{"paper_id":"b7d1dc64-b081-4fc8-9c91-bb8bbea0abda","body_text":"|\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 1 of 46\nEndometrial epithelial cells with high\nALDH activity control uterine\ndevelopment and regeneration\nSuni Tang1,2, Anna Catherine Unser1,2, Peixin Jiang1, Sydney E Parks1,2, Genesis J Herrera1,2, Ting Geng1,2,\nLinda Alpuing-Radilla3, Brooke A Thigpen3, Xiaoming Guan3, Diana Monsivais1,2\n1Department of Pathology & Immunology, Baylor College of Medicine, Houston, United States • 2Center for Drug\nDiscovery, Baylor College of Medicine, Houston, United States • 3Department of Obstetrics and Gynecology, Baylor\nCollege of Medicine, Houston, United States\neLife Assessment\nThis valuable study reports that the ALDH-abundant cells display stem cell properties and\nmay play a key role in the endometrial epithelial development in the mouse. The data\nsupporting the main conclusion are solid, although further improvements are needed to\nstrengthen the conclusions. This work will be of great interest to reproductive biologists\nand biomedical researchers working on women's reproductive health.\nhttps://doi.org/10.7554/eLife.110975.1.sa3\nAbstract\nAdult stem cells are thought to drive the regenerative potential of the endometrium and\ncontribute to the pathogenesis of endometriosis, however, their identity and defining features\nremain to be characterized. Here, we used in vivo and in vitro approaches to demonstrate that cells\nwith high aldehyde dehydrogenase 1 activity (ALDHHI cells) were long lived progenitors in the\nendometrial epithelium with a higher organoid formation capacity, long-term passaging potential,\nand stemness gene signatures. Using lineage tracing with an Aldh1a1cre/ERT2; ROSA26tdTomato\nreporter mouse, Aldh1a1+ cells expanded during postnatal development, estrus cycling, and\nfollowing post-partum repair. In response to ovariectomy or exogenous estradiol, we found that\nALDH1A1+ cells localized to glandular crypts of the endometrium or throughout the luminal\nepithelium, respectively, indicating that their spatial localization is hormone sensitive.\nFunctionally, we found that selective ablation of ALDH1A1+ cells in Aldh1a1cre/ERT2; ROSA26-\nDTRflox/flox mice decreased endometrial gland number and FOXA2 expression. These findings\nwere recapitulated in the human endometrium, where endometrial epithelial organoids with high\nALDH activity (ALDHHI cells) showed a higher organoid formation capacity than ALDHLO cells and\ndisplayed unique transcriptomes with fewer luminal-like ciliated cells. Overall, our studies\nindicate that ALDH1A1+ cells are hormone-sensitive adult stem cells in the endometrium with\nregenerative potential that are critical for endometrial development and function.\nIntroduction\nThe endometrium is the inner lining of the uterine cavity whose unique regenerative potential is\nfueled by stem cells (Cousins et al., 2021     ). The location and identity of these adult stem cells is an\nactive area of investigation due to the high prevalence of endometriosis, adenomyosis, and other\nmenstrual-related pathologies that arise from endometrial tissue. Recent studies have used\nclassical stem cell assays to identify CDH2+, SSEA1+, and SUSD2+ progenitors that are enriched in\nthe basalis endometrium and localize to the perivascular regions of the endometrium (Masuda et\nal., 2012     ; Nguyen et al., 2017     ; Valentijn et al., 2013     ). Using these markers, several groups\nhave detected CDH2+, SSEA1+, SOX9+, and SUSD2+ stem cells in the menstrual effluent and\nFor correspondence:\ndmonsiva@bcm.edu\nCompeting interests: Additional\nDeclarations: The authors declare no\ncompeting interests.\nFunding: See page 30\nReviewing editor: Wei Yan,\nWashington State University, United\nStates\n© 2026, Tang et al. This article is\ndistributed under the terms of the\nCreative Commons Attribution\nLicense, which permits unrestricted\nuse and redistribution provided that\nthe original author and source are\ncredited.\nReviewed Preprint\nv1 • May 6, 2026\nNot revised\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 2 of 46\nperitoneal fluid of women with endometriosis (Hapangama et al., 2019     ; Masuda et al., 2021     ).\nThese studies support the hypothesis that stem cells present in menstrual effluent enter the\nperitoneal cavity during retrograde menstruation and are a key factor in the establishment of\nendometriosis (Bulun, 2022     ; Sampson, 1927     ). Therefore, defining stem cell identity and\nsignaling networks within the eutopic endometrium is key to developing novel targeted\napproaches for the treatment of ectopic endometriotic lesions and their associated pain,\ninflammation, and infertility.\nThe mouse has been used to identify stem cell markers driving the regenerative potential of the\nendometrium. Specifically, lineage tracing studies identified a putative stem cell niche that is\nenriched at the junctional zone between the luminal and glandular epithelium in the\nendometrium (Jin, 2019     ). Studies tracing the fate, ablation, and proliferative capacity of Lgr5+\ncells in the uterus identified an Lgr5+ niche that is enriched in the crypts of the glandular\nepithelium and promotes endometrial regeneration (Seishima et al., 2019     ). Similarly, using\nlineage tracing, ablation, and organoid models, Axin2+ cells were shown to be localized within the\nglandular crypts of the mouse endometrium, driving normal homeostasis and oncogenic\ntransformation in the endometrium (Syed et al., 2020     ).\nMaintenance of endometrial architecture during the estrus cycle and the structural remodeling of\nthe postpartum period involves the coordinated expansion and differentiation of stem cells. We\nidentified that conditional inactivation of the TGFβ receptor, ALK5, impairs postpartum\nendometrial remodeling, leading to structural defects, tumors, and lung metastases (Monsivais et\nal., 2019     ). We also showed that conditional inactivation of the downstream effectors of TGFβ,\nthe SMAD2 and SMAD3 transcription factors, resulted in aggressive and metastatic endometrial\ntumors (Kriseman et al., 2019     ; Kriseman et al., 2023     ). When cultured in vitro, organoids\nderived from mice with conditional SMAD2/3 inactivation, as well as control organoids treated\nwith A83-01 (an ALK4/5/7 inhibitor), developed an altered morphology and elevated expression of\nretinoic acid signaling molecules, including elevated expression of aldehyde dehydrogenase 1a1\n(Aldh1a1) (Kriseman et al., 2023     ) (Tojo et al., 2005     ). Furthermore, endometrial epithelial\norganoids from mice lacking TGFBR2 become stratified and abnormally express keratin 5 and p63,\nhighlighting its critical role in epithelial cell type specification (Parks et al., 2025     ). Thus,\ndisruption of TGFβ signaling impaired organoid and endometrial homeostasis through alterations\nin the retinoic acid pathway.\nOne important enzyme in retinoic acid metabolism is aldehyde dehydrogenase 1a1 (ALDH1A1),\nwhich catalyzes the conversion of retinaldehyde into retinoic acid (Haselbeck et al., 1999     ).\nPrevious studies suggested that high expression and activity of ALDH1A1 is a putative stemness\nmarker in the endometrium of women and mice (Wu et al., 2017     ). In the postnatal mouse\nendometrium, ALDH1A1 is abundantly expressed throughout the epithelium suggesting a role in\ndevelopmental uterine maturation (Spencer et al., 2023     ). In humans, ALDH1A1 is enriched in\nthe basalis endometrial epithelium, where it colocalizes with endometrial stem cell marker CDH2\nand is expressed in ectopic endometriotic lesions on the ovary (Ma et al., 2020     ). Here, we used\nlineage tracing, organoid formation assays, and transcriptomic analyses to characterize the\ncontribution of ALDH1A1+ stem cells in the endometrium.\nResults\nMouse ALDHHI cells have a higher organoid formation capacity\nand stem-like signatures than ALDHLO cells\nTo define the stem-like characteristics of mouse endometrial epithelial cells with high aldehyde\ndehydrogenase activity, we used the ALDEFLUOR assay (Storms et al., 1999     ), which separates\ncells by their ability to metabolize a BODIPY-labeled ALDH substrate (Figure 1A-B     ). We collected\nadult female WT mice during the estrus phase and dissociated their endometrial epithelial cells to\nsort by FACS into ALDHHI and ALDHLO populations using the ALDEFLUOR assay (Figure 1A-C     ).\nBecause organoid formation ability is indicative of stemness, we expanded the cells and subjected\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 3 of 46\nALDHHI and ALDHLO cells to organoid formation assays and transcriptomic profiling by RNAseq.\nCompared to ALDHLO organoids, ALDHHI organoids had a higher organoid formation rate (0.01%\nvs. 2.69%, n=3 and n=5, p<0.05), perimeter (664 ±119 vs. 936 ±117 μm, p<0.05) and area (33,017\n±10,580, vs. 65,068 ±17,014 μm2, p<0.05) (Figure 1D-H     ).\nUsing RNAseq we identified that 346 transcripts were increased and 674 transcripts were\ndecreased (>1, <-1 log2FC, Adj. p-value <0.005) in ALDHHI vs. ALDHLO organoids (Figure 1I     ).\nGene ontology analysis of the DE genes showed that compared to ALDHLO cells, the ALDHHI cells\nhad decreased expression of genes involved in ‘Aurora B signaling’ and ‘E2F transcription factor\nnetwork,’ with genes such as Ccnb2, Ccna2, Ccne1, Aurka, Mki67, and Bub1, being significantly\ndecreased in ALDHHI vs. ALDHLO cells (Figure 1I-J     , Supplemental Figure 1A     , Supplemental\nTable S1     ). Genes involved in categories related to stem cell activity, such as ‘BMP receptor\nsignaling’ were increased in the ALDHHI vs. ALDHLO cells, with genes such as Bmp4, Bmp5, Bmp6,\nFst, Cdh2, Lgr5, Wnt9a, and Fzd3 showing higher expression. To identify similarities with other\nstemness cells in the mouse endometrium, we compared the DE genes in the ALDHHI vs. ALDHLO\nlist with the DE genes from the Axin2HI vs. Axin2LO mouse endometrial epithelial cells (Syed et al.,\n2020     ). We found that 19 genes were increased, and 19 genes were decreased in both ALDHHI\nand AXIN2HI cell populations (Supplemental Figure 1B     , Supplemental Table S1     ). Specifically,\nLgr5, Calb1, and Msx2, were all identified as increased in both cell types (Figure 1K     ).\nKeratin genes encode intermediate filament proteins that are used to classify epithelial subtypes\n(i.e., basal vs. simple columnar epithelium) but can also indicate progenitor state, differentiation\nstatus, or wound-healing response (Cohen et al., 2022     ). We found differences in keratin gene\nexpression between ALDHHI and ALDHLO cells (Supplemental Figure 1C     , Supplemental Table\nS1     ), with ALDHHI cells showing higher expression of Krt17, which is associated with cell\ndifferentiation, wound healing responses, and localizes with SOX9+/LGR5+ progenitors in the\nhuman endometrium (Cohen et al., 2024     ; Garcia-Alonso et al., 2021     ). ALDHLO cells had higher\nexpression of several other keratin-related genes (i.e., Krt15, Krt4, Krt12) when compared to\nALDHHI cells, suggesting unique differentiation potential between the two cell types. Because of\nthe critical roles of the steroid hormones on epithelial cell proliferation, we also analyzed the\nexpression levels of the estrogen (Esr1) and progesterone receptors (Pgr) and observed\ncomparable levels between ALDHLO and ALDHHI cells (Supplemental Table S1     ). Overall, these\nstudies show that ALDHHI cells display enhanced clonogenic and regenerative capacity in\norganoid assays, consistent with an adult stem cell state.\nEndometrial Aldh1a1+ cells display gene expression signatures\nconsistent with a stem cell state\nPrevious studies used scRNAseq analyses of postnatal uteri to identify a niche of potential\nendometrial stem cells that repopulate the regenerating endometrium throughout life (Spencer et\nal., 2023     ; Wu et al., 2017     ). Other studies have analyzed endometrial epithelial cells of adult\ncycling mice but have captured only few epithelial cells, preventing in-depth analyses of cells with\nstem/progenitor signatures (Winkler et al., 2024     ). We enriched endometrial epithelial cells from\nadult cycling wild-type (WT) mice and analyzed the expression of approximately 5,984 total cells\nfrom mice during the estrus phase and 3,995 cells from mice in diestrus phase using scRNAseq on\nthe 10X Genomics platform (Figure 2     ). We obtained between 55,000-98,000 reads per cell and\ndetected 18,000-24,000 genes per cell. Cells were clustered on Seurat version 5.2.0 using uniform\nmanifold approximation and projection (UMAP). We then classified cell types by identifying\ndifferentially expressed (DE) genes between the clusters and using markers that were previously\ndescribed (Wang et al., 2023     ; Wang et al., 2020     ; Winkler et al., 2024     ). This classification\nmethod identified eight different cell types (epithelial, stromal, macrophages, mesothelial, natural\nkiller cells (NK), eosinophils, endothelial, and T-cells) (Figure 2A-B     ) within our samples. Of these\ncells, approximately 7,569 (4,488 from estrus, 3,081 from diestrus) were classified as epithelial\ncells, indicating that we effectively enriched the epithelial cell population.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 4 of 46\nFigure 1.Mouse endometrial epithelial ALDHHI cells have higher organoid formation capacity and\nstemness transcriptomes.\nA-C) Strategy to isolate and establish ALDHHI and ALDHLO cells from adult WT mouse epithelial organoids using the\nALDEFLUOR assay. D-E) Organoid formation assay performed by plating equal numbers of viable ALDHLO (D) and ALDHHI (E)\nepithelial cells and culturing for two weeks. F-H) Organoid formation rate (F), organoid perimeter (G) and area (H) were\nassessed by quantifying the total number of organoids formed per 100 cells seeded. Assays were performed using the cells\npooled from 7-9 WT adult mice at estrus three independent times. I-J) Transcriptomic profiling of ALDHHI vs. ALDHLO mouse\norganoids was performed, and the total number of differentially expressed genes (DEG) was determined (I). The total\nnumber of up- and down-regulated genes was displayed as a volcano plot. J) Gene ontology analysis was performed on the\nup- and down-regulated genes between the ALDHLO vs. ALDHHI organoids. K) Comparison of total genes that are conserved\nas up- or down-regulated between ALDHHI/AXIN2HI and ALDHLO/AXIN2LO cell, selected genes involved in stemness\nsignatures are displayed. Graphs show mean ± SEM and analyzed using a non-parametric Mann-Whitney Test, *, p<0.05; **,\nP<0.01; ***, p<0.001.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 5 of 46\nFigure 2.ALDH1A1 is expressed in cells with epithelial stem cell characteristics.