of these cells, they successfully \nidentified and characterized 14 distinct clusters belong -\ning to the following five major cell type categories: (a) \nepithelial cells (b) endothelial cells, (c) immune cells, (d) \nstromal cells, and (e) supporting cells (including smooth \nmuscle cells, perivascular cells, and fibroblasts expressing \nthe cell marker C7) [11]. Recent advancements in single-\ncell technologies have facilitated more detailed investiga -\ntions into cellular composition and heterogeneity within \nthe endometrium, providing valuable insights into previ -\nously unrecognized cell subsets and their functional roles \n(Fig. 2).\nIdentification of rare stem cell subpopulations \nwithin endometrial tissue\nThe human endometrium possesses a remarkable regen -\nerative capacity, undergoing significant remodeling and \nregeneration during each menstrual cycle, resulting in the \ngeneration of 4–10 mm of a mucus layer [39]. The ability \nof the endometrium to undergo such extensive regen -\neration is attributed to the presence of resident epithelial \nprogenitors and stromal-like endometrial stem cells. [2, \n40]. The identification of endometrial stem cells with the \ncapacity to generate substantial colonies is a rare occur -\nrence, comprising only a small fraction of the overall cell \npopulation. Specifically, the presence of these stem cells \nhas been documented at frequencies of approximately \n0.08% for epithelial cells and 0.02% for stromal cells [41].\nIn spite of significant advancements in the study of \nendometrial stem cells, our understanding of their spe -\ncific identity, localization, and regulatory mechanisms \nremains largely elusive. Conventional approaches such as \nbulk RNA sequencing, while valuable, often obscure the \npresence and distinct gene expression patterns of these \ninfrequent cells within the complex endometrial tissue. \nHowever, unlike traditional methods, recent application \nof single-cell analysis enables identification and quan -\ntification of these rare stem cell subpopulations within \nthe endometrial tissue by assessing gene expression, epi -\ngenetic modifications, and cell surface markers at the \nindividual cell level without needing enrichment or iso -\nlation. For example, Garcia-Alonso et al. have employed \nscRNA-Seq to investigate epithelial cell populations \nwithin primary endometrial tissues and cells and identi -\nfied potential epithelial stem cell populations and their \nassociated markers. Notably, they revealed that a  SOX9+ \nepithelial subpopulation was a potential driver of epithe -\nlial regeneration within the endometrium [11].\nThey discovered distinct subsets within the identified \n SOX9+ epithelial subpopulation, including noncycling \n SOX9+/LGR5+ cells located in the surface epithelium, \n SOX9+/LGR5− cells present in the basal glands, and \nproliferating  SOX9+ cells found in the regenerating \nsuperficial endometrial layer. Such cellular dynamics \nand heterogeneity within the  SOX9+ subpopulation pro -\nvide valuable insights into the mechanisms of epithelial \nregeneration and underscore the complexity of endome -\ntrial stem cell behavior in different regions of the endo -\nmetrium. Queckbörner et  al. have found the presence \nof multiple stromal cell subpopulations in addition to \nknown endometrial cell types. These stromal subpopu -\nlations exhibited distinct characteristics, suggesting the \nexistence of specific stromal niches with potential regula-\ntory roles in inflammation and extracellular matrix com -\nposition [13]. They also identified ten different stromal \ncell clusters and two subsets of pericytes, highlighting the \ndiversity within the stromal compartment. Furthermore, \nthey delineated the diversity of cell clusters and estab -\nlished lineage trajectories utilizing various analytical \nplatforms, including SingleR, Seurat, and Velocyto [13]. \nWang et  al. have also undertaken comprehensive analy -\nsis of human endometrium at single-cell level throughout \nthe menstrual cycle. Notably, they identified transcrip -\ntomic transformations associated with critical events \nsuch as opening of the window of implantation, a piv -\notal phase in the endometrial preparation for embryo \nattachment. Additionally, they provided a systematic \nsingle-cell transcriptomic delineation of endometrial \ntransformation, enabling a detailed understanding of \ndiverse changes observed in various cell types, cellular \nstates, growth patterns, and differentiation processes \nthroughout the entirety of the human menstrual cycle \n[42]. Cao et  al. have characterized specific endometrial \ncell subpopulations as stem cell entities with regenera -\ntive potential by performing single-cell expression profil -\ning analysis. However, their results raised uncertainties \nabout the extent to which cultured putative endometrial \nstem cell population could faithfully represent the in vivo \npopulation of stromal cells expressing both biomarkers \nPDFGRB and MCAM [9].