Mouse endometrial epithelial ALDHHI cells have higher organoid formation capacity and stemness transcriptomes.

A-C) Strategy to isolate and establish ALDHHI and ALDHLO cells from adult WT mouse epithelial organoids using the ALDEFLUOR assay. D-E) Organoid formation assay performed by plating equal numbers of viable ALDHLO (D) and ALDHHI (E) epithelial cells and culturing for two weeks. F-H) Organoid formation rate (F), organoid perimeter (G) and area (H) were assessed by quantifying the total number of organoids formed per 100 cells seeded. Assays were performed using the cells pooled from 7-9 WT adult mice at estrus three independent times. I-J) Transcriptomic profiling of ALDHHI vs. ALDHLO mouse organoids was performed, and the total number of differentially expressed genes (DEG) was determined (I). The total number of up- and down-regulated genes was displayed as a volcano plot. J) Gene ontology analysis was performed on the up- and down-regulated genes between the ALDHLO vs. ALDHHI organoids. K) Comparison of total genes that are conserved as up- or down-regulated between ALDHHI/AXIN2HI and ALDHLO/AXIN2LO cell, selected genes involved in stemness signatures are displayed. Graphs show mean ± SEM and analyzed using a non-parametric Mann-Whitney Test, *, p<0.05; **, P<0.01; ***, p<0.001.

ALDH1A1 is expressed in cells with epithelial stem cell characteristics.

A-B) UMAP displaying the various cell types identified by scRNAseq of enriched endometrial epithelium from WT 6-week-old mice in the estrus (A) and diestrus (B) phases. Epithelial cells were sub-clustered to identity different cell identities (C) and classified into luminal, glandular, and epithelial stem cell (EpSCs) based on the expression of key markers (D, estrus; E, diestrus). F-I) Pseudotime analysis of the epithelial cell types in estrus (F, G) and diestrus (H,I) was performed to identify the trajectory of differentiation, which shows that EpSCs give rise to glandular and epithelial cell lineages. J-L) Dual feature plots showing the overlapping and unique expression patterns of Aldh1a1/Lgr5 (J), Aldh1a1/Axin2 (K), and Lgr5/Axin2 (L) in the epithelial cell clusters in estrus. M-O) A signature score was assigned to the epithelial cells from the estrus phase to determine how strongly genes involved in ‘Glandular Epithelial Development’ (M), ‘BMP signaling’ (N), and ‘Stereocilium’ (O) are expressed. P-S) ALDH1A1 immunohistochemistry in the uterus of adult WT mice during the diestrus (P-P’) and estrus (Q-Q’) phases, or in WT ovariectomized mice without (R-R’) or with an E2 pellet for 90 days (S-S’). T) Aldh1a1 was also quantified in the uterus of 6-8-week-old WT mice collected at different times during the estrous cycle. Experiments were repeated in more than three mice per group. Data in T are displayed as mean ± SEM analyzed by a One-Way ANOVA test with a Tukey’s post-hoc test. *, p<0.05; **, P<0.01; ***, p<0.001. UMAP, uniform manifold approximation and projection; EpSC, epithelial stem cell; BMP, bone morphogenetic protein.

Lineage tracing reveals the contribution of ALDH1A1+ cells to endometrial integrity.

A-C’) ALDH1A1 immunohistochemistry in the WT mouse uterus at PND7 (A-A’), PND14 (B-B’), and 3-weeks (C-C’). D) Schematic of the tamoxifen-inducible ALDH1A1 reporter allele and experimental scheme used for lineage tracing in the postnatal endometrium. E-N’) RFP immunohistochemistry was used to detect the ALDH1A1-tdTomato-expressing cells in the uterus when tracing was performed from PND7 to PND8 (E-E’), PND8 to PND14 (F-F’), PND8 to PND28 (G-G’), PND14 to PND15 (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 tamoxifen (0.15 mg/g body weight). H, L, O) Quantification of RFP+ cells was performed and are presented as total RFP+ endometrial epithelial or stromal cells. Black arrows (E’, I’, M’) indicate singly labeled RFP+ cells, red arrow (K’) indicates RFP+ stromal cell. Images represent staining that was performed in 3 or more mice per timepoint. Data are presented as mean ± SEM analyzed by a Two-Way ANOVA with a Sidak test for multiple comparisons. *, p<0.05; **, P<0.01; ***, p<0.001. PND, postnatal day; TAM, tamoxifen; ORF, open reading frame.

Tracing of ALDH1A1+ cells in the adult cycling uterus identified positive cells in the epithelium and stroma.