\nA-B) UMAP displaying the various cell types identified by scRNAseq of enriched endometrial epithelium from WT 6-week-old\nmice in the estrus (A) and diestrus (B) phases. Epithelial cells were sub-clustered to identity different cell identities (C) and\nclassified into luminal, glandular, and epithelial stem cell (EpSCs) based on the expression of key markers (D, estrus; E,\ndiestrus). F-I) Pseudotime analysis of the epithelial cell types in estrus (F, G) and diestrus (H,I) was performed to identify the\ntrajectory of differentiation, which shows that EpSCs give rise to glandular and epithelial cell lineages. J-L) Dual feature plots\nshowing the overlapping and unique expression patterns of Aldh1a1/Lgr5 (J), Aldh1a1/Axin2 (K), and Lgr5/Axin2 (L) in the\nepithelial cell clusters in estrus. M-O) A signature score was assigned to the epithelial cells from the estrus phase to\ndetermine how strongly genes involved in ‘Glandular Epithelial Development’ (M), ‘BMP signaling’ (N), and ‘Stereocilium’ (O)\nare expressed. P-S) ALDH1A1 immunohistochemistry in the uterus of adult WT mice during the diestrus (P-P’) and estrus (Q-\nQ’) phases, or in WT ovariectomized mice without (R-R’) or with an E2 pellet for 90 days (S-S’). T) Aldh1a1 was also quantified\nin the uterus of 6-8-week-old WT mice collected at different times during the estrous cycle. Experiments were repeated in\nmore than three mice per group. Data in T are displayed as mean ± SEM analyzed by a One-Way ANOVA test with a Tukey’s\npost-hoc test. *, p<0.05; **, P<0.01; ***, p<0.001. UMAP, uniform manifold approximation and projection; EpSC, epithelial\nstem cell; BMP, bone morphogenetic protein.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 6 of 46\nWe performed further analyses in the epithelial cells by sub-clustering them with previously\nannotated markers of the luminal (Ifi203, Pla2g2e, Itgam, Cdc42ep2, Lrrc26, Irag2, Cyp21a1, Adrg7)\nand glandular epithelium (Sult1d1, Napsa, Gpx3, Klk1, Foxa2) (Padilla-Banks et al., 2023     ) (Figure\n2C     , Supplemental Figure S2D-E     ). To identify whether a niche of epithelial stem cells (EpSC)\nwas represented in each phase, we analyzed the expression of Aldh1a1, Axin2, Lgr5, Gstm7, En2,\nand Wnt7a in the epithelial cells and found that Aldh1a1, Axin2 and Lgr5 were enriched in clusters\n4, 25, and 13 (Figure 2D-E     , Supplemental Figure S2A-B     ). Coexpression of Aldh1a1, Lgr5, and\nAxin2 in these cells could be observed in the dual feature plots of epithelial cell subclusters from\nmice in estrus (Figure 2J-L     ). EpSCs present in clusters 4 and 25 had lower expression of\nproliferation markers Top2a and Mki67 relative to other clusters, suggesting that they were in a\nquiescent state (Figure 2D-E     , Supplemental Table S2     ). In addition to expressing Aldh1a1,\nAxin2, and Lgr5, cluster 13 also expressed Top2a and Mki67, indicating that these cells are less\nquiescent (Figure 2D-E     ). Other notable differences included expression of Aldh1a1 in the\nluminal cell cluster 14 in the diestrus phase epithelium (Figure 2E     ), a shift that we also observed\nby ALDH1A1 IHC (Figure 2P-Q’     ).\nTo further characterize the transcriptomes of quiescent EpSCs, we analyzed differentially\nexpressed (DE) genes in clusters 4 and 25 versus the other epithelial cells in our dataset\n(Supplemental Table S2     ), which showed that approximately 170 conserved genes were\nincreased (>1 log2FC, Adj. p-value <0.01) and 52 were decreased (<-1 log2FC, Adj. p-value, 0.01) in\nthe EpSC of mice analyzed during both the estrus and diestrus phases. Differentially expressed\ngenes included Calb1, Lpar3, Cited4, and Tgfbi, whose expression increases in the epithelium\nduring postnatal uterine maturation (Spencer et al., 2023     ). Susd2, a marker of endometrial stem\ncells (Masuda et al., 2012     ), was also increased in the EpSC clusters, along with Cyp26a1, a\nmember of retinoic acid signaling (Isoherranen and Zhong, 2019     ), and Tgfbi, which is induced\nby the transforming growth factor beta signaling pathway, stimulates NOTCH signaling, and\nmaintains glioma stem cell identity (Chen et al., 2024     ; Corona and Blobe, 2021     ; Lee et al.,\n2023     ). Comparison with a previously published scRNAseq dataset (Padilla-Banks et al., 2023     )\nalso showed that clusters 4 and 25 shared transcriptomic signatures with the EpSC cluster\nidentified in their analysis (Supplemental Figure S2C     ).\nWe then determined the dynamic continuum of the EpSCs using trajectory analysis of the\nepithelial subclusters using Slingshot pseudotime analysis (Figure 2F-I     , Supplemental Figure S2F-\nL     ). This analysis showed that the EpSC clusters from the estrus phase transitioned from the\nquiescent Aldh1a1, Lgr5, Axin2 expressing cells in clusters 4 and 25 toward the proliferative\nAldh1a1, Lgr5, Axin2-expressing cluster 13, which expressed the proliferative Mki67 and Top2a\nmarkers (Figure 2F-G     ). The trajectory then progressed toward the glandular epithelial cells and\nended with the cells in the luminal epithelium (Figure 2F-G     ), with additional projected\ntrajectories shown in Supplemental Figure S2F-G     . The trajectory for epithelial cells in the\ndiestrus phase had more branching points than those of the estrus phase cells (Figure 2H-I     ,\nSupplemental Figure S2H-L     ), suggesting that Aldh1a1-expressing cells are controlled by the\ndynamic levels of sex hormones during the estrus cycle (Nilsson et al., 2015     ). Accordingly, gene\nenrichment analysis of the epithelial clusters showed that cells in the EpSC clusters displayed high\nsignature scores for the categories of ‘Glandular Epithelial Development’ and ‘BMP Signaling,’\nwhile luminal cells had higher scores of genes enriched in ‘Stereocilium’ categories (Figure 1M-\nO     ). Our analyses of adult cycling endometrial epithelium suggest that ALDH1A1+ cells are\nenriched in cell types with stem cell signatures.\nExpression and localization of ALDH1A1 is controlled by estrogen\nand progesterone in the adult cycling uterus\nWe validated the dynamic localization of Aldh1a1-expressing cells identified by scRNAseq using\nIHC in the WT uterus of adult mice in estrus and diestrus (Figure 2P-T     ). Expression of ALDH1A1\nwas detected in both the luminal and glandular epithelium during diestrus (Figure 2P-P’     ) but\nlocalized to the glandular crypts during the estrus phase (Figure 2Q-Q’     ). We also observed this\nhormone-dependent expression of Aldh1a1 at the mRNA level, where Aldh1a1 was highest during\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 7 of 46\nthe diestrus phase compared to the pro-estrus and estrus phases (Figure 2T     ). Ovariectomy led to\ndiffusing ALDH1A1 expression throughout the luminal and glandular epithelium, while a long-\nterm E2 treatment restricted ALDH1A1 expression to the glandular crypts (Figure 2RS’     ). These\nresults confirmed that the localization of ALDH1A1-expressing cells is controlled by steroid\nhormones in the adult uterus.\nTo further investigate the hormone-dependent expression patterns of ALDH1A1, we treated\novariectomized mice with a series of hormones (Figure S3A     ). Ovariectomy, and thus depletion of\nendogenous estrogen (E2) and progesterone (P4), led to expression of ALDH1A1 in both the\nluminal and glandular epithelium (Figure S3B-B’     ). In contrast, treatment with P4, E2, or a\ncombination of P4+E2, increased localization toward the glandular crypts (Figure S3C-E’     ).\nTreatment with P4 alone decreased the gene expression levels of Aldh1a1 when compared to\nuterine tissues from ovariectomized or E2-treated mice (Figure S3F     ). Thus, in the absence of\nhormones, ALDH1A1 was diffusely expressed throughout the luminal and glandular epithelium.\nOn the other hand, E2 and P4 directly impacted the localization of ALDH1A1+ cells in the\nepithelium.\nLineage tracing of Aldh1a1+ cells in the postnatal period reveals\nthey are long-lived cells of the endometrium\nALDH1A1 has a dynamic expression pattern in the postnatal endometrium, displaying prominent\nexpression throughout the epithelium at postnatal day 7 (PND7) (Figure 3A-A’     ), gradual\naccumulation in the endometrial glands as their development progresses at PND14 (Figure 3B-\nB’     ), and restriction to the glandular crypts of more mature glands by PND21 (Figure 3C-C’     ).\nThis expression pattern resembles WNT-related signaling molecules, such as AXIN2 and LGR5,\nwhich are previously characterized drivers of endometrial regeneration (Seishima et al., 2019     ;\nSyed et al., 2020     ). To define the long-term contributions of ALDH1A1+ cells in the endometrium,\nwe generated an ALDH1A1tdTomato reporter mouse by crossing an Aldh1a1cre/ERT2/+ mouse to a\nROSA26tdTomato/TdTomato cre-reporter line, which would label ALDH1A1+ cells at the time of\ninduction and their subsequent progeny with red fluorescent protein (RFP) (Figure 3D     )\n(Madisen et al., 2010     ; Poulin et al., 2018     ).\nSingly labeled cells were obtained by using a low-dose of 4-OHT (0.5μg/g) and verified following 1\nday of tracing (PND7→ PND8) (Figure 3E-E’     ). Increasing numbers of luminal and glandular cells\nwere detected 6 days later (PND8→ PND14) (Figure 3F-F’     ) with increased expansion in both the\nglandular and luminal epithelium following long-term labeling (PND8 → PND56) (Figure 3G-G’     ).\nThis gradual increase of RFP-labeled epithelial cells was quantified, showing that ALDH1A1+ cells\nexpanded and remained as long-term resident cells of the luminal and glandular endometrial\nepithelium (Figure 3H     ). When tracing was begun at PND14, a time when endometrial glands\nbegin to invaginate into the underlying stroma (Figure 3I-L     ), singly labeled RFP+ cells were\ndetected in luminal and glandular epithelium following a short-term trace (PND14 → PND15,\nFigure 3I-I’     ). Increasing numbers of cells were observed in the mice traced from PND14 →\nPND28 and PND14 → PND56, showing that at this timepoint, ALDH1A1+ cells contribute to both\nluminal and glandular cells of the endometrium (Figure 3J-J’, K-K’, L     ). When ALDH1A1+ cells\nwere labeled at PND21, single glandular epithelial cells were RFP+ (Figure 3M-M’     ), and these\nwere detected in larger patches of glandular and luminal epithelial cells when traced from PND21\n→ PND56 (Figure 3N-N’, O     ). Total RFP+ cells per visual field across both the stromal and\nepithelial compartments showed that stromal cells were labeled at each time point we analyzed\nwith no significant increase in ALDH1A1+ stromal cells over time (Figure 3H,L,O     ). Thus, our\nlineage tracing studies indicate that ALDH1A1+ cells can be detected in glandular, luminal, and\nstromal compartments within the endometrium during short and long labeling periods.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 8 of 46\nFigure 3.Lineage tracing reveals the contribution of ALDH1A1+ cells to endometrial integrity.\nA-C’) ALDH1A1 immunohistochemistry in the WT mouse uterus at PND7 (A-A’), PND14 (B-B’), and 3-weeks (C-C’). D)\nSchematic of the tamoxifen-inducible ALDH1A1 reporter allele and experimental scheme used for lineage tracing in the\npostnatal endometrium. E-N’) RFP immunohistochemistry was used to detect the ALDH1A1-tdTomato-expressing cells in the\nuterus when tracing was performed from PND7 to PND8 (E-E’), PND8 to PND14 (F-F’), PND8 to PND28 (G-G’), PND14 to\nPND15 (I-I’), PND14 to PND28 (J-J’), PND14 to PND56 (K-K’) or PND21 to PND22 (M-M’) or PND21 to PND56 (N-N’) with\ntamoxifen (0.15 mg/g body weight). H, L, O) Quantification of RFP+ cells was performed and are presented as total RFP+\nendometrial epithelial or stromal cells. Black arrows (E’, I’, M’) indicate singly labeled RFP+ cells, red arrow (K’) indicates RFP+\nstromal cell. Images represent staining that was performed in 3 or more mice per timepoint. Data are presented as mean ±\nSEM analyzed by a Two-Way ANOVA with a Sidak test for multiple comparisons. *, p<0.05; **, P<0.01; ***, p<0.001. PND,\npostnatal day; TAM, tamoxifen; ORF, open reading frame.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 9 of 46\nALDH1A1+ cells are dynamically expressed in the adult\nendometrium of cycling mice\nTo determine the fate of ALDH1A1+ cells in the adult cycling endometrium, we began tracing\nALDH1A1+ cells during the estrus phase of their cycle (Figure 4A-B     ), a time when ALDH1A1 cells\nwere enriched in the glandular crypts (Figure 2Q     ). We verified the number of epithelial and\nstromal cells that were labeled in the endometrium following one day of tracing (Figure 4C, F-G     ).\nAfter 7 and 28 days of tracing, several glandular, luminal and sub-epithelial stromal cells were\nlabeled (Figure 4D-E’     ). When tracing was carried out for 28 days, the number of stromal but not\nepithelial labeled cells expanded relative to one day of tracing (Figure 4F-G     ). Hence, when\nlineage tracing studies were performed in adult mice, expansion of ALDH1A1+ stromal cells was\nmore readily observed than expansion of ALDH1A1+ epithelial cells. This suggested that\nALDH1A1+ cells play a critical role in stromal cell expansion in the adult endometrium.\nALDH1A1+ cells are detected in the postpartum endometrium\nWe traced the fate of ALDH1A1+ cells in the postpartum endometrium to observe their fate during\nendometrial regeneration (Figure 5     ). We administered tamoxifen to mice to initiate tracing two\nmonths prior to mating and then collected their uterine tissues 1, 3 and 5 days postpartum (PPD1,\nPPD3 and PPD5) (Figure 5A-B     ). By analyzing the uterine tissues near the placental detachment\nsite, we observed that RFP positive cells were present in the epithelial folds of the endometrium,\nwith some stromal cell expression (Figure 5C-E’     ). At PPD5, the proportion of RFP+ epithelial cells\nhad expanded relative to PPD1 and PPD3 (Figure 5E-E’     ). When we performed\nimmunofluorescence imaging of CK8 (epithelial cell marker), VIM (stromal cell marker), and RFP,\nwe observed that the total number of VIM+CK8+ transitional stromal/epithelial cells was\nsignificantly higher in the PPD3 endometrium when compared to PPD5 (19.4 ± 7.48 vs. 3.5 ± 0.5,\np<0.05), suggesting the presence of transitional VIM+CK8+ cells is abundant at PPD3, with a subset\nof these transitional cells also being RFP+ (Figure 5F-K     ). Our results showed that ALDH1A1+ cells\nwere involved in postpartum endometrial regeneration, with some also displaying expression of\ntransitional CK8+ and VIM+ cell markers.