\nFunctional changes of endometrial stem cells \nduring the development of various endometrial diseases\nCurrently, the presence of abnormalities and mutations \nin endometrial stem cells is believed to be a crucial fac -\ntor in the initiation and progression of various endome -\ntrial diseases, including infertility associated with a thin \nendometrium, endometriosis, and endometrial cancers \n[43]. The achievement of a successful pregnancy hinges \n\nPage 6 of 15Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \non the growth of an embryo within an accommodating \nendometrium of sufficient thickness. Clinically, a thin \nendometrium is acknowledged as a contributing factor \nto infertility, recurrent pregnancy loss, and complications \nin placental development [44]. Consistently, Tewary and \ncolleagues presented their research findings, highlighting \nFig. 2 Profiling diverse endometrial cell types at single‑cell resolution throughout the menstrual cycle. Schematic outlining the sequential \nsteps involved in the isolation and individual‑level analysis of various cell populations from healthy endometrial tissue (A). UMAP visualization \ndepicting distinct cell clusters identified in endometrial samples collected during both the proliferative and secretory phases of the menstrual \ncycle. A total of 14 distinct endometrial cell clusters representing different menstrual cycle stages are highlighted on the UMAP plot (B). Distinctive \nexpression patterns of multiple biomarkers for each cell type were explored. Each data point on the visualization corresponds to an individual gene, \nwith the intensity of color reflecting the average expression level. Additionally, the size of each data point corresponds to the proportion of cells \nwithin the cell type that exhibit expression of the given gene (C). Heatmap of top differentially expressed genes in each endometrial consistent \ncell types during both the proliferative and secretory phases (D). Visualization of expression patterns for differentially expressed genes (DEGs) \namong different endometrial cell types during both the proliferative and secretory phases, presented through violin plots (E). The figures presented \nin this article were crafted by our group\n\nPage 7 of 15\nLee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n \na connection between reduced clonogenic populations of \nendometrial cells at the baseline and the varying degrees \nof recurrent pregnancy loss associated with impaired \nendometrial growth [45].\nEndometriosis is a common and non-malignant \ngynecological disease frequently characterized by pelvic \npain and infertility. This disease is defined by the exist -\nence of tissue resembling the endometrium in locations \noutside the normal uterus, and these ectopic tissues \nundergo substantial alterations influenced by hormonal \nfluctuations during the menstrual cycle [46, 47]. Indeed, \nMoggio et al. have observed that noted endometrial stem \ncells derived from endometriosis tissues demonstrated \nmarkedly enhanced self-renewal capacity, migratory \npotential, and angiogenesis when compared to stem cells \nfrom the same individual’s normal endometrial tissue or \nthose from healthy controls [48]. This result indicates \nthat the aberrant behavior exhibited by endometrial stem \ncells could potentially play a role in the development of \nendometriosis. Uzan et al. have observed a notable down-\nregulation in the expression levels of ARID1A, PTEN, \nand TNFα within  CD73+/CD90+/  CD105+ endometrial \nstem/progenitor cells in ectopic endometriosis tissue \nsamples when compared to normal endometrial tissue \nsamples [49]. This result indicates that irregular expres -\nsion of particular genes may make endometrial stem cells \nmore susceptible to the development of endometriosis.\nMoreover, there is a potential involvement of endo -\nmetrial stem cells in the development and progression \nof endometrial cancer [6], as they exhibit heightened \nself-renewal capacity and genetic instability in certain \ninstances. A suggested mechanism posits that genetic \nmutations may accumulate in endometrial stem cells, ini-\ntiating the transformation of these cells from their nor -\nmal state into cancerous cells. For instance, Syed et  al. \nhave proposed a hypothesis proposing the existence of \nstem/progenitor cells within endometrial glands that \nrespond to Wnt signaling pathway. They identified Axin2, \na recognized Wnt reporter gene, as a biomarker indica -\ntive of epithelial-like stem/progenitor cells located in the \nendometrial glands. [50].