A) Schematic diagram demonstrating the reabsorption and reorganization that occurs between estrus phases in the adult mouse uterus. Uterine structures are labelled as: M for mesometrial, AM for anti-mesometrial, Myo for myometrium, S for stromal compartment, and LE for luminal epithelium. B) Timeline for tamoxifen-dependent tomato labeling in ALDH1A1-expressing cells in the adult mice when traced for one day, seven days, or 28 days after Tamoxifen administration. C-E’) RFP immunohistochemistry was used to detect labeled cells when tracing was performed in the adult mouse for 1 day (C-C’), seven days (D-D’), or 28 days (E-E’). Red arrows indicate detection of RFP in the sub-epithelial stromal cells. F-G) Quantification of RFP+ cells in the epithelial and stromal compartments one day, seven days, or four days after tamoxifen administration. Data are presented as mean ± SEM analyzed by a Two-Way ANOVA with a Sidak test for multiple comparisons. *, p<0.05; **, P<0.01; ***, p<0.001. TAM, tamoxifen.

ALDH1A1+ cells contribute to post-partum endometrial regeneration.

A) Schematic highlighting post-partum repair occurring at PPD1, PPD3, and PPD5. Uterine structures are labeled as: M for mesometrial, AM for anti-mesometrial, S for stromal compartment, LE for luminal epithelium, and GE for glandular epithelium. B) ALDH1A1 lineage tracing was begun in the Aldh1a1cre/ERT2;ROSA26tdTomato/tdTomato mice at 2 months of age. Two months after TAM administration, the mice were mated, and their uteri were collected at PPD1, PPD3, and PPD5. C-E’) RFP immunohistochemistry was performed in uterine cross-sections obtained from the placental detachment sites at PPD1 (C-C’), PPD3 (D-D’) and PPD5 (E-E’). Red arrows indicate the RFP+ subepithelial stromal cells. F-J) Immunofluorescence staining of uterine cross-sections with cytokeratin 8 (CK8, cyan), red fluorescence protein (RFP, yellow), vimentin (VIM, magenta), and DAPI (white). Yellow arrowheads (F, G-J) show the presence of cells that are CK8+/RFP+/VIM+; White arrowheads indicate cells that are CK8+/VIM+. Images represent groups of more than three animals analyzed per group. J) Quantification of RFP+ cells in the stromal and epithelial cells at PPD3. K) Analysis of the cells expressing CK8+/VIM+ in the uterine cross-sections at PPD3 and PPD5. Data are presented as mean ± SEM of positive cells per imaged field and analyzed by a Mann-Whitney test (M). *, p<0.0033; **, P<0.002; ***, p<0.001. TAM, tamoxifen; PPD, post-partum day.

Ablation of ALDH1A1+ cells impairs organoid expansion and reduces endometrial glands in adult mice.

A-C) Diphtheria toxin-mediated ablation of ALDH1A1+ cells was obtained in vitro (A) and in vivo (B) by crossing Aldh1a1cre/ERT2;ROSA26tdTomato/tdTomato mice to a line containing a conditional diphtheria toxin receptor (DTR). D-I) Organoids from adult control (DTRf/f) and experimental (Aldh1a1cre/ERT2;DTRf/f) mice were established and expanded in culture for two passages. Once established (D-E), the organoids were treated with DT (F-G) and visualized, fixed and stained with cleaved caspase-3 antibody (H-I). J-K) The impact of DT-mediated ablation of ALDH1A1+ cells was determined in mice treated with TAM at PND7, and with DT at PND10. Uterine tissues were collected and analyzed at PND56 using immunohistochemistry to detect ALDH1A1 (J-K’). N-O) Glands were visualized in the control mice (N) and experimental (O) by staining with FOXA2 (green), cytokeratin 8 (red), and DAPI (white) using confocal imaging. P-Q) FOXA2 intensity per gland (P) and glandular number (Q) were quantified in the uterine cross-sections of >3 mice per genotype. The results are displayed as mean ± SEM and analyzed using a two-tailed t-test, *, p<0.05; **, P<0.01; ***, p<0.001.

ALDHHI cells from eutopic endometrium display organoid formation capacity and different transcriptomic signatures than ALDHLO cells.

A) Schematic approach to isolate ALDHHI and ALDHLO cells from human endometrial epithelial organoids with the ALDEFLUOR assay. B-C) Organoid establishment was assessed in freshly isolated ALDHLO and ALDHHI epithelial cells plated at equal densities. D-F) Organoid formation assay was performed by plating equal numbers of viable ALDHLO (D) and ALDHHI (E) cells followed by quantifying the total number of organoids that were established per 100 cells plated (F). Graph displays the 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) Images comparing the growth of ALDHLO (G,I) and ALDHHI (H,J) organoids at passage 11 (G-H) or passage 16 (I,J). K) Volcano plot showing the total number of differentially expressed transcripts in the ALDHHI vs ALDHLO eutopic organoids from three different patients. L-M) Gene enrichment analysis of increased (L) and decreased (M) genes in ALDHHI eutopic vs ALDHLO eutopic organoids. N) Immunostaining of eutopic ALDHLO and ALDHHI organoids stained with cytokeratin 8 (CK