\nAblation of ALDH1A1+ cells disrupts endometrial epithelial\nexpansion in vitro and in vivo\nTo investigate how ablation of the ALDH1A1+ cell population affects epithelial expansion, we\nablated ALDH1A1+ cells in vitro and in vivo using diphtheria toxin (DT)-mediated ablation by\ncrossing ROSA26DTR/DTR mice harboring conditionally expressed diphtheria toxin receptors (DTR)\nto Aldh1a1cre/ERT2/+ mice (Figure 6A-C     ). We then established endometrial epithelial organoids\nfrom adult female ROSA26DTR/DTR;Aldh1a1cre/ERT2/+ mice. Once mature organoids were obtained,\nCre activity was induced by treating with 4-OHT for two days followed by DT treatment (Figure\n6A     ). Organoids from the control (ROSA26DTR/DTR) and experimental mice\n(ROSA26DTR/DTR;Aldh1a1cre/ERT2/+) were intact under phase contrast microscopy prior to DT\ntreatment (Figure 6D-E     ). After DT treatment, the organoids from the control ROSA26DTR/DTR\nmice were intact, while those from the experimental group, ROSA26DTR/DTR;Aldh1a1cre/ERT2/+,\nbegan to disintegrate, appeared dark, and were positive for cleaved caspase-3 (Figure 6F-I     ).\nTo examine the effects of ALDH1A1 ablation in vivo, we treated control and experimental mice\nwith tamoxifen at PND7 to induce Cre activity in the ALDH1A1+ cells, followed by treatment with\nDT at PND10. When we analyzed the mice at P56, we observed decreased ALDH1A1 expression in\nthe experimental mice (Figure 6J-K’     ). We then immunostained uterine cross-sections with CK8\nand FOXA2, which showed that the experimental mice had fewer glands compared to controls\n(11.75 ± 8.2 vs. 22 ± 3.4, p<0.05) and decreased FOXA2 intensity per gland (10,434 ± 2,136 vs. 15,079\n± 3,006, p<0.001) relative to the controls (Figure 6L-O     ). These results indicated that the ablation\nof ALDH1A1 cells decreases epithelial organoid expansion in vitro and FOXA2 expression in vivo.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 10 of 46\nFigure 4.Tracing of ALDH1A1+ cells in the adult cycling uterus identified positive cells in the epithelium and\nstroma.\nA) Schematic diagram demonstrating the reabsorption and reorganization that occurs between estrus phases in the adult\nmouse uterus. Uterine structures are labelled as: M for mesometrial, AM for anti-mesometrial, Myo for myometrium, S for\nstromal compartment, and LE for luminal epithelium. B) Timeline for tamoxifen-dependent tomato labeling in ALDH1A1-\nexpressing cells in the adult mice when traced for one day, seven days, or 28 days after Tamoxifen administration. C-E’) RFP\nimmunohistochemistry was used to detect labeled cells when tracing was performed in the adult mouse for 1 day (C-C’),\nseven days (D-D’), or 28 days (E-E’). Red arrows indicate detection of RFP in the sub-epithelial stromal cells. F-G)\nQuantification of RFP+ cells in the epithelial and stromal compartments one day, seven days, or four days after tamoxifen\nadministration. Data are presented as mean ± SEM analyzed by a Two-Way ANOVA with a Sidak test for multiple comparisons.\n*, p<0.05; **, P<0.01; ***, p<0.001. TAM, tamoxifen.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 11 of 46\nFigure 5.ALDH1A1+ cells contribute to post-partum endometrial regeneration.\nA) Schematic highlighting post-partum repair occurring at PPD1, PPD3, and PPD5. Uterine structures are labeled as: M for\nmesometrial, AM for anti-mesometrial, S for stromal compartment, LE for luminal epithelium, and GE for glandular\nepithelium. B) ALDH1A1 lineage tracing was begun in the Aldh1a1cre/ERT2;ROSA26tdTomato/tdTomato mice at 2 months of age.\nTwo months after TAM administration, the mice were mated, and their uteri were collected at PPD1, PPD3, and PPD5. C-E’)\nRFP immunohistochemistry was performed in uterine cross-sections obtained from the placental detachment sites at PPD1\n(C-C’), PPD3 (D-D’) and PPD5 (E-E’). Red arrows indicate the RFP+ subepithelial stromal cells. F-J) Immunofluorescence\nstaining of uterine cross-sections with cytokeratin 8 (CK8, cyan), red fluorescence protein (RFP, yellow), vimentin (VIM,\nmagenta), and DAPI (white). Yellow arrowheads (F, G-J) show the presence of cells that are CK8+/RFP+/VIM+; White\narrowheads indicate cells that are CK8+/VIM+. Images represent groups of more than three animals analyzed per group. J)\nQuantification of RFP+ cells in the stromal and epithelial cells at PPD3. K) Analysis of the cells expressing CK8+/VIM+ in the\nuterine cross-sections at PPD3 and PPD5. Data are presented as mean ± SEM of positive cells per imaged field and analyzed\nby a Mann-Whitney test (M). *, p<0.0033; **, P<0.002; ***, p<0.001. TAM, tamoxifen; PPD, post-partum day.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 12 of 46\nFigure 6.Ablation of ALDH1A1+ cells impairs organoid expansion and reduces endometrial glands in adult\nmice.\nA-C) Diphtheria toxin-mediated ablation of ALDH1A1+ cells was obtained in vitro (A) and in vivo (B) by crossing\nAldh1a1cre/ERT2;ROSA26tdTomato/tdTomato mice to a line containing a conditional diphtheria toxin receptor (DTR). D-I)\nOrganoids from adult control (DTRf/f) and experimental (Aldh1a1cre/ERT2;DTRf/f) mice were established and expanded in\nculture for two passages. Once established (D-E), the organoids were treated with DT (F-G) and visualized, fixed and stained\nwith cleaved caspase-3 antibody (H-I). J-K) The impact of DT-mediated ablation of ALDH1A1+ cells was determined in mice\ntreated with TAM at PND7, and with DT at PND10. Uterine tissues were collected and analyzed at PND56 using\nimmunohistochemistry to detect ALDH1A1 (J-K’). N-O) Glands were visualized in the control mice (N) and experimental (O) by\nstaining with FOXA2 (green), cytokeratin 8 (red), and DAPI (white) using confocal imaging. P-Q) FOXA2 intensity per gland (P)\nand glandular number (Q) were quantified in the uterine cross-sections of >3 mice per genotype. The results are displayed as\nmean ± SEM and analyzed using a two-tailed t-test, *, p<0.05; **, P<0.01; ***, p<0.001.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 13 of 46\nALDHHI epithelial cells of the human endometrium display\nproperties and signatures of adult stem cells\nPrevious studies showed that ALDH1A1 is enriched in the glandular epithelial cells in the basalis\nhuman endometrium, a site considered to be enriched with adult stem cells (Ma et al., 2020     ). To\ndetermine the regenerative potential of ALDHHI cells in the human endometrium, we established\norganoids from the eutopic endometrium of donors. We separated ALDHHI and ALDHLO cells\nusing the ALDEFLUOR assay and expanded them in vitro (Figure 7A     ). Organoid formation assays\nidentified that ALDHHI cells had a higher capacity to form organoids than ALDHLO cells (1.98 ±\n0.38 vs 0.58 ± 0.055, p=0.022; 9.04 ± 0.72 vs 1.7 ± 0.38, p=0.0008) (Figure 7B-F     , Supplemental\nFigure S4A-C     ). ALDHHI cells were also maintained in culture over longer passages, though the\ndifference was not statistically significant (23 ± 3 vs. 14 ± 2 passages, p=0.0576, n=3 donors) (Figure\n7G-J     ).\nTo determine the gene expression differences between the two cell types, we performed\ntranscriptomic profiling in the ALDHHI and ALDHLO organoids from the eutopic endometrium of\nthree donors. Analyses of the differentially expressed genes showed that 50 genes were increased\n(>0.5 log2FC, Adj. p-value <0.05) and 98 genes were decreased (<-0.5 log2FC, Adj. p-value <0.05) in\nALDHHI vs. ALDHLO organoids (Figure 7K     , Supplemental Table S3     ). Gene ontology analyses\nshowed that ALDHLO cells had higher expression of genes enriched in ciliated cells, such as\nRSPH4A, CFAP73, DNAI1, SPAG17, and several others, while ALDHHI had higher expression of\ngenes involved in epithelial cell proliferation and gland development, such as GATA2,VEGFA and\nIGFBP3 (Figure 7K-M     , Supplemental Figure S5     , and Supplemental Table S3     ). Genes that\nwere increased in the ALDHHI cells also included genes involved in stemness, such as BMP3,\nADH1C, KCP, and PLA2R1 (Figure 7K     , Supplemental Table S3     ). We did not identify any\ndifferences in the expression of ESR1, PGR, or other nuclear hormones between groups\n(Supplemental Table S3     ). Immunostaining of ALDHHI and ALDHLO organoids from eutopic\norganoids confirmed that significantly more ciliated cells were present in ALDHLO organoids\nwhen compared to ALDHHI eutopic organoids (7.2 ± 1.56 vs. 0.77 ± 0.42, p<0.001) (Figure 7N     ).\nThus, in human endometrial epithelial cells, ALDHHI cells displayed a higher organoid formation\nrate, expressed fewer ciliated cell-associated genes, and had increased levels of stemness genes,\nconsistent with characteristics of adult stem cells of the human basalis endometrium.\nDiscussion\nKey studies have identified and characterized endometrial stem cells by analyzing them in the\nendometrial tissues of postmenopausal women, a tissue that is enriched in basalis cells. Through\nstudies of the postmenopausal basalis, AXIN2, SOX9, SSEA1, and CDH2-positive cells are now\nwidely accepted as epithelial cell progenitors localized in the human basalis endometrium\n(Nguyen et al., 2012     ) (Nguyen et al., 2017     ; Valentijn et al., 2013     ). Additionally, SUSD2+\nstromal cells located in the perivascular areas possess more colony formation capacity than\nSUSD2- cells. This population of cells also expresses CD140b (PDGFRb) and CD146, which are\nconsidered to be endometrial mesenchymal stem-like cells (Masuda et al., 2012     ). Due to its\nenriched location in the basalis endometrium and colocalization with CDH2, ALDH1A1 has also\nbeen proposed as a stem cell marker in the endometrium (Ma et al., 2020     ).\nRecent scRNAseq and spatial transcriptomic analyses of human endometrium have confirmed\nmany of the proposed endometrial stem cell markers, with SOX9+ cells enriched in the basal\nendometrium which give rise to SOX9+/LGR5+ cells (Garcia-Alonso et al., 2021     ; Wang et al.,\n2020     ). WNT and NOTCH were also identified as critical growth factors controlling endometrial\nstem cell differentiation, with WNT activator signals controlling luminal epithelial cell\ndevelopment, and NOTCH maintaining stemness in the basalis (Garcia-Alonso et al., 2021     ). In a\nhigh-resolution single cell reference atlas of the human endometrium, a population of\nCDH2+/SOX9+/AXIN2+/ALDH1A1+ cells was identified in the basalis endometrium using spatial\ntranscriptomics, further suggesting the identity of stem-like ALDH1A1+ cells (Mareckova et al.,\n2024     ). Our analyses of ALDHHI vs ALDHLO human eutopic endometrial cells are in line with\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 14 of 46\nFigure 7.ALDHHI cells from eutopic endometrium display organoid formation capacity and different\ntranscriptomic signatures than ALDHLO cells.\nA) Schematic approach to isolate ALDHHI and ALDHLO cells from human endometrial epithelial organoids with the\nALDEFLUOR assay. B-C) Organoid establishment was assessed in freshly isolated ALDHLO and ALDHHI epithelial cells plated at\nequal densities. D-F) Organoid formation assay was performed by plating equal numbers of viable ALDHLO (D) and ALDHHI\n(E) cells followed by quantifying the total number of organoids that were established per 100 cells plated (F). Graph displays\nthe mean ± SEM of organoids from one patient and analyzed using a two-tailed t test, *, p<0.05; **, P<0.01; ***, p<0.001. G-J)\nImages comparing the growth of ALDHLO (G,I) and ALDHHI (H,J) organoids at passage 11 (G-H) or passage 16 (I,J). K) Volcano\nplot showing the total number of differentially expressed transcripts in the ALDHHI vs ALDHLO eutopic organoids from three\ndifferent patients. L-M) Gene enrichment analysis of increased (L) and decreased (M) genes in ALDHHI eutopic vs ALDHLO\neutopic organoids. N) Immunostaining of eutopic ALDHLO and ALDHHI organoids stained with cytokeratin 8 (CK8, green),\nAcetylated-a-tubulin (AcTub, red), and DAPI (white).\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 15 of 46\nthese previous findings, given that ALDHLO cells displayed more luminal-like gene expression\npatterns, with prominent expression of cilia-related genes. ALDHHI cells, on the other hand,\ndisplayed greater organoid formation capacity than ALDHLO cells.\nEndometrial stem cells are implicated in endometriosis pathogenesis due to previous studies that\nhave identified abnormalities in the menstrual effluent of women with endometriosis. These\ninclude the presence of more basalis-like tissues in the effluent of women with endometriosis,\ncontaining peristromal muscular tissue markers (Leyendecker et al., 2002     ). More recent studies\nshow that menstrual cells from patients with endometriosis express higher SSEA+/SOX9+ stemness\nmarkers (Hapangama et al., 2019     ). Furthermore, exome sequencing studies have identified\nmatching DNA mutations to be present in the eutopic and ectopic endometrium, suggesting that\nendometrial tissue from women with endometriosis inherently has a selective growth advantage\n(Suda et al., 2018     ). Our studies show that eutopic ALDHHI epithelial organoids have a higher\norganoid formation capacity and organoid formation rate than epithelial organoids established\nfrom ALDHLO cells, supporting the hypothesis that endometrial stem cells within menstrual\neffluent are implicated in endometriosis. While further investigations are warranted to specify the\nrole of ALDHHI cell populations in endometriosis, our studies suggest that the presence of\nALDH1A1+ cells in endometrial cells or menstrual effluent could be a biomarker for predicting a\npropensity for endometriosis.\nFew mouse models have been used to further define and characterize stem and progenitor cells in\nthe adult endometrium. Luminal epithelium, glandular structures, and stromal cells are induced\nto proliferate by the mitogenic potential of E2, while P4 induces differentiation in preparation for\na pregnancy. At the end of the 4-5 day estrus cycle, the glandular folds are resorbed through\nprocesses involving apoptosis or autophagy (Dharma et al., 2001     ; Popli et al., 2022     ; Wood et\nal., 2007     ) (Popli et al., 2023     ). Single cell analyses of the postnatal uterus have identified key\nputative stemness genes that are critical for patterning of the endometrium (Fu et al., 2020     ;\nSpencer et al., 2023     ; Wu et al., 2017     ). Additionally, lineage tracing studies have identified that\nLgr5+ and Axin2+ are long lived progenitors in the endometrium that are enriched in the crypts of\nendometrial glands and display stemness characteristics (Seishima et al., 2019     ; Syed et al.,\n2020     ). More recently, Nestin+ perivascular cells were shown to contribute to endometrial re-\nepithelialization in the adult mouse uterus (Li et al., 2025     ).