\nDynamic changes in gene expression and epigenetic \nmodifications throughout the menstrual cycle \nand the differentiation process\nThe human endometrium is a remarkable tissue that \nundergoes dynamic cyclic changes characterized by grad-\nual shedding of the surface epithelium and subsequent \nrapid restoration of tissue homeostasis. Apart from other \ntissues such as the skin, endometrium has a unique ability \nto efficiently repair itself without leaving any scar forma -\ntion. Dynamic interactions of various endometrial cel -\nlular components within the microenvironment further \nadd to the challenge of comprehending how different \ncell types are mobilized and coordinated to facilitate \nthese dynamic cyclic changes. In addition, endometrial \nstem cells involved in endometrial regeneration undergo \ndynamic changes in gene expression and epigenetic mod-\nifications throughout the menstrual cycle and the differ -\nentiation process.\nIn this context, single-cell analysis is a valuable tech -\nnique for investigating endometrial stem cells within \nthe endometrium due to their dynamic changes during \nthe menstrual cycle and high heterogeneity. For exam -\nple, Kirkwood et  al. have analyzed endometrial tissues \nof mice throughout the normal cycle by integrating \nscRNA-Seq and lineage tracing analysis. They identified \na previously unrecognized population of  PDGFRb+ mes-\nenchymal stem-like cells, referred to as repair-specific \nfibroblasts [14]. These cells exhibited the capability to \nundergo a transformative process from a mesenchymal \nstate to an epithelial state, allowing them to integrate \ninto the re-epithelialized luminal surface of the repaired \ntissue. This integrated analysis revealed the existence of \na unique population of wound-responsive, plastic endo -\nmetrial stromal fibroblasts. Remarkably, these fibroblasts \ndemonstrated their crucial role in rapid restoration of a \nfully functional luminal epithelium during the process \nof endometrial repair [14]. Similarly, Queckbörner et al. \nhave investigated endometrial samples obtained from \nhealthy fertile women during the proliferative phase \nof the menstrual cycle by employing single-cell analy -\nsis combined with advanced bioinformatics techniques. \nAlthough the sample size was limited to n = 3, results \nprovided valuable insights into the diverse landscape of \nstromal subsets within the endometrium [13]. These stro-\nmal cell subtypes exhibited different surface markers, cell \nstates, ECM compositions, and immune responses. For \nexample,  ISG15+ stromal subtype exhibited an expres -\nsion profile indicative of interferon-regulated genes and \n ACTA2+ stromal subtype displayed a consistent state \ncharacterized by a lower capacity for differentiation com-\npared to other cell populations in the perivascular envi -\nronment [13]. In addition, Kirkwood et al. have employed \na transgenic reporter mouse model along with single-cell \ntranscriptomics to establish a comprehensive repertoire \nof cell-specific markers for endometrial progenitor cell \npopulations and successfully identified three distinct \nsubpopulations of putative endometrial mesenchymal \nprogenitors [51]. These three identified mesenchymal \nprogenitor cell subtypes shared characteristic expression \nof PDGFRα and CD34 markers. However, they exhib -\nited distinct gene expression profiles, highlighting their \nunique functional attributes within the endometrium. \nThe first population demonstrated notable expression of \nNgfr, Spon2, and Angptl7 genes. The second population \n\nPage 8 of 15Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \nexhibited distinct expression patterns of Cxcl14, Smoc2, \nand Rgs2 genes. The third population, also identified as \ntype 1, showed specific expression of Clec3b, Col14a1, \nand Mmp3 genes. These genes are involved in the organi-\nzation and remodeling of the extracellular matrix, sug -\ngesting the involvement of this progenitor population in \nmaintaining structural integrity and functionality of the \nendometrial tissue [51].\nIn a previous study, Lucas et  al. have observed a loss \nof clonal mesenchymal stem-like cells in endometrium \nspecifically during the midluteal phase of patients with \nrecurrent pregnancy loss [52]. However, the underlying \nmechanisms linking this stem cell loss to the subsequent \ninfertility remained unclear. To address this gap, they \nfurther investigated and demonstrated that deficiency in \nstem/progenitor cells could contribute to a pro-senescent \ndecidual response during the peri-implantation window \nby performing scRNA-Seq for decidualizing primary \nendometrial stromal-like stem cell cultures. This in turn \nled to chronic inflammation in early pregnancy, prote -\nolysis of the decidual-placental interface, and ultimately \nmiscarriage. They identified SCARA5 as a biomarker \ngene for decidual cells, providing a valuable tool for dis -\ntinguishing and studying these specialized cells. On the \nother hand, DIO2 emerged as a marker gene for pro -\ngesterone-resistant senescent decidual cells [53]. These \nfindings contribute to our understanding of the complex \ninteractions and molecular processes involved in decidu -\nalization, implantation, and early pregnancy.\nVarious single‑cell analysis methods for studying \nendometrial stem cells\nSingle-cell RNA sequencing (scRNA-seq)\nIn the last two decades, extensive research has revealed \nthat numerous coding genes undergo dynamic changes \nin their expression levels within the endometrium dur -\ning different phases of the endometrial cycle [42, 54, 55]. \nWhile achieving reproducible data across studies has \nbeen challenging due to technical constraints, limitations \nin sample availability, high heterogenicity, and dynamic \nchange during menstrual cycle, our understanding of \ntranscriptional networks governing functional changes \nin the endometrium has significantly advanced. Notably, \na recent study has employed single-cell transcriptomic \nanalysis to provide crucial insights into transcriptional \nprofiles of individual cell types constituting the endome -\ntrium. By capturing and analyzing gene expression pro -\nfiles at the single-cell level, researchers have uncovered \nintricacies of transcriptional dynamics within the endo -\nmetrium [56]. scRNA-Seq has emerged as a powerful and \ntransformative tool that enables comprehensive evalua -\ntion of gene expression profiles and unravels intricate cel-\nlular compositions of the endometrium to comprehend \nits molecular complexity in thousands of individual cells \n[57]. By capturing transcriptomes of individual cells, \nresearchers have gained insights into unique molecular \nsignatures that define different cell types, states, and sub -\npopulations [58]. This technique has proven instrumental \nin characterizing heterogeneity of the endometrium as \nit enables identification and characterization of previ -\nously unrecognized subpopulations that might play criti -\ncal roles in endometrial physiology and pathology [59] \n(Fig. 3).\nFor example, Wang et  al. have characterized tran -\nscriptomic changes occurring in the functionalis layer \nof the endometrium, which undergoes cyclic shedding \nand regeneration throughout the menstrual cycle. They \nfocused on dynamic gene expression alterations at the \nsingle-cell level in stromal and epithelial cell compo -\nnents [42]. They observed notable enhanced expression \nof PAEP , GPX3, and CXCL14 in epithelial cells. These \ngenes serve as potential biomarkers for regulating the \nreceptive state of the endometrium during the window \nof implantation. On the other hand, gene expression \nchanges observed in stromal cells were more gradual and \ncontinuous, with genes such as FOXO1 and IL15 show -\ning notable upregulation. Interestingly, these alterations \nwere already detected earlier in the menstrual cycle, sug -\ngesting their involvement in preparing the endometrium \nfor embryo implantation even before the receptive win -\ndow. These transcriptomic markers could serve as valua -\nble tools for diagnosing impaired endometrial receptivity \nand improving successful implantation rates in in  vitro \nfertilization (IVF) treatments. Ren et al. have investigated \ndynamic changes in various cell components within \nendometrium during the transition from normal to endo-\nmetrial cancer to provide valuable insights into cellu -\nlar origins of endometrial cancer and identified specific \nsubpopulations associated with the tumorigenic process \nusing scRNA-Seq [60]. Through analysis, they discovered \nthat endometrial cancer originated from epithelial cells \nrather than stromal cells. More specifically, they identi -\nfied unciliated glandular epithelium as cellular source of \nendometrial cancer [60]. Furthermore, they identified a \ndistinctive subpopulation of cells that might play a cru -\ncial role in tumor development. These cells were char -\nacterized by the expression of LCN2, SAA1, and SAA2 \ngenes. These findings contribute to the broader under -\nstanding of endometrial cancer pathogenesis with poten -\ntial to inform the development of targeted therapies. \nKirkwood et al. have employed scRNA-Seq to investigate \nthe process of endometrial repair in a mouse model. They \nexamined transcriptomic profiles of three different trans-\ngenic mouse models and identified a previously unknown \nsubpopulation of  PDGFRb+ stromal-like stem cells that \nexhibited distinct transcriptomic changes specifically \n\nPage 9 of 15\nLee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n \nin response to endometrial dysfunction or damage [14]. \nThey also demonstrated that the plasticity and versatility \nof stromal fibroblasts could contribute to the restoration \nof the endometrium’s structural integrity by undergoing \na mesenchyme to epithelial transformation. Jiang et  al. \nhave explored dynamic changes occurring in mouse \nendometrial tissues during the post-implantation stage. \nThey made significant discoveries regarding the involve -\nment of different subtypes of endometrial stromal-like \ncells that played crucial roles in extracellular remodeling \nduring implantation [61]. Furthermore, their study shed \nlight on communication and interactions between endo -\nmetrial stromal cells, epithelial cells, and immune cells \nduring the implantation process. They also revealed that \nthese stromal cells engaged in communication with epi -\nthelial cells and immune cells through nectin and ICAM \nsignaling pathways during implantation.