\nPreviously, there was a lack of functional assays and lineage tracing studies to further characterize\nand confirm ALDH1A1+ endometrial stem cell populations. Using an inducible fluorescent\nALDH1A1 reporter mouse, we characterized the presence and contribution of ALDH1A1+ cells\nthroughout endometrial glandular development, the murine hormonal cycle, and in postpartum\nregeneration. By lineage tracing ALDH1A1+ cells at different developmental and adult timepoints,\nour results show that they give rise to cells that repopulate and persist in the glandular and\nluminal epithelium in the long-term. Endometrial glandular development in the mouse is a\npostnatal process that begins at approximately postnatal day 5 (PND5) and is completed by PND21\n(Hayashi et al., 2011     ) (Vue et al., 2018     ). Glandular patterning in the postnatal period is\nattributed to signaling pathways that involve WNT/β-catenin, estrogen receptor (ESR1) signaling,\nBMPs, and other complex networks (Rizo et al., 2023     ) (Mericskay et al., 2004     ; Miller and\nSassoon, 1998     ; Nanjappa et al., 2015     ). Transcriptomic analyses of the developing uterus have\nshown that genes involved in retinoic acid (RA) metabolism peak from PND0 to PND14 and then\nbegin to gradually decline at PND28, suggesting that this process is critical in glandular patterning\nin the postnatal period (Wu et al., 2017     ). In line with these findings, our study showed that\nALDH1A1 is highly expressed throughout the luminal uterine epithelium at PND7, with a gradual\nshift to the glandular crypts as glandular development progressed at PND14.\nTo further address the impacts of ALDH1A1+ cells on glandular development, we used an\ninducible DT ablation model where DTR was conditionally expressed in the ALDH1A1-expressing\ncells. DT-mediated ablation of ALDH1A1+ cells was performed at PND7, led by the hypothesis that\nablation of ALDH1A1+ cells would impair glandular development in adult mice. We analyzed\nuterine tissues of mice 56 days after administration of DT, where we indeed found fewer glands\nand reduced FOXA2 intensity in the mice with DT-mediated ablation of ALDH1A1+ cells. Similar\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 16 of 46\nresults were obtained in vitro, where DT-mediated ablation of ALDH1A1+ cells caused organoid\ndeath. The observed partial ablation of glands that we found in vivo is consistent with the\nphenotype of ALDH1A1 KO mice, which are viable and fertile, suggesting that compensation by\nadditional ALDH isozymes can rescue glandular development and function (Matt et al., 2005     ).\nInterestingly, we also found the pattern of ALDH1A1 expression to be dynamic in the adult murine\nuterus, with restricted expression in the crypts during estrus and more diffuse staining\nthroughout the luminal and glandular compartments in the diestrus phases. We confirmed that\nthis dynamic ALDH1A1 expression was hormone driven by using an ovariectomized model treated\nwith exogenous hormones. In this model, ovariectomy without hormones led to ALDH1A1\nexpression throughout the luminal uterine epithelium, while E2 and P4 treatment caused dynamic\nshifting of expression between the glandular crypts and luminal epithelium. Ovariectomized mice\ntreated with 90-day E2 pellets, on the other hand, showed a complete restriction of ALDH1A1 to\nthe glandular crypts, while the ovariectomized controls had ALDH1A1 expression throughout the\nluminal and glandular epithelium. Thus, it is possible that in the absence of hormones, the uterine\nepithelium takes on a more plastic state with both glandular and luminal cells displaying stemness\nqualities. Conversely, under the mitogenic actions of E2, when epithelial cell turnover is higher,\nALDH1A1+ cells are restricted to the glandular crypts to remain as a reservoir for subsequent\nproliferative cycles.\nThe conversion of retinaldehyde to retinoic acid is driven by the ALDH1A enzymes, which are\nexpressed in a spatiotemporally restricted pattern within the developing and adult endometrium\n(Vermot et al., 2000     ; Wu et al., 2017     ). In the adult, we observed that ALDH1A1 localized to the\ncrypts of the endometrial glands of mice during the estrus phase and in ovariectomized mice\nsupplemented with estradiol pellets (Supplemental Figure S1     ). The local synthesis and activity of\nretinoic acid via the retinoic acid receptor (RAR) may be critical for maintaining the stemness of\nadult endometrial epithelial cells, allowing for cellular proliferation and differentiation when\nexposed to estradiol at key phases of the cycle. This idea is supported by studies showing estrogen\ninduces epithelial cell stratification in the vagina and cervix and increases expression of RAR (and\nits heterodimeric partner, RXR) in basal epithelial cells, suggesting that the two pathways of\nestrogen and retinoic acid signaling converge (Celli et al., 1996     ; Tannous-Khuri and Talmage,\n1997     ). Further, recent studies using uterine conditional ablation of RARA/RARB/RARG with the\nprogesterone receptor cre show that these mice develop excessive stratification of the uterine\nluminal epithelium upon RAR ablation (Yin et al., 2025     ). Because improper epithelial cell\nstratification is counteracted by administration of Fulvestrant, a potent estrogen receptor\nantagonist, the authors conclude that RA/RAR signaling antagonizes E2/ER action and is required\nfor epithelial cell fate maintenance in the adult. Thus, it is plausible that ALDH1A1 activity,\nthrough its impact on RA/RAR activity and E2/ER signaling, is driving endometrial cell\ndifferentiation and maintaining a reservoir of quiescent stem-like cells in the endometrial\nepithelium.\nAdditionally, our previous studies showed that conditional ablation of the downstream effectors of\nthe transforming growth factor β (TGFβ) signaling pathway in the uterus, SMAD2 and SMAD3,\ndisrupted epithelial cell homeostasis, leading to excessive estrogen-dependent cell proliferation,\nendometrial tumors, and disrupted retinoic acid metabolism (Kriseman et al., 2019     ; Kriseman et\nal., 2023     ; Monsivais et al., 2019     ). Hence, it is also plausible that TGFβ/SMAD2/3 are critical for\nintegrating the RA/RAR-dependent antagonism of E2 action into the epithelium, thereby directing\nproliferation and differentiation programs in the adult endometrium. The exact mechanisms\ncontrolling this antagonism, however, are not yet known, and likely involve paracrine signaling\nnetworks between the endometrial stroma and epithelium. Whether ALDH1A1 and other ALDH\nisozymes control similar proliferative and stemness programs in the stroma remains to be\nevaluated.\nWe also found ALDH1A1+ stromal cells were more prevalent when tracing began in adult mice.\nOther studies have shown that mesenchymal cells contribute to endometrial regeneration in the\npostpartum phase or after induced menses through a process of MET (Cousins et al., 2014     ;\nKirkwood et al., 2022     ; Li et al., 2025     ). This prompted us to determine whether the stromal\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 17 of 46\nALDH1A1+ cells were contributing to epithelial regeneration in the postpartum phase. In cycling\nmice, we found sporadic cells that expressed both stromal and epithelial markers in the ALDHA1+\ncells. However, analyses of PPD3 regenerating endometrium showed a greater number of cells\nexpressing VIM/CK8/RFP compared to the PPD5 endometrium, suggesting that the presence of\nthese transitional cells was more frequent at PPD3. We noted that not all the Vim+/CK8+ cells\nexpressed ALDH1A1+, suggesting that the cells marked by all three represent a subpopulation of\ncells that contribute to MET in the postpartum phase. However, because at the time of labeling\nboth epithelial and stromal cells express ALDH1A1, this does not exclude the possibility that the\ntransitional VIM/CK8/RFP cells we observed were undergoing EMT and not MET.\nOverall, our lineage tracing, ablation, and regeneration models show that ALDH1A1+ endometrial\ncells display characteristics of an adult stem cell. Organoid formation assays in ALDHHI vs.\nALDHLO endometrial cells from both human and mice support these findings and place the\nactivity of ALDH1 enzymes as central regulators of regenerative potential in the endometrium.\nThis is also observed in the scRNAseq analyses of the adult cycling mouse uterus, where\nALDH1A1+ cells that lacked proliferative markers clustered with Lgr5 and Axin2- expressing cells\nand displayed a trajectory of EpSCs giving rise to both glandular and luminal cells. By integrating\nwith scRNA profiles of EpSCs characterized in previous studies (Padilla-Banks et al., 2023     ;\nWinkler et al., 2024     ), this population of cells and datasets can be used to identify and\ncharacterize additional stem cells in the adult endometrial epithelium. Additionally, our studies in\nhuman endometrium extend our characterization of ALDH1A1 as an adult endometrial stem cell\nmarker and emphasize the importance of ALDH1A1+ in the regerenerative potential of the\nendometrium.\nMethods\nHuman sample collection\nTissues were collected from patients after obtaining informed written consent and following the\nguidelines as approved by the Baylor College of Medicine IRB protocol (H-21138). Tissues and any\ncells derived from them were de-identified prior to use to ensure patient confidentiality\nguidelines. Donor age and clinical information is reported in Supplemental Table S5     .\nAnimal ethics statement\nAll animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC)\nof Baylor College of Medicine (BCM) and guidelines established by the National Institutes of Health\nGuide for the Care and Use of Laboratory Animals. All the mice were housed under standard\nconditions of a 12 h light/dark cycle in a vivarium that maintained a controlled ambient\ntemperature of 70 °C ± 2 °C and a relative humidity of 2070%.\nMouse models and genotyping\nThe Aldh1a1cre/ERT2 mice were obtained from Dr. Raj Awatramani, Department of Neurology,\nNorthwestern University Feinberg Medical School (Azcorra et al., 2023     ), while the Ai9 tdTomato\nmice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J) were provided by Dr. Stephanie Pangas,\nDepartment of Pathology and Immunology, Baylor College of Medicine. The ROSA26iDTR mice\n(C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J, JAX strain # 007900) were purchased from the Jackson\nLaboratory. All experimental mice were either homozygous for Ai9 tdTomato or iDTR. Genotyping\nwas performed using DNA extracted from 2-3 mm tail snips. Tail samples were digested in 200 μL\nof 50 mM NaOH at 95°C for 30 minutes. Following digestion, 100 μL of 1 M Tris-HCl (pH 8.0) was\nadded to neutralize the solution, and the mixture was centrifuged at maximum speed for 5\nminutes to pellet any debris. The supernatant containing the isolated DNA (1-2 μL) was used as a\ntemplate for PCR amplification. Amplification was performed using amfiSure PCR Master Mix\n(GenDepot) with the primer sequences provided in Supplementary Table S4      and following the\ncycling conditions detailed in Supplementary Figure S6     .\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 18 of 46\nAldh1a1 lineage tracing mouse experiments\n4OH-Tamoxifen (Sigma, H7904) and tamoxifen (Sigma, T5648) powders were initially dissolved in\n100% ethanol and then diluted in corn oil at a concentration of 10 mg/mL. For Aldh1a1 lineage\ntracing at early stage, Ai9/Ai9 and Ai9/Ai9; Aldh1a1cre/ERT2 female mice received a single\nintraperitoneal (IP) injection of either 4OH-Tamoxifen at 0.5 μg/g body weight on postnatal day 7,\n8, or tamoxifen at 0.15mg/g body weight on postnatal day 14 or 21. Mouse uteri were then\nharvested on postnatal days 8,14, 15, 21, 28, and 56 respectively. For Aldh1a1 lineage tracing across\nestrus cycles, Ai9/Ai9; Aldh1a1cre/ERT2 female mice at the age of 6 weeks in estrus were injected\nwith a single dose of tamoxifen at 0.15mg/g body weight. Their uteri were then collected either in\nthe next estrus or estrus one month later. To trace ALDH1a1+ cells in endometrial remodeling and\nregeneration during pregnancy, Ai9/Ai9; Aldh1a1cre/ERT2 femalemice were given a single dose of\ntamoxifen at 0.15mg/g body weight at the age of two months and then mated with WT male adult\nmice one week later. Mouse uteri were collected on postpartum days (PPD) 1, 3 and 5.\nSurgeries and hormone treatments\nSix-week-old female mice were ovariectomized and given a two-week period to ensure the\ncomplete clearance of residual ovarian hormones. The ovariectomized mice received two doses of\nestradiol-17β (E2, 100ng/mouse, Sigma, E8875) through subcutaneous injection. After two days’\nrest, the mice were randomly divided into four groups: Vehicle group received 4 doses of sesame\noil, P4 group received 4 doses of progesterone (P4, 1mg/mouse, Sigma, P0130), E2 group received 3\ndoses of sesame oil followed by one dose of E2 (100ng/mouse), P4+E2 group received 3 doses of P4\n(1 mg/mouse) followed by one combined dose of 100ng E2 and 1mg P4. The uterine horns from\nthese four groups were collected 15 hours after receiving the last dose. Six-week-old female mice\nwere ovariectomized and implanted with a placebo or estradiol pellet (17β-ESTRADIOL, 0.025 mg,\n90 days, Innovative Research of America, NE-121) and their uteri were collected after 90 days.\nRNA extraction and quantitative real-time PCR\nOrganoids or tissue samples were homogenized in Trizol reagent (Life Technology, 15596018) and\ntotal RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research, R2052) according\nto the manufacturer’s protocol. Reverse transcription was performed using the qScript cDNA\nSuperMix (Quantabio, 95048-100) with 200ng of total RNA as template, following the\nmanufacturer’s instructions. cDNA was diluted three times in water. Quantitative real-time PCR\n(qPCR) was carried out on a BioRad CFX Real-Time PCR System using SYBR Green Master Mix (Life\nTechnology, 4364346). Each 10 μL reaction contained 10 μL of cDNA, 0.5 μM of each gene-specific\nprimer, and 1X SYBR Green mix. The PCR primers are listed in Supplementary Table S4     . All\nreactions were performed in duplicate with three biological replicates. Gene expression levels\nwere normalized to the housekeeping gene Rpl17 or Hprt and relative quantification was\ncalculated using the 2^(- ΔΔCt) method (Schmittgen and Livak, 2008     ). Data were presented as the\nmean fold change ± SEM and analyzed using a two-tailed t-test in GraphPad Prism.\nMouse endometrial epithelium dissociation for organoids and\nsingle-cell RNA sequencing (scRNAseq)\nEpithelial cells were isolated from mouse endometrial tissue using a combination of mechanical\nand enzymatic dissociation as previously described (Tang et al., 2023b     ). Briefly, uterine horns\nwere dissected from six-week-old female wild-type (WT) mice at the estrus stage, as confirmed by\nvaginal cytology, and were then cut into small fragments (4-5 mm) using sterile scissors. Tissue\nfragments were moved to a digestion solution containing 1% Trypsin (Sigma, T1426) in HBSS\n(ThermoFisher, 14170112) and incubated at 37°C for 45 minutes. Following incubation, the uterine\nfragments were moved to a 35 mm tissue culture plate containing 1 mL of Dulbecco’s Phosphate-\nBuffered Solution (DPBS), where epithelial sheets were separated mechanically from the uterine\ntubes using a 1 mL pipette. The epithelial sheets were then collected and pelleted by centrifugation\nat 2000 rpm for 5 minutes at 4 °C. The pellets were resuspended in HBSS containing collagenase I\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 19 of 46\n(5mg/mL, Sigma, C0130) and DNase I (0.2mg/mL, Sigma, DN25) and subsequently filtered through\n100 pm cell strainers. The red blood cells were lysed in 0.2% NaCl for 20 seconds followed by 1.6%\nNaCl. The resulting single cells were then used for organoid culture or live cell sorting using DAPI.\nHuman endometrial epithelium dissociation\nEndometrial organoids were established from human fresh endometrial tissue samples obtained\nwith informed consent following ethical approval. Tissue samples were minced into small\nfragments and enzymatically digested using a combination of 5mg/mL collagenase I (Sigma,\nC0130) and 0.2mg/mL DNase I (Sigma, DN25) in HBSS (ThermoFisher, 14170112) for 1 hour at 37°C\nwith gentle agitation. The resulting cell suspension was filtered through a 100 μm cell strainer to\nremove undigested tissue, and the filtrate was centrifuged at 2000 rpm for 5 minutes to pellet the\ncells. The red blood cells were lysed in 0.2% NaCl for 20 seconds followed by 1.6% NaCl. The\nresulting single cells were then used for organoid culture.\nMouse and human endometrial organoid culture\nThe isolated mouse epithelial cells were resuspended in ice-cold Matrigel (Corning, 354230) and\nseeded as 30 μL droplets onto 12-well culture plates. After allowing the Matrigel to solidify at 37°C\nfor 15 minutes, the mouse organoid culture medium, composed of Advanced DMEM/F12 (Life\nTechnologies, 12634010) supplemented with 1× B27 (Life Technologies, 12587010), 1× N2 (Life\nTechnologies, 17502048), 100 μg/mL primocin (Invivogen, ant-pm-1), 1.25mM N-Acetyl-L-cysteine\n(Sigma, A9165), 2mM L-glutamine (Life Technologies, 25030024), 50 ng/mL EGF (PeproTech, AF-\n100-15), 100ng/mL FGF-10 (PeproTech, 100-26), 50ng/mL HGF (PeproTech, 100-39), 10% Noggin\n(BCM Digestive Diseases Center), 10% R-spondin (BCM Digestive Diseases Center), 10% WNT3a\n(BCM Digestive Diseases Center), 10nM Nicotinamide (Sigma, N0636), and 10 μM Y-27632 (ROCK\ninhibitor, Sigma, Y0503), was gently added to each well. Human isolated epithelial cells were also\nresuspended in ice-cold Matrigel (Corning, 354230), seeded as 30 μL droplets onto 12-well culture\nplates, and fed with human organoid culture medium containing complete mouse organoid\nmedium supplemented with 1 μM A83-01 (Tocris, 2939). Organoids were maintained at 37°C in a\nhumidified incubator with 5% CO2, and the medium was refreshed every 2−3 days. Organoid\ngrowth and morphology were monitored under an inverted microscope, and passaging was\nperformed every 7-10 days by mechanically disrupting the matrigel and reseeding them in fresh\nmatrigel. For experimental assays, organoids were dissociated into single cells using Accutase cell\ndissociation reagent (Life Technologies, A1110501), followed by further processing or analysis as\nrequired.\nALDEFLUOR assay and fluorescence-activated cell sorting\nCell sorting based on aldehyde dehydrogenase (ALDH) activity was performed using the\nALDEFLUOR kit (StemCell Technologies, 01700) according to the manufacturer’s instructions.\nBriefly, single-cell suspensions were prepared from cultured organoids or dissociated tissues and\nresuspended in ALDEFLUOR assay buffer at a concentration of 1 × 10^6 cells/mL. The cell\nsuspension was divided into two aliquots: one for the experimental sample and the other for the\nnegative control containing the ALDH inhibitor diethylaminobenzaldehyde (DEAB). To each tube,\nactivated ALDEFLUOR reagent was added, and the samples were incubated at 37°C for 30−45\nminutes in a CO2 incubator. During this incubation, cells with high ALDH activity converted the\nALDEFLUOR substrate into a fluorescent product that accumulates intracellularly. Following\nincubation, cells were centrifuged at 2000 rpm for 5 minutes, resuspended in fresh ALDEFLUOR\nassay buffer, and kept on ice until sorting. Flow cytometry was performed using a BD FACSAria\nsorter equipped with appropriate filters to detect ALDH-dependent fluorescence (FITC channel) by\nthe Cytometry and Cell Sorting Core at Baylor College of Medicine. Cells treated with DEAB served\nas a baseline to define the ALDH-negative population (ALDHLO), ensuring accurate gating of\nALDH-positive cells (ALDHHI). ALDHLO and ALDHHI cell populations were collected in culture\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 20 of 46\nmedium supplemented with 10% fetal bovine serum (FBS) to preserve viability and immediately\nprocessed for downstream applications. All flow cytometry data were analyzed using FlowJo\nsoftware to confirm sorting accuracy and purity.\nOrganoid Formation Assay\nSorted ALDHLO and ALDHHI cells from human organoids or pools of mouse endometrial epithelial\ncells at the estrus stage were directly seeded in equal numbers onto 40μL Matrigel domes in a 24\nwell plate and cultured in organoid medium for ~2 weeks. To perform organoid formation assays\nwith both the mouse and human organoids, organoids were first resuspended in ice-cold\nAdvanced DMEM/F12 to break up organoids from the Matrigel. The organoids were spun down at\n600 xg for 5 minutes, the supernatant was removed, and the Matrigel and organoid layer\nremaining were again resuspended 2-3 more times until the organoids were separated from the\nMatrigel layer. Next, the Matrigel layer was removed, and the organoid pellet was resuspended in\n5 mL of StemPro Accutase Cell Dissociation Reagent (ThermoFisher Scientific, A1110501). The\nAccutase-organoid mixture was incubated on a shaker in the 37 T tissue culture incubator for 30-\n40 minutes until a single-cell suspension was achieved. The suspension was then spun down at 600\nxg for 5 minutes and filtered through a sterile 40 μm filter (Corning, 352340) followed by a 20 μm\nsterile filter (PluriSelect, 43-50020-03) to ensure only single cells remain. The flow through was\nspun-down as previously mentioned and resuspended in a cell-counting volume. A small volume\nof the suspension was then mixed 1:1 with Trypan-Blue (Gibco, 15250061) to count alive cells with\na manual hemocytometer. Cells were resuspended in a ratio of 100 cells/1 μL of Matrigel and\nplated in triplicate with one 40 μL Matrigel dome plated in the center of the well in a 24-well plate.\nThe Matrigel domes were then allowed to solidify in the 37 °C tissue culture incubator for 10\nminutes before 750 μL of complete organoid media (+A83-01 for human, -A83-01 for mouse) with\n10 μM of Y-27632 dihydrochloride (Sigma, Y0503) was added. Media was changed every 2 days,\nand only the first two days of media had the addition of Y-27632 to help the organoids reconstitute.\nWells were imaged on Day 7 with the Yokogawa CV8000 or the BioTek Cytation 5. Images were\nthen tiled and stacked to create z-projections of the full Matrigel dome plated. For the human\norganoids, organoid quantification and analysis were completed using a trained-AI model on the\nfinal z-projection images through BioDock (Biodock, AI Software Platform. Biodock 2024. Available\nfrom www.biodock.ai     .) For mouse organoids, organoid quantification and analysis were\ncompleted manually using ImageJ.\nIn vitro and in vivo Aldhlal ablation\nDiphtheria toxin (DT, Sigma, D0564) was resuspended in sterile water at a concentration of\n1mg/mL. Endometrial epithelial cells were isolated from the uteri of six-week-old female\nROSA26DTR/DTR and ROSA26DTR/DTR; Aldh1a1cre/ERT2 mice at the estrus stage and cultured in\norganoid medium for one week to establish organoids. After the initial culture, the organoids were\npassaged with an equal number of cells seeded into 40μL Matrigel domes in a 24 well plate and\nfurther cultured for an additional week. On day 7, the organoids were treated with 0.01mg/mL\n4OH-T followed by exposure to 0.25 μg/mL DT on day 9. The organoids were harvested on day 10,\nfixed in 4% paraformaldehyde, and subsequently analyzed for apoptotic marker, cleaved caspase-\n3. To ablate ALDH1A1+ cells in the mouse endometrium, ROSA26DTR/DTR and ROSA26DTR/DTR;\nAldh1a1cre/ERT2 mice were administered a single dose of 4OH-Tamoxifen at 0.5 μg/g body weight\non postnatal day 7 (PND7), following by a single dose of DT at 16.6 μg/g body weight on PND10.\nMouse uteri were collected on PND 56 and analyzed to evaluate glandular development.\nImmunohistochemistry and immunofluorescent staining\nMouse uteri were fixed in 10% neutral-buffered formalin for 24 hours and then stored in 70%\nethanol. Human or mouse organoids were fixed in 4% paraformaldehyde for 24 hours, mounted in\nhistogel processing gel (Thermo Scientific, HG-4000-012), and stored in 70% ethanol. Tissue\nsamples were processed and embedded in paraffin using standard histological procedures.\nSections of 5 pm thickness were mounted onto adhesive microscope slides. Prior to staining, the\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 21 of 46\nslides were deparaffinized in xylene, rehydrated through a series of decreasing concentrations of\nethanol, and rinsed in distilled water. Antigen retrieval was performed by heating the slides in a\ncitrate buffer (pH 6.0) using a microwave. For IHC staining, sections were incubated in 3%\nhydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. Non-specific binding\nwas blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST)\nfor 60 minutes at room temperature. The sections were then incubated overnight at 4°C with\nprimary antibodies (Supplementary Table S4     ) diluted in blocking buffer according to the\nmanufacturer’s recommendations. Following this, the slides were washed in TBST and incubated\nwith a biotinylated secondary antibody (Supplementary Table S4     ) for 60 minutes, followed by\nthe incubation with the horseradish peroxidase (HRP)-conjugated streptavidin complex (Vector\nLaboratories, PK-6100). Visualization was achieved using 3,3′-diaminobenzidine (DAB, Sigma,\nD5637) as the chromogen, which produced a brown precipitate at the site of antibody binding. The\nsections were counterstained with Harris hematoxylin (Sigma# HHS32) to visualize nuclei,\ndehydrated, and mounted with Permount mounting medium (Fisher Scientific, SP15). Negative\ncontrols were prepared by omitting the primary antibody. All stained slides were scanned at 40x\nby the Digital Pathology Service from the BCM Department of Pathology & Immunology. For IF\nstaining, after antigen retrieval sections were permeabilized with 0.1% Triton X-100 in TBST for 10\nminutes to allow intracellular antibody access. Nonspecific binding was blocked with 3% bovine\nserum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) for 60 minutes at room\ntemperature. The sections were then incubated overnight at 4°C in a humidified chamber with\nprimary antibodies (Supplementary Table S4     ) diluted in blocking buffer according to the\nmanufacturer’s recommendations. The next day, the sections were washed and incubated with\nsecondary antibodies conjugated to fluorophores (Supplementary Table S4     ) for 1 hour at room\ntemperature, protected from light. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole\n(DAPI) for 5 minutes, followed by thorough washing with TBST. The coverslips were mounted onto\nglass slides using Vectasheild anti-fade mounting medium (Vector Laboratories, H-1000) to\npreserve fluorescence. Slides were stored in the dark at 4°C until imaging. Negative controls were\nprepared by omitting the primary antibody. All fluorescently labeled slides were evaluated and\nimaged at the Optical Imaging and Vital Microscopy Core Facility Laboratory at Baylor College of\nMedicine using an LSM880 confocal microscope.\nTranscriptomic Profiling of Human Eutopic and Ectopic Organoids\nby RNA Sequencing\nTotal RNA was extracted from cultured organoids or tissue samples using the DirectZol kit (Zymo\nResearch, R2052) following the manufacturer’s protocol, with an additional on-column DNase I\ndigestion step to remove genomic DNA contamination. Sorted ALDHHi and ALDHLO organoids\nfrom eutopic endometrium samples of three patients with endometriosis were analyzed. Three\nwells of a 12-well plate of ALDH sorted organoids containing two 30μl domes of Matrigel from\neach patient were pooled (n=3 patients) and total mRNA was extracted using DirectZol Kit from\nZymo. RNA with a high integrity (RIN >8) was used for library preparation using the Illumina\nTruSeq RNA Library Prep Kit and sequenced with Illumina Novaseq (Novogene, Inc., Sacramento,\nCA). Differentially expressed genes (DEGs) between the ALDHHI and ALDHLO groups were\ncalculated using DEseq2 (version 1.42.1) with a log2FC > 1 and < -1 and an adjusted p value < 0.05.\nThe DEGs were plotted and visualized using SRplot (Tang et al., 2023a     ). Biological process gene\nontologies of the separated up-regulated or down-regulated DEGs were identified using enrichGO\nin clusterProfiler (Yu et al., 2012     ) (version 4.10.1) and then replotted using SRplot. The three\neutopic ALDHHI organoid RNA sequencing data were then compared in the same manner with two\npatients’ ectopic endometriotic lesion derived ALDHHI organoid RNA sequencing to identify DEGs\nbetween eutopic and ectopic ALDHHI organoids. The same cutoff values and programs for data\nanalysis and visualization as mentioned above were used in this analysis.\nSequencing data of all human organoid samples are available at GSE294342.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 22 of 46\nTranscriptomic Profiling and Comparison of Mouse Endometrial\nOrganoids by RNA Sequencing\nTotal RNA was extracted from cultured mouse endometrial organoids at passage 3-5 using the\nDirectZol kit (Zymo Research, R2052) following the manufacturer’s protocol, with an additional on-\ncolumn DNase I digestion step to remove genomic DNA contamination. Endometrial epithelial cells\nfrom ~13 WT (randomly cycling) were subjected to the ALDEFLUOR assay and sorted into ALDHLO\nand ALDHHI populations. After sorting, the cells were plated and expanded for 3-5 passages. The\nexperiment was repeated three different times. The ALDHLO and ALDHHI organoids were\ncollected in Trizol, and profiled using RNAseq. RNA with a high integrity (RIN >8) was used for\nlibrary preparation using the Illumina TruSeq RNA Library Prep Kit and sequenced with Illumina\nNovaseq (Novogene, Inc., Sacramento, CA). DEGs between the mouse ALDHHI and ALDHLO groups\nwere calculated using DEseq2 (version 1.42.1) with a log2FC > 1 and < -1 and an adjusted p value <\n0.005. The DEGs were plotted and visualized using SRplot (Tang et al., 2023a     ). Syed et al.’s\nAXIN2HI RNA sequencing data was acquired from Series GSE140222 where the gene list was\nfiltered according to log2FC > 1 and < -1 and an adjusted p value < 0.005 to identify significantly up\nand down genes to compare to the ALDHHI gene lists. Overlap of the up and down genes between\nthe ALDH and AXIN2 lists was done with Gene List Venn Diagram available at\nhttps://www.bioinformatics.org/gvenn/     . Sequencing data of the ALDHHI and ALDHLO mouse\nendometrial organoids are available at GSE294342.