\nAssay for transposase-accessible chromatin using \nsequencing (ATAC-seq)\nIn eukaryotic cells, DNA is packaged and organized by \nhistones to form chromatin, a highly dynamic struc -\nture that undergoes reversible chemical modifications. \nThese modifications primarily include DNA methyla -\ntion and histone post-translational modifications. They \nplay crucial roles in various biological processes, includ -\ning gene regulation, genomic imprinting, and chromatin \nstability [62]. Gene expression is regulated by accessibil -\nity of chromatin, which is achieved through modulating \ninteractions between their target DNAs and transcrip -\ntion factors. Chromatin modifications play a crucial role \nin determining the packing level of chromatin, thereby \ninfluencing its accessibility. These modifications include \nDNA methylation, histone acetylation, methylation, \nphosphorylation, and many others [63]. By altering the \nchromatin structure, these modifications can regulate the \naccessibility of transcriptional factors to their target DNA \nsequences, ultimately affecting gene expression patterns \n[64]. Euchromatin characterized by open and accessible \nregions is particularly associated with the pluripotency \nof embryonic stem cells. In contrast, heterochromatin \nregions tend to increase during cellular differentiation \nprocesses, leading to a more compact and repressed \nchromatin state [65, 66]. Understanding the dynamic \nnature of epigenetic modifications and their impact on \ngene expression is crucial for unraveling complexities of \ndevelopmental processes, cellular differentiation, and dis-\nease etiology [67]. Advancements in epigenomic profiling \ntechnologies such as DNA methylation sequencing and \nchromatin immunoprecipitation sequencing (ChIP-Seq) \nhave provided valuable tools to investigate the epigenetic \nlandscape and its functional implications. In addition to \nthese conventional epigenomic profiling platforms, assay \nfor transposase-accessible chromatin using sequencing \nFig. 3 Comparison between Whole‑Genome RNA Sequencing (bulk RNA‑Seq) and Single‑Cell RNA Sequencing (scRNA‑seq) strategies. The top \npanel of the schematic illustrates the concept of bulk RNA‑Seq, which involves sequencing the entire transcriptome of a mixed population of cells. \nThis method provides an average measure of gene expression across all cells within the sample. In contrast, the bottom panel demonstrates \nthe methodology of scRNA‑seq, which enables a more precise exploration of cellular heterogeneity within a tissue or a specific cell subset. By \nindividually sequencing the RNA of single cells, scRNA‑seq uncovers the distinct gene expression patterns inherent to each cell. This approach, \ntherefore, mitigates the bias introduced by bulk RNA Seq and empowers high‑throughput molecular investigations with unprecedented single‑cell \nresolution. The figures presented in this article were crafted by our group\n\nPage 10 of 15Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n(ATAC-seq) has emerged as a transformative technique \nfor characterizing the gene regulatory landscape and \nquantifying chromatin accessibility at a single-cell level. \nIts ability to quantify chromatin accessibility at high reso-\nlution, its applicability to heterogeneous samples, and its \ncompatibility with other single-cell techniques make it \nan invaluable tool for deciphering complex mechanisms \ngoverning gene regulation in health and diseases [38] \n(Fig. 4).\nDecidualization, a crucial process in mammalian preg -\nnancy, involves transformation of undifferentiated endo -\nmetrial stem cells into specialized decidual cells. This \ntransformation occurs when the implanting embryo \nbreaches the luminal endometrial epithelium. Decid -\nual cells play a vital role in establishing a protective and \nnutritive environment around the developing embryo, \nfacilitating controlled trophoblast invasion and ensuring \nmaternal immune tolerance of the antigenically distinct \nfetus [ 68]. Despite the significance of decidualization, \nthe precise mechanisms governing the dynamic change \nof undifferentiated endometrial stem cells to decidual \ncells in the chromatin landscape during this process \nremain largely uncharacterized. In this context, Vrljicak \net  al. have investigated chromatin accessibility profiles \nin undifferentiated endometrial stem cells and upon \ndecidualization using ATAC-Seq. They revealed a notable \nreduction in chromatin accessibility during the process of \ndecidualization [69]. This reduced accessibility was spe -\ncifically associated with loss of binding motifs for certain \ntranscription factors (TFs) known to be repressed upon \ndecidualization. Runt-related transcription factors 1 and \n2 (RUNX1 and RUNX2), ETS Proto-Oncogene 1 (ETS1), \nand SRY-box  12 (SOX12) are among TFs with dimin -\nished binding motifs [69]. Their study provided valuable \ninsights into chromatin-level changes underlying the pro-\ncess of decidualization in human endometrial stem cells.