\nSingle-cell RNA sequencing (scRNAseq)\nEndometrial epithelial cells were isolated from ~6 adult WT mice uteri at the stages of estrus and\ndiestrus as described above (“Mouse endometrial epithelium dissociation for organoids and single-\ncell RNA sequencing.”) Epithelial cells were viably frozen in 90% FBS and 10% DMSO until the day\nof analysis. Single viable cells from the 6 adult WT mice were obtained by sorting on a BD FACS\nDIVA using a 100pm nozzle, low pressure, and using DAPI and capturing in 100% FBS to increase\nviability. After the sorting, live cells we centrifuged and resuspended in PBS to adjust the\nconcentration to ~ 1600 cells/μL and a targeted cell recovery of 20000 total cells. Single-cell RNA\nsequencing libraries were prepared using the Chromium Single Cell 3′ Reagent Kits v4 (10x\nGenomics) according to the manufacturer’s instructions. In brief, single-cell suspensions were\nloaded onto the Chromium Controller to generate Gel Bead-In-Emulsions (GEMs), followed by\nreverse transcription and barcoded cDNA-library construction via T100 Thermal Cycler (Bio-Rad\nLaboratories). The resulting libraries were assessed and confirmed to pass quality controls using\nthe Agilent TapeStation system with High Sensitivity D1000 ScreenTape assays (Agilent\nTechnologies). The sequencing was performed on the Illumina NovaSeq X platform (Novogene,\nInc) using paired-end 150 bp reads, targeting approximately 24,000 reads per cell to ensure\nsufficient coverage for robust downstream analysis. Raw sequencing data were processed using\nCell Ranger 9.0.1. The raw reads were aligned to the GRCh38 reference genome, and feature-\nbarcode matrices were subsequently generated. Raw count matrices were imported into Seurat\n5.2.0 for filtering, scaling, normalization, dimensional reduction, and clustering. The analysis of\ntrajectory inference was performed using Slingshot 2.16.0 for constructing developmental lineages\nand identifying dynamic marker genes. The single cell RNA sequencing data for the enriched\nepithelial and stromal populations from mouse uteri in both estrus and diestrus phases are\navailable at GSE294342.\nStatistics and analysis\nStatistical analyses were performed using GraphPad Prism. Data are presented as mean ±\nstandard error of the mean (SEM) or standard deviation (SD), as indicated in the figure legends.\nFor comparisons between two groups, unpaired or paired Student’s t-tests were used for normally\ndistributed data, while Mann-Whitney U or Wilcoxon signed-rank tests were applied for non-\nparametric data. For multiple group comparisons, one-way or two-way ANOVA was performed,\nfollowed by appropriate post hoc tests to adjust for multiple comparisons. A p-value < 0.05 was\nconsidered statistically significant, and all statistical tests were two-tailed unless otherwise\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 23 of 46\nspecified. Outliers were identified and excluded only if justified by experimental or technical\nreasons. All graphs were generated using GraphPad Prism and raw data were maintained for\nreproducibility and transparency.\nData availability\nSequencing data are available in the Gene Expression Omnibus under the superseries accession\nnumber GSE294342.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 24 of 46\nSupplementary figures\nFigure S1.Analysis of DEG in ALDHHI vs. ALDHLO mouse organoids. Clustering analysis of stemness-related (A)\nand keratin genes (C) in the mouse epithelial cells established from ALDHHI or ALDHLO organoids. B) Overlap of\nALDHHI genes that are increased and decreased with AXIN2HI endometrial epithelial stem cells in mouse.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 25 of 46\nFigure S2.Identification and characterization of epithelial stem cells in the adult WT mouse uterus at\nestrus and diestrus.\nA) Dot plots showing the expression of Aldh1a1, Lgr5, and Axin2 across the epithelial cell subclusters from the estrus and\ndiestrus phases. B) Estrus and diestrus phase expression of genes identifying epithelial, luminal, and epithelial stem cell\nclusters across each cluster of epithelial cells. C) Heatmap (row for gene, column for individual cell) comparing epithelial cell\nsubclusters from our dataset to DEGs identified from EpSC DEGs of cluster 12 (Padilla-Banks et al., 2023     ). D) UMAP of\nepithelial cell subclusters from mouse samples obtained during the estrus (E) or diestrus phases (F). F-L) Lineage trajectories\nusing pseudotime analysis of the epithelial cell types in estrus (F,G) and diestrus (H-L).\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 26 of 46\nFigure S3.Estrogen and progesterone impact the expression and localization of ALDH1A1 in the WT mouse\nuterus.\nA) Description of the experimental scheme used to ovariectomize and administer hormonal treatments to 6-8-week-old WT\nmice. B-E’) ALDH1A1 immunohistochemistry in the uterine cross-sections of ovariectomized mice treated with vehicle (B-B’),\n1mg P4 (C-C’), 50ng E2 (D-D’), or 1mg P4 + 50ng E2 (E-E’). F) Aldh1a1 gene expression was quantified in the uterine tissues of\nthe ovariectomized mice treated with Vehicle, P4, E2 or E2 + P4. Experiments were repeated in more than three mice per\ngroup. Data in F displayed as mean ± SEM analyzed by a One-Way ANOVA test with a Tukey’s post-hoc test. *, p<0.05; **,\nP<0.01; ***, p<0.001.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 27 of 46\nFigure S4.Comparison of ALDHHI and ALDHLO formation in the organoids from human eutopic endometrial\nepithelium.\nA-B) Phase contrast confocal images and quantification of an organoid formation assay comparing ALDHHI and ALDHLO cells.\nData in C are displayed as mean ± SD analyzed by a two-tailed t-test. *, p<0.05; **, P<0.01; ***, p<0.001.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 28 of 46\nFigure S5.Gene expression pathways are differentially regulated between ALDHHI and ALDHLO cells in the\neutopic endometrial organoids.\nClustering of the top 100 differentially expressed genes in ALDHHI eutopic vs ALDHLO human endometrial epithelial\norganoids.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 29 of 46\nFigure S6.Genotyping strategy for the various mouse lines used in the study.\nA-C) Gel electrophoresis results showing the PCR results for the Aldh1a1cre/ERT2 allele (A), for the Ai9/Ai9tdTomato reporter (B)\nand for the DTR knock-in and WT alleles (C).\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 30 of 46\nAcknowledgements\nStudies were supported by Eunice Kennedy Shriver National Institute of Child Health and Human\nDevelopment grants R01-HD105800, R01-HD099341 (D.M.), R01-HD032067 and R01-HD110038\n(M.M.M.). Diana Monsivais, Ph.D. holds a Next Gen Pregnancy Award (NGP10125) from the\nBurroughs Wellcome Fund. Human Tissue Acquisition and Pathology (HTAP) Core is supported by\na P30 Cancer Center Support Grant (NCI-CA125123). We also thank BCM Department of Pathology\n& Immunology for access to the Sectra Digital Pathology Services. This project was supported by\nthe Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT\nCore Facility Support Award (CPRIT-RP240432), the NIH (CA125123 and OD036336) and the\nassistance of Joel M. Sederstrom. Imaging for this project was supported by the Integrated\nMicroscopy Core at Baylor College of Medicine with funding from the NIH (DK56338, CA125123,\nP30 CA125123, S10OD030414).\nAdditional information\nAuthor contributions\nDesigned experiments (ST, ACU, PJ, DM). Conducted experiments, analyzed and interpreted data\n(ST, ACU, GJH, SP, TG). Collected experimental samples (LAR, BT, XG). Wrote and edited manuscript\n(ST, ACU, PJ, GJH, SP, TG, LAR, BT, XG, DM).\nFunding\nFunder Grant reference number Author\nHHS | NIH | Eunice Kennedy Shriver National\nInstitute of Child Health and Human\nDevelopment (NICHD)\nHD099341 Diana Monsivais\nHHS | NIH | Eunice Kennedy Shriver National\nInstitute of Child Health and Human\nDevelopment (NICHD)\nHD105800 Diana Monsivais\nAuthor ORCID iDs\nDiana Monsivais:  https://orcid.org/0000-0001-5660-6392\nAdditional files\nTable S1.     Differentially expressed genes and gene enrichment analysis of ALDHHI vs. ALDHLO\nmouse endometrial epithelial organoids.\nTable S2.     Differentially expressed genes between clusters 4 and 25 versus other epithelial cell\nclusters in the scRNAseq dataset.\nTable S3.     Differentially expressed genes and gene enrichment analysis of ALDHHI vs. ALDHLO\nhuman endometrial epithelial organoids.\nTable S4.     List of primers and antibodies.\nTable S5.     Patient sample information.\nReferences\nAzcorra M., Gaertner Z., Davidson C., He Q., Kim H., Nagappan S., Hayes C.K., Ramakrishnan C., Fenno L.,\nKim Y.S., et al. 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Omics 16:284 ‑ 287 https://doi.org/10.1089/omi.2011.0118 | PubMed\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 35 of 46\nPeer reviews\nReviewer #1 (Public review):\nThe manuscript by Tang et al. characterizes the expression dynamics and functional roles of\naldehyde dehydrogenase 1 activity in uterine physiology. Using a combination of in vivo\nlineage tracing and cell ablation coupled with organoid culture, the authors propose that\nAldh1a1 lineage-marked cells contribute to uterine gland development and epithelial\nregeneration. The descriptive data will be of interest to reproductive biologists and clinicians\nand will build on established hypotheses in the field. The manuscript is well written and\nscientifically sound; however, several experimental limitations and interpretation caveats\nshould be addressed.\nThe methods surrounding the passage number and duration of culture following sorting\nprior to transcriptomic profiling should be clarified in the figure legends. Related to this, the\nrepresentative images in Figures 1D and 1E do not appear consistent with the quantification\npresented in Figures 1F-H and should be reconciled.\nThe conclusion that ALDH1A1+ cells are enriched in populations with stem cell\ncharacteristics relies primarily on transcriptomic analysis. Protein-level co-localization\nshould be performed to strengthen this claim.\nThe overlap of 19 genes between the data set here and AXIN2 HI data is presented as\nevidence of shared stemness identity, but no statistical assessment of this overlap is provided.\nA hypergeometric test should be performed to determine whether this overlap is greater than\nexpected by chance.\nThe impact of tamoxifen injection on Aldh1a1 expression should be characterized in the\nneonatal uterus, as tamoxifen itself has known estrogenic activity that could confound\ninterpretation of the lineage tracing results at early postnatal timepoints. Related to this,\nwhile low-dose tamoxifen is shown to label individual cells within 24 hours of injection, the\ntranslation dynamics of the label following Cre-mediated recombination can require up to 72\nhours. The presence of only a few labeled clones at PND8 but multiple separate clones per\ncross-section at later timepoints warrants discussion and may reflect labeling kinetics rather\nthan clonal expansion.\nIt would strengthen the in vivo ablation data to validate the degree of cell death following\ndiphtheria toxin treatment directly. It is possible that a general decrease in cell number\nrather than specific loss of a stem cell population is responsible for the observed reduction in\ngland number and FOXA2 expression (Tongtong et al 2017).\nThe lineage tracing data in the postpartum endometrium demonstrate that Aldh1a1-marked\ncells are present during regeneration, but it remains unclear whether these cells are\npreferentially activated or expanded in response to tissue injury. Coupling these studies with\ndiphtheria toxin-mediated ablation during active regeneration would more directly test the\nproposed regenerative role of this population.\nThe contribution of stromal Aldh1a1 lineage-positive cells is underexplored in the discussion,\ngiven the lineage tracing data showing stromal labeling across multiple timepoints and its\npotential relevance to mesenchymal-to-epithelial transition.\nFinally, the word 'control' may overstate the functional evidence presented. 'Contribute' may\nbe more accurate given the partial and context-dependent nature of the phenotypes\nobserved.\nhttps://doi.org/10.7554/eLife.110975.1.sa2\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 36 of 46\nReviewer #2 (Public review):\nTang et al. investigated the contribution of Aldh1a1+ cells, as putative stem/progenitor cells,\nto endometrial development, maintenance during the estrous cycle, and postpartum repair in\nmouse models. They employed in vitro organoid formation and in vivo lineage tracing\nmodels coupled with RNA-seq to test the stem-ness of Aldh1a1+ cells. They found that mouse\nendometrial cells with high ALDH activity (using the ALDEFLUOR assay) formed more and\nlarger organoids and were enriched for stem/progenitor cell gene signatures. Similar results\nwere shown using endometrial cells from a human patient sample. Epithelial ALDH1A1\nexpression was shown to be hormonally regulated, becoming more restricted to the glands, a\nputative epithelial stem cell niche, under estrogen stimulation. Using lineage-tracing initiated\npostnatally/prepubertally, Aldh1a1+ epithelial cells were shown to expand, contributing to\nboth the luminal and glandular epithelium into adulthood, whereas adult initiation of\nlabeling showed expansion of stromal Aldh1a1+ cells but not epithelial. Postnatal ablation of\nsingle-labeled Aldh1a1+ epithelial cells resulted in impaired gland development. Lastly,\nAldh1a1-lineage traced cells (adult labeled) were present during postpartum endometrial\nrepair as were epithelial/mesenchymal transitional cells.\nThis study addresses an important area of research in the field of endometrial\nstem/progenitor cell biology. The authors are commended for their use of multiple\ncomplementary methods, including lineage tracing, DTR-mediated cell ablation, organoid\nassays, and RNA-seq in mouse and human models to assess the stem-like nature of Aldh1a1+\ncells. The data support the stem/progenitor phenotype of Aldh1a1+ epithelial cells during\nendometrial development; however, there are noted discrepancies between organoid\nformation assays and lineage tracing experiments regarding the stemness of Aldh1a1+\nepithelial cells in adults. Specifically, organoids were generated from adult cells and\ndemonstrated in vitro stem cell activity; however, in vivo lineage-tracing of adult cells either\nduring the estrous cycle or postpartum repair does not show expansion of Aldh1a1+ cells,\nsuggesting they do not have stem/progenitor activity. Additionally, the stem-ness of epithelial\nvs stromal Aldh1a1+ cells is confounded in the study because epithelial cells were not\npurified for organoid experiments, epithelial cells were not exclusively lineage-traced as\nstromal cells were also labeled, and mesenchymal-epithelial transition was suggested to\noccur during postpartum repair. The following specific comments are presented to detail\nthese concerns:\n(1) The statement in the brief summary, \"...critical for lifelong endometrial regeneration,\" is\nnot supported by the data provided.\n(2) AlDH1A1 is not restricted to the endometrial epithelium, and epithelial cells were not\npurified by flow cytometry for experiments in Figure 1. Figure 2 clearly shows the presence\nof mesenchymal cells, even using the described method for enriching for epithelial cells.\nTherefore, contaminating mesenchymal cells with high ALDH activity may confound the\nexperimental results in Figure 1, either through promoting epithelial cell growth or through\nMET. The authors should provide clear evidence of epithelial purity in organoid experiments\nor that mesenchymal cells are not contained in the ALDHhi population. These comments also\napply to the human organoid experiments in Figure 7.\n(3) Lines 186-187: Susd2 was increased in EpSC clusters, yet this is a mesenchymal\nstem/progenitor marker in humans. The authors should discuss the implications of this.\n(4) In Figure 5, RFP+ epithelial cells should be quantified as in previous figures to substantiate\nthe statement in lines 279-280, \"At PPD5, the proportion of RFP+ epithelial cells had expanded\nrelative to PPD1 and PPD3 (Figure 5E-E').\" Especially because in the low mag images (C-E),\nRFP+ epithelial cells appear to be most abundant at PPD1 and decrease at PPD3 and PPD5,\nsuggesting that they may not be involved in endometrial regeneration/repair (contradicting\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 37 of 46\nthe interpretation in line 285). Further, if there is in fact a decrease over postpartum repair,\nthen regeneration should be removed from the title of the manuscript. RFP+ stromal cells\nshould also be quantified.\n(5) For Figure 7F, it should be clearly stated in the main text that the results are from one\npatient sample and the data presented are experimental replicates, so as not to be confused\nwith biological replicates (the same for Supplementary Figure S4). Were B and G in Figure 7\nalso from one patient?\n(6) Lines 425-427: \"Ovariectomized mice treated with 90-day E2 pellets, on the other hand,\nshowed a complete restriction of ALDH1A1 to the glandular crypts.\" In Figure 2 S' ALDH1A1+\ncells are visible in the LE (the staining is lighter than in the GE but looks real), contradicting\nthis statement.\n(7) Lines 466-467: \"In cycling mice, we found sporadic cells that expressed both stromal and\nepithelial markers in the ALDHA1+ cells.\" These data are not presented.\n(8) These data support the role of Aldh1a1+ cells in endometrial epithelial development, but\nconclusions about their role in repair/regeneration should be tempered as the data are much\nweaker here.\nhttps://doi.org/10.7554/eLife.110975.1.sa1\nReviewer #3 (Public review):\nSummary:\nTan et al demonstrated the importance of ALDH-high cells in the epithelial development in\nthe mouse endometrium, and these cells displayed properties of stem cells.\nStrengths:\nThe findings are solid, supported and validated through a combination of technical methods.\nI appreciated this combined use of mouse and human endometrial cells to strengthen the\nfindings. Genomic results from a single-cell sequencing dataset were informative as they\ndepicted the different stages of the estrus cycle during the regeneration process. Verification\nwith immunostainings with various markers made it convincing for readers to visualize the\ncell's location, progression, and status at different timepoints. Utilizing human endometrial\ncells further demonstrated that the phenomenon observed in mice can be translated to\nhumans.\nThis work will greatly advance the understanding of endometrial regeneration for\nreproductive biologists.\nWeaknesses:\nNo major weaknesses were identified by this reviewer.\nhttps://doi.org/10.7554/eLife.110975.1.sa0\nAuthor response:\neLife Assessment\nThis valuable study reports that the ALDH-abundant cells display stem cell properties and\nmay play a key role in the endometrial epithelial development in the mouse. The data\nsupporting the main conclusion are solid, although further improvements are needed to\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 38 of 46\nstrengthen the conclusions. This work will be of great interest to reproductive biologists\nand biomedical researchers working on women's reproductive health.\nWe thank the reviewers and editor for their critical reading and assessment of our\nmanuscript. We carefully considered each of the points raised by the reviewers. In this\ndocument and in the edited manuscript and figures, we have carefully addressed each of the\ncomments and requested modifications. In light of these changes, we expect that you will find\nthat the manuscript has improved.\nWe indicate our responses to the reviewers below in blue font and highlight the changes in\nthe manuscript using the line numbers corresponding to the tracked version of the revised\ndocument.\nPublic Reviews:\nReviewer #1 (Public review):\nThe manuscript by Tang et al. characterizes the expression dynamics and functional roles\nof aldehyde dehydrogenase 1 activity in uterine physiology. Using a combination of in\nvivo lineage tracing and cell ablation coupled with organoid culture, the authors propose\nthat Aldh1a1 lineage-marked cells contribute to uterine gland development and\nepithelial regeneration. The descriptive data will be of interest to reproductive biologists\nand clinicians and will build on established hypotheses in the field. The manuscript is well\nwritten and scientifically sound; however, several experimental limitations and\ninterpretation caveats should be addressed.\nWe thank the reviewer for their comments and expert assessment of our paper.\n(1) The methods surrounding the passage number and duration of culture following\nsorting prior to transcriptomic profiling should be clarified in the figure legends. Related\nto this, the representative images in Figures 1D and 1E do not appear consistent with the\nquantification presented in Figures 1F-H and should be reconciled.\nThanks for this comment. We have now clarified this in the Figure 1 legend as follows,\nLines 1026-1029: “Organoid formation assay performed immediately after luminal epithelial\ncell isolation and by plating equal numbers of viable ALDHLO (D) and ALDHHI (E) epithelial\ncells. ALDHLO and ALDHHI organoids were cultured for two weeks and passaged once prior\nto the organoid formation assays and transcriptomic analyses.”\nRegarding the second comment, we recognize that the images we showed may not have been\nthe most representative of our quantification. As such, we replaced them with the organoid\nimages below so that they better reflect the quantification outlined in Figure 1F-H.\n(2) The conclusion that ALDH1A1+ cells are enriched in populations with stem cell\ncharacteristics relies primarily on transcriptomic analysis. Protein-level co-localization\nshould be performed to strengthen this claim.\nWe thank the reviewer for this comment. Unfortunately, the antibodies for many of these\nstem cell markers (such as LGR5, AXIN2, and SUSD2) are not well-suited for immunostaining.\nOthers that have been proposed in human and are amenable to immunostaining are not\nsuitable markers for mouse endometrial stem cells (such as CDH2). We hope that by showing\nthat ALDH1A1 is expressed in patterns that are similar to the previously published stem cell\nmarkers LGR5 and AXIN2 (i.e., throughout the epithelium in the developing uterus and\nsubsequently enriched in the tips of the endometrial glands of adult mice), along with\ntranscriptomic studies, we can demonstrate its utility as a marker for mouse endometrial\nstem cells.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 39 of 46\n(3) The overlap of 19 genes between the data set here and AXIN2 HI data is presented as\nevidence of shared stemness identity, but no statistical assessment of this overlap is\nprovided. A hypergeometric test should be performed to determine whether this overlap\nis greater than expected by chance.\nThank you for this suggestion. We have performed a hypergeometric test and determined\nthat the reported shared genes between the two datasets are greater than is expected by\nchance. We have updated the results section to state the following:\nLines 133-141: \"We determined that the overlap between ALDHHI and Axin2+ stemness\nmarker genes was significantly greater than expected by chance for both upregulated (21/346\ngenes, 1.81-fold enrichment, p = 0.0067) and downregulated (19/674 genes, 1.67-fold\nenrichment, p = 0.021) gene sets (hypergeometric test, universe = 23,182 genes).\"\n(4) The impact of tamoxifen injection on Aldh1a1 expression should be characterized in\nthe neonatal uterus, as tamoxifen itself has known estrogenic activity that could\nconfound interpretation of the lineage tracing results at early postnatal timepoints.\nAlthough we took measures to control for this possibility by using multiple time-points and\nmodels to trace the impact of Aldh1a1+ cells in development and adulthood, we recognize the\nimportance of this comment and acknowledge that this is a limitation in the design of our\nstudy. We have included the following text to the Discussion acknowledging this point:\nLines 434-442: “Given the well-documented impacts of tamoxifen for lineage tracing studies,\nit is imperative to use doses of tamoxifen that will minimize estrogenic impacts and result in\noff-target effects (Rios et al., 2016). This often requires administration at doses that will\nachieve maximal recombination of the desired gene, while ensuring that the potential\ndeleterious impacts of tamoxifen are minimized (Chen et al., 2023; Pimeisl et al., 2013). The\ncre/ERT2 tamoxifen inducible model is widely used to study uterine biology where it serves\nas a useful tool to interrogate the spatiotemporal impact of key genes, either through\ninactivation or for lineage tracing. Despite its widely documented utility across many tissue\ntypes and developmental timepoints, the use of tamoxifen and its impacts on the\nendometrium remain a limitation of our study, which we tried to address by implementing\nmultiple timepoints, doses, and orthogonal assays in our experimental design.”\n(4b) Related to this, while low-dose tamoxifen is shown to label individual cells within 24\nhours of injection, the translation dynamics of the label following Cre-mediated\nrecombination can require up to 72 hours. The presence of only a few labeled clones at\nPND8 but multiple separate clones per cross-section at later timepoints warrants\ndiscussion and may reflect labeling kinetics rather than clonal expansion.\nThe reviewer raises an important point. We agree that the 72hr-translation kinetics of the cre-\nmediated recombination is a legitimate consideration for interpreting our data and we have\nadded the text below to the Discussion section acknowledging this point.\nWe have addressed this by adding the following text to the discussion:\nLines 418-423: We hypothesized that the singly labeled cells observed from one day tracing\nexperiments expanded in a clonal fashion during the various timepoints we measured. We\nnote that the translation kinetics of the labeled cells following cre-mediated recombination\nmay contribute to the limited labeling observed at PND8/PND15 and there is a potential for\ndelayed labeling of cells between 24 and 72 hours of tamoxifen administration. However, the\ncontinuous increase in labeled cells at the subsequent timepoints favors our interpretation of\nclonal expansion as the primary explanation.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 40 of 46\n(5) It would strengthen the in vivo ablation data to validate the degree of cell death\nfollowing diphtheria toxin treatment directly. It is possible that a general decrease in cell\nnumber rather than specific loss of a stem cell population is responsible for the observed\nreduction in gland number and FOXA2 expression (Tongtong et al 2017).\nWe agree that this is an important control to incorporate into our experimental design. To\nrule out this possibility, we performed immunohistochemistry of cleaved caspase 3 in the\nuterine tissues of DTRflox/flox and DTRflox/flox;Aldh1a1cre/ERT2 mice 4 days after\nadministration of diphtheria toxin. The results indicate similar levels of cleaved caspase 3\ndetection in both genotypes, suggesting that the decrease in FOXA2+ cells is not due to non-\nspecific cell death, but rather the result of ALDH1A1+ cells. These data and the following text\nhave been added to the manuscript:\nLines 321-325: “We determined that the decreased in FOXA2+ cells in the experimental mice\nwas not the result of non-specific DT-mediated cell death, as similar levels of cleaved caspase\n3-positive cells were detected in the DT-treated control ROSA26DTR/DTR and\nROSA26DTR/DTR;Aldh1a1cre/ERT2/+ mice 4 days post-diphtheria toxin administration (Figure\nS3G-H’).”\n(6) The lineage tracing data in the postpartum endometrium demonstrate that Aldh1a1-\nmarked cells are present during regeneration, but it remains unclear whether these cells\nare preferentially activated or expanded in response to tissue injury. Coupling these\nstudies with diphtheria toxin-mediated ablation during active regeneration would more\ndirectly test the proposed regenerative role of this population.\nThis is a great point and one that we would be very interested in pursuing as follow-up\nstudies in our future work. Regretfully, due to the long generation time and experimental\nprocedures associated with these proposed studies, we are not able to include these\nexperiments in the current manuscript. Thus, we have changed our wording and conclusions\nthroughout the manuscript to be less definitive in terms of the role of Aldh1a1 in\nregeneration, since this will be the focus of future studies\nThe contribution of stromal Aldh1a1 lineage-positive cells is underexplored in the\ndiscussion, given the lineage tracing data showing stromal labeling across multiple\ntimepoints and its potential relevance to mesenchymal-to-epithelial transition.