\nThe combination of scRNA-Seq and scATAC-Seq rep -\nresents a powerful approach that allows for high-resolu -\ntion investigation of complex epigenetic events in tumor \nbiology. This integrated approach not only enables iden -\ntification and classification of distinct cell types within a \ntumor, but also provides insights into underlying mecha -\nnisms and pathways driving tumorigenesis beyond tradi -\ntional taxonomic classifications. Therefore, Regner et al. \nwere able to gain insights into the intratumoral hetero -\ngeneity and its impact on gene expression regulation by \ngenerating matched transcriptome and chromatin acces -\nsibility profiles at the single-cell level using scRNA-Seq \nFig. 4 Illustrative depiction of the methodology behind single‑cell ATAC sequencing (scATAC‑Seq) and the intricate mechanisms impacting \nchromatin accessibility. Chromatin accessibility, a pivotal indicator of a cell’s regulatory landscape, is profoundly shaped by a convergence \nof molecular events. At the DNA level, the methylation and acetylation of specific sites intricately modulate the affinity of diverse proteins, \nincluding transcription factors and enzymes involved in histone modification. The cumulative effect of these molecular interactions contributes \nto the selective silencing of particular genomic regions, orchestrating the cell’s gene expression program. The scATAC‑Seq employs a hyperactive \nvariant of the Tn5 transposase to elucidate accessible chromatin regions. Consequently, during scATAC‑Seq, the genome is treated with this \nmodified Tn5 transposase to identify open chromatin regions, revealing the dynamic accessibility of various genomic loci. The figures presented \nin this article were crafted by our group\n\nPage 11 of 15\nLee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n \nand scATAC-Seq combination [10]. They observed sub -\nstantial variations in chromatin accessibility among \nmalignant cells derived from the same patients. This vari-\nation in chromatin accessibility was found to be directly \nlinked to transcriptional output, indicating that changes \nin chromatin structure might play a crucial role in driv -\ning gene expression patterns within tumors.\nSpatial transcriptomics\nRecent advancements in scRNA-Seq technologies have \nenabled exploration of single-cell transcriptome in vari -\nous contexts, including human endometrial tissue and \nmouse uterus throughout different menstrual cycle and \npre-/post-implantation [42, 51, 70, 71]. While scRNA-\nSeq provides valuable insights into cellular heterogene -\nity and gene expression profiles at the single-cell level, it \nlacks spatial information, which is lost during single-cell \nisolation process [72]. This limitation hinders compre -\nhensive understanding of cellular interactions within the \ntissue. To overcome this challenge, spatial transcriptomic \ntechnologies, which allow for spatial assessment of gene \nexpression patterns at a single-cell levels within intact tis-\nsue sections, have garnered significant attention [73]. By \npreserving the spatial context of cells, spatial transcrip -\ntomics not only provides insights into the localization \nand heterogeneity of cell populations within the endo -\nmetrium, but also reveals how neighboring cells influ -\nence the behavior and function of individual cells [74, 75] \n(Fig. 5).\nGarcia-Alonso et al. have performed an in-depth inves -\ntigation into cellular states and spatial organization of \nhuman endometrial cells during different phases of the \nmenstrual cycle in women of reproductive age integrat -\ning scRNA-Seq and spatial transcriptomic profiling [11]. \nThey identified specific spatial coordinates associated \nwith distinct subsets of cells expressing transcription \nfactor SOX9. They observed that noncycling  SOX9+/\nLGR5+ cells were predominantly enriched in the sur -\nface epithelium of the endometrium. On the other hand, \nnoncycling  SOX9+/LGR5− cells were primarily located \nwithin basal glands. Additionally, their study revealed \nthat cycling  SOX9+ cells were predominantly mapped to \nglands within the growing superficial layer of the endo -\nmetrium. These findings have implications for under -\nstanding dynamic changes occurring in the endometrium \nthroughout the menstrual cycle and provide a foundation \nfor further investigations into roles of these specific cell \nsubsets in endometrial function, regeneration, and estab -\nlishment of receptive conditions for embryo implan -\ntation. Li et  al. have employed a combination of spatial \ntranscriptomics and scRNA-Seq analyses to investigate \nlocal gene expression patterns at the site of implanta -\ntion on pregnancy [76]. This approach allowed them to \ngain a comprehensive understanding of gene expression \npatterns and cellular compositions within the micro -\nenvironment at this critical stage of pregnancy. Spatial \ntranscriptomic analysis enabled the characterization of \n11 distinct domains, each characterized by unique gene \nFig. 5 A comprehensive depiction of spatial transcriptomic analysis. Spatial Transcriptomics represents a pioneering approach that facilitates \na meticulous exploration of gene expression patterns within the context of tissue sections. By capturing high‑resolution gene expression data \nwhile preserving the intricate tissue architecture, spatial transcriptomic datasets not only provide precise gene expression measurements \nbut also impart a profound understanding of gene activity within its native tissue microenvironment. The figures presented in this article were \ncrafted by our group\n\nPage 12 of 15Lee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \nsignatures [76]. Yu et al. have also utilized a combination \nof spatial transcriptomics data scRNA-Seq datasets to \ninvestigate cellular compositions and molecular interac -\ntions within endometrial cancer tissue slices [77]. They \nobserved that two subclusters of epithelial cells, namely \nblood endothelial cells and lymphatic endothelial cells, \nexhibited a more malignant phenotype. Their malig -\nnant phenotype might be conferred through activation \nof the MK pathway by MDL-NCL signal cascades [77]. \nThis signaling mechanism potentially plays a role in pro -\nmoting malignancy in endothelial cells associated with \nendometrial carcinoma. Notably, NCL was found to be \nassociated with suppressed immune activity, indicating \na potential mechanism through which endometrial car -\ncinoma cells could inhibit immune cells within the tumor \nmicroenvironment. The integration of spatial transcrip -\ntomics and scRNA-Seq data provides valuable insights \ninto molecular interactions and cellular heterogeneity \nwithin endometrial cancer, contributing to our under -\nstanding of tumor progression and potential therapeutic \ntargets.\nConclusions\nSingle-cell analysis has emerged as a powerful tool for \ninvestigating properties of endometrial stem cells. For \ninstance, scRNA-Seq has unveiled dynamic changes in \nthe functionalis layer during the menstrual cycle, provid -\ning potential biomarkers for endometrial receptivity and \naiding in IVF treatments. In the context of endometrial \ncancer, scRNA-Seq identified specific cellular origins and \nsubpopulations associated with tumorigenesis, inform -\ning potential targeted therapies. Additionally, scRNA-\nSeq has been instrumental in studying endometrial \nrepair, post-implantation dynamics, and communica -\ntion between stromal cells, epithelial cells, and immune \ncells. Combining scRNA-Seq and scATAC-Seq offers a \npowerful approach to investigating epigenetic events in \ntumor biology, uncovering intratumoral heterogeneity \nand its impact on gene expression regulation. Integrat -\ning scRNA-Seq and spatial transcriptomics has identi -\nfied specific cellular states and spatial coordinates in \nthe endometrium throughout the menstrual cycle. This \napproach has also been applied to study implantation \nsites during pregnancy and explore molecular interac -\ntions and cellular heterogeneity in endometrial cancer, \ncontributing to our understanding of tumor progression \nand potential therapeutic targets.\nHowever, there are still some limitations that need to \nbe addressed to fully harness the potential of single-cell \nanalysis in endometrial stem cell research. One major \nlimitation is the lack of standardization in sample prepa -\nration and data analysis. Single-cell analysis involves iso -\nlation of individual cells, which can be challenging due to \ntechnical variations and heterogeneity of endometrial tis-\nsues that limit the accuracy and reproducibility of down -\nstream analyses. To overcome these challenges, several \nstrategies have been developed for cell isolation, includ -\ning fluorescence-activated cell sorting (FACS) and mag -\nnetic-activated cell sorting (MACS). FACS and MACS \nrelying on labeling of cells with fluorescent or magnetic \nmarkers, respectively, are widely used methods for cell \nisolation. These methods enable the selection and isola -\ntion of specific cell populations based on their surface \nmarkers or other molecular features. In addition, the use \nof multiple complementary cell isolation methods such as \nFACS and MACS can help validate and cross-reference \nresults from single-cell analysis studies. Furthermore, the \nintegration of cell isolation with other single-cell analy -\nsis techniques such as spatial transcriptomics and multi-\nomics approaches can provide a more comprehensive \nand detailed understanding of molecular and functional \nproperties of endometrial stem cells.