\nThank you for the suggestion. We have now expanded this section in the Discussion to\ninclude the following:\nLines 497-505: We also found ALDH1A1+ stromal cells were more prevalent when tracing\nbegan in adult mice. Other studies have shown that mesenchymal cells contribute to\nendometrial regeneration in the postpartum phase or after induced menses through a\nprocess of MET (Cousins et al., 2014; Kirkwood et al., 2022; Li et al., 2025). Similarly, lineage\ntracing studies have shown that MET is an active process and contributes to epithelial cell\nregeneration in the post-partum phase (Huang et al., 2012; Patterson et al., 2013). Although\nthis is an area of active investigation in the field, with some contradicting reports, it is\nplausible to hypothesize that endometrial tissue has the capacity to undergo wound-healing\nand regeneration via several mechanisms (Ang et al., 2023; Ghosh et al., 2020). The process of\nMET in wound healing is widely documented in other organs, such as the kidney, liver and\nlung, where MET is associated with depletion of the resident epithelial cell pool (Bi et al.,\n2012; Niayesh-Mehr et al., 2024; Zeisberg et al., 2005).\nFinally, the word 'control' may overstate the functional evidence presented. 'Contribute'\nmay be more accurate given the partial and context-dependent nature of the phenotypes\nobserved.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 41 of 46\nWe agree with the reviewer’s point that control may overstate the evidence that we provide\nin the manuscript. To reflect this, we have edited the manuscript title and text to address this\nsuggestion.\nReviewer #2 (Public review):\nTang et al. investigated the contribution of Aldh1a1+ cells, as putative stem/progenitor\ncells, to endometrial development, maintenance during the estrous cycle, and\npostpartum repair in mouse models. They employed in vitro organoid formation and in\nvivo lineage tracing models coupled with RNA-seq to test the stem-ness of Aldh1a1+ cells.\nThey found that mouse endometrial cells with high ALDH activity (using the ALDEFLUOR\nassay) formed more and larger organoids and were enriched for stem/progenitor cell\ngene signatures. Similar results were shown using endometrial cells from a human\npatient sample. Epithelial ALDH1A1 expression was shown to be hormonally regulated,\nbecoming more restricted to the glands, a putative epithelial stem cell niche, under\nestrogen stimulation. Using lineage-tracing initiated postnatally/prepubertally, Aldh1a1+\nepithelial cells were shown to expand, contributing to both the luminal and glandular\nepithelium into adulthood, whereas adult initiation of labeling showed expansion of\nstromal Aldh1a1+ cells but not epithelial. Postnatal ablation of single-labeled Aldh1a1+\nepithelial cells resulted in impaired gland development. Lastly, Aldh1a1-lineage traced\ncells (adult labeled) were present during postpartum endometrial repair as were\nepithelial/mesenchymal transitional cells.\nThis study addresses an important area of research in the field of endometrial\nstem/progenitor cell biology. The authors are commended for their use of multiple\ncomplementary methods, including lineage tracing, DTR-mediated cell ablation,\norganoid assays, and RNA-seq in mouse and human models to assess the stem-like\nnature of Aldh1a1+ cells. The data support the stem/progenitor phenotype of Aldh1a1+\nepithelial cells during endometrial development; however, there are noted discrepancies\nbetween organoid formation assays and lineage tracing experiments regarding the\nstemness of Aldh1a1+ epithelial cells in adults. Specifically, organoids were generated\nfrom adult cells and demonstrated in vitro stem cell activity; however, in vivo lineage-\ntracing of adult cells either during the estrous cycle or postpartum repair does not show\nexpansion of Aldh1a1+ cells, suggesting they do not have stem/progenitor activity.\nAdditionally, the stem-ness of epithelial vs stromal Aldh1a1+ cells is confounded in the\nstudy because epithelial cells were not purified for organoid experiments, epithelial cells\nwere not exclusively lineage-traced as stromal cells were also labeled, and mesenchymal-\nepithelial transition was suggested to occur during postpartum repair. The following\nspecific comments are presented to detail these concerns:\nWe thank the reviewer for their critical reading of our manuscript and constructive\ncomments.\n(1) The statement in the brief summary, \"...critical for lifelong endometrial regeneration,\"\nis not supported by the data provided.\nWe have edited the brief summary to exclude this statement, it now reads as follows:\nLines 4-5: “We uncover ALDH1A1+ cells as a group of hormone sensitive stem cells\ncontributing to endometrial development and regeneration.”\n(2) AlDH1A1 is not restricted to the endometrial epithelium, and epithelial cells were not\npurified by flow cytometry for experiments in Figure 1. Figure 2 clearly shows the\npresence of mesenchymal cells, even using the described method for enriching for\nepithelial cells. Therefore, contaminating mesenchymal cells with high ALDH activity may\nconfound the experimental results in Figure 1, either through promoting epithelial cell\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 42 of 46\ngrowth or through MET. The authors should provide clear evidence of epithelial purity in\norganoid experiments or that mesenchymal cells are not contained in the ALDHhi\npopulation. These comments also apply to the human organoid experiments in Figure 7.\nWe thank the reviewer for raising this important point. Our group has been using the\nenzymatic method to routinely separate epithelial from stromal cell populations from the\nmouse uterus (see references dating back to 2015, PMID 26721398, 28324064, 34099644). In\nthese experiments we typically obtain >98% purity in the epithelial and stromal cell\ncompartments, respectively. We can directly observe this purity in the immunofluorescence\nimages shown below, where mouse endometrial epithelial cells and stromal cells were\nenzymatically separated and immunostained with E-cadherin and vimentin antibodies to\ndetect epithelial and mesenchymal cells in both cell preparations. The images show very few\ncontaminating epithelial and stromal cells in either cell preparation. We have observed\nsimilar results when preparing epithelial and stromal cell preparation from the human\nendometrium, where the epithelial cell organoids display high purity with ~100% epithelial\ncell expression when we perform immunostaining.\nAuthor response image 1.Purity of mouse endometrial epithelial cells obtained via enzymatic and\nmechanical dissociation. A-B) Shows the epithelial (A) and stromal (B) cells plated on glass coverslips and\nimmunostained with an epithelial cell marker (cytokeratin 8, red), a stromal cell marker (vimentin, green), and\nDAPI.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 43 of 46\nAuthor response image 2.Human endometrial epithelial organoids were fixed and immunostained with\ncytokeratin 8 (green) and DAPI. The images are typical for our epithelial cell cultures and demonstrate that all\nepithelial cells are CK8-positive.\n(3) Lines 186-187: Susd2 was increased in EpSC clusters, yet this is a mesenchymal\nstem/progenitor marker in humans. The authors should discuss the implications of this.\nWe thank the reviewer for highlighting this. We have now included the following in our\nDiscussion to address this point:\nLines 528-533: Clustering with this population of EpSCs were Susd2+ cells, which are well-\ncharacterized mesenchymal progenitors that are enriched in the perivascular regions of the\nhuman endometrium (Darzi et al., 2016; Khanmohammadi et al., 2021). The presence of\nSusd2+ cells, while unexpected in an epithelial stem cell niche, could indicate the presence of\na transitional mesenchymal or perivascular cell that is differentiating into epithelium.\nEvidence for both mesenchymal and Nestin2+ pericytes have been recently described in the\nmouse endometrial epithelium (Kirkwood et al., 2022; Li et al., 2025).\n(4) In Figure 5, RFP+ epithelial cells should be quantified as in previous figures to\nsubstantiate the statement in lines 279-280, \"At PPD5, the proportion of RFP+ epithelial\ncells had expanded relative to PPD1 and PPD3 (Figure 5E-E').\" Especially because in the\nlow mag images (C-E), RFP+ epithelial cells appear to be most abundant at PPD1 and\ndecrease at PPD3 and PPD5, suggesting that they may not be involved in endometrial\nregeneration/repair (contradicting the interpretation in line 285). Further, if there is in\nfact a decrease over postpartum repair, then regeneration should be removed from the\ntitle of the manuscript. RFP+ stromal cells should also be quantified.\nWe appreciate this reviewer’s comment and agree that as stated, the conclusion is not fully\nsupported by the data. To address this comment, we have edited the results so that they\nclearly indicate the results and remove any ambiguity:\nAs requested, we quantified the number of RFP+ stromal and epithelial cells during the\npostpartum phase and noted that RFP+ cells were prominent in the stromal compartment of\nthe endometrium. While RFP+ epithelial were also observed during these timepoints, they\nwere less abundant than RFP+ stromal cells. Because the number of RFP+ cells did not\nsignificantly change over the postpartum phases in neither the stromal nor epithelial\ncompartment, we have modified our conclusion to state that ALDH1A1+ cells are transiently\ndetected in the regenerating endometrium.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 44 of 46\nResults:\nLines 286-295: “By analyzing the uterine tissues near the placental detachment site, we\nobserved that RFP positive cells were prominent in the endometrial stromal cells that were\nadjacent to the luminal epithelium (Figure 5C-C’, green arrows). RFP+ cells were also observed\nin the stromal cells near the placental detachment sites at PPD1 and PPD3 (Figure 5D’-E’, red\n& blue arrows) and in limited luminal epithelial cells (Figure 5D”,E”). Quantification of RFP+\ncells throughout these postpartum phases indicated that stromal cells had more frequent\nALDH1A1+ stromal cells (360 ± 103, PPD1, n=3; 217 ± 107, PPD3, n=3; 254 ± 32, PPD5, n=4) than\nALDH1A1+ epithelial cells in the regenerating endometrium (65 ± 65, PPD1, n=3; 20 ± 10,\nPPD3, n=3; 114.25 ± 39, PPD5, n=4) (Figure S4).”\nDiscussion:\nLines 513-521: “We also noted that a majority of ALDH1A1+ cells were localized to the active\nareas of endometrial regeneration near the placental detachment sites at PPD1 with a\npronounced expression in the sub-epithelial stromal cells. As regeneration progressed, we\ncontinued to observe ALDH1A1+ cells in the stromal compartment within the placental\ndetachment sites at PPD3 and PPD5, with a progressive, but not statistically significant,\nincrease in ALDH1A1+ epithelial cells. Collectively, our data demonstrate that ALDH1A1+\nlineage cells participate in the restoration of endometrial architecture and functional\ncompartments in the postpartum phase, even if their direct contribution is transient. Future\ndetailed and mechanistic studies will be necessary to fully characterize their role in this\nprocess and their long-term consequence in postpartum regeneration.”\n(5) For Figure 7F, it should be clearly stated in the main text that the results are from one\npatient sample and the data presented are experimental replicates, so as not to be\nconfused with biological replicates (the same for Supplementary Figure S4). Were B and\nG in Figure 7 also from one patient?\nThanks for pointing this out. We have edited the figure legends in the main text and\nsupplemental figures to indicate this.\nLines 337-338: “…main figures show representative results from one patient sample\nperformed in technical replicates, with additional patient samples included in the\nsupplement…”\n(6) Lines 425-427: \"Ovariectomized mice treated with 90-day E2 pellets, on the other\nhand, showed a complete restriction of ALDH1A1 to the glandular crypts.\" In Figure 2 S'\nALDH1A1+ cells are visible in the LE (the staining is lighter than in the GE but looks real),\ncontradicting this statement.\nThis is an important distinction. We have now edited this part of the manuscript to state:\nLines 459-462: “Ovariectomized mice treated with 90-day E2 pellets, on the other hand,\nshowed enriched ALDH1A1 in the glandular crypts with weak luminal epithelial staining,\nwhile the ovariectomized controls had strong ALDH1A1 expression throughout the luminal\nand glandular epithelium.”\n(7) Lines 466-467: \"In cycling mice, we found sporadic cells that expressed both stromal\nand epithelial markers in the ALDHA1+ cells.\" These data are not presented.\nWe apologize for the confusion, this sentence has been removed from the discussion.\n(8) These data support the role of Aldh1a1+ cells in endometrial epithelial development,\nbut conclusions about their role in repair/regeneration should be tempered as the data\nare much weaker here.\nStem Cells and Regenerative Medicine\n\nTang et al., 2026 eLife 15:RP110975.  https://doi.org/10.7554/eLife.110975.1 45 of 46\nWe thank the reviewer for their overall assessment. To address this point, we have\nthoroughly edited the appropriate areas to temper the conclusions and ensure that they are\nstrongly supported by our data. We have also edited the manuscript’s title to reflect this.\nReviewer #3 (Public review):\nSummary:\nTan et al demonstrated the importance of ALDH-high cells in the epithelial development\nin the mouse endometrium, and these cells displayed properties of stem cells.\nWe thank the reviewer for their assessment of our manuscript.\nStrengths:\nThe findings are solid, supported and validated through a combination of technical\nmethods. I appreciated this combined use of mouse and human endometrial cells to\nstrengthen the findings. Genomic results from a single-cell sequencing dataset were\ninformative as they depicted the different stages of the estrus cycle during the\nregeneration process. Verification with immunostainings with various markers made it\nconvincing for readers to visualize the cell's location, progression, and status at different\ntimepoints. Utilizing human endometrial cells further demonstrated that the\nphenomenon observed in mice can be translated to humans.\nThis work will greatly advance the understanding of endometrial regeneration for\nreproductive biologists.\nWe thank the reviewer for their expert assessment and positive comments regarding our\nmanuscript.\nWeaknesses:\nNo major weaknesses were identified by this reviewer.\nReference\nAng, C.J., Skokan, T.D., and McKinley, K.L. (2023). Mechanisms of Regeneration and Fibrosis in\nthe Endometrium. Annu Rev Cell Dev Biol 39, 197-221.\nBi, W.R., Jin, C.X., Xu, G.T., and Yang, C.Q. (2012). Bone morphogenetic protein-7 regulates Snail\nsignaling in carbon tetrachloride-induced fibrosis in the rat liver. Exp Ther Med 4, 1022-1026.\nChen, M.Y., Zhao, F.L., Chu, W.L., Bai, M.R., and Zhang, D.M. (2023). 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J Biol Chem 280, 8094-8100.\nhttps://doi.org/10.7554/eLife.110975.1.sa4\nStem Cells and Regenerative Medicine","source_license":"CC0","license_restricted":false}