\nOther limitations of single-cell analysis include its \nhigh cost and complexity. The equipment and reagents \nrequired for single-cell analysis can be expensive and \nthe data generated from single-cell analysis can be com -\nplex and difficult to interpret. One major cost driver in \nsingle-cell analysis is the cost of sequencing, which can \nbe a significant expense for studies that require large-\nscale sequencing of individual cells. To overcome these \ncost limitations, several strategies have been developed \nto reduce the cost of single-cell analysis. One approach \nis to use targeted sequencing methods such as single-cell \ntargeted sequencing (scTSS) to enable sequencing of a \nfocused set of genes or genomic regions at a lower cost \nthan whole-genome or whole-transcriptome sequenc -\ning. Another approach is to use pooling strategies such as \ncell hashing or split-pool barcoding to enable sequencing \nof multiple cells in a single sequencing reaction, thereby \nreducing the cost per cell. In addition, the use of efficient \ndata processing and analysis pipelines, such as those \nbased on machine learning or deep learning algorithms, \ncan help reduce computational resources required for \ndata analysis.\nDespite these limitations, various single-cell analysis \nplatforms have been used to investigate endometrial \nstem cells. One of the main advantages of single-cell \nanalysis is that it can reveal cell-to-cell variations that \nmight be masked by bulk analysis. By analyzing indi -\nvidual cells, researchers can identify rare cell types or \nsubpopulations that might be missed by bulk analy -\nsis. They can also identify cell-to-cell variations in \ngene expression or other cellular features that might \nbe important for understanding stem cell function. \nIn addition, by analyzing gene expression patterns of \nindividual cells over time, researchers can identify key \n\nPage 13 of 15\nLee and Lee  Stem Cell Research & Therapy          (2023) 14:379 \n \nsignaling pathways and regulatory factors that drive \nstem cell differentiation. They can also investigate the \nrole of environmental cues in modulating stem cell \nfate. In the future, there is a need for more compre -\nhensive and integrated single-cell analysis approaches \nthat can simultaneously measure multiple aspects of \nendometrial stem cells, such as their gene expression, \nepigenetic modifications, protein expression, and func -\ntional properties. Additionally, the development of new \ntechnologies for single-cell analysis, such as spatial \ntranscriptomics and multi-omics approaches, will be \nimportant for advancing our understanding of endo -\nmetrial stem cell biology and their roles in reproductive \nhealth and disease.\nAbbreviations\nChIP‑Seq  Chromatin immunoprecipitation sequencing\nCXCL14  C‑X‑C motif chemokine ligand 14\nDIO2  Iodothyronine deiodinase 2\nECM  Extracellular matrix\nETS1  ETS proto‑oncogene 1\nFACS  Fluorescence‑activated cell sorting\nGPX3  Glutathione peroxidase 3\nICAM  Intercellular adhesion molecule\nIVF  In vitro fertilization\nLCN2  Lipocalin‑2\nLGR5  Leucine rich repeat containing G protein‑coupled receptor 5\nMACS  Magnetic‑activated cell sorting\nMCAM  Melanoma cell adhesion molecule\nPAEP  Progestagen associated endometrial protein\nPDFGRB  Platelet derived growth factor receptor Beta\nRPL  Recurrent pregnancy loss\nRUNX1  RUNX family transcription factor 1\nSAA1  Serum amyloid A1\nscATAC‑Seq  Single‑cell ATAC sequencing\nscRNA‑Seq  Single‑cell RNA sequencing\nscTSS  Single‑cell targeted sequencing\nSCARA5  Scavenger receptor class A member 5\nSOX9  SRY‑box transcription factor 9\nTFs  Transcription factors\nWOI  Window of implantation\nAcknowledgements\nWe would like to acknowledge the reviewers for their helpful comments on \nthis paper.\nAuthor contributions\nJWL and HYL designed manuscript, organized manuscript, and wrote the \npaper. All authors read and approved the final manuscript.\nFunding\nThis work was supported by the National Research Foundation of Korea (NRF) \ngrant funded by the Korea government (MSIP) (NRF‑2020R1I1A2061281).\nAvailability of data and materials\nNot applicable.\nDeclarations\nEthics approval and consent to participate\nNot applicable.\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no conflicts of interest.\nReceived: 21 August 2023   Accepted: 14 December 2023\nReferences\n 1. Fitzgerald HC, Schust DJ, Spencer TE. In vitro models of the human \nendometrium: evolution and application for women’s health. Biol Reprod. \n2021;104(2):282–93.\n 2. Cousins FL, Filby CE, Gargett CE. Endometrial stem/progenitor cells‑\ntheir role in endometrial repair and regeneration. Front Reprod Health. \n2021;3:811537.\n 3. Cheung VC, Peng CY, Marinic M, Sakabe NJ, Aneas I, Lynch VJ, Ober C, \nNobrega MA, Kessler JA. 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Front Immunol. \n2023;14:1145300.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in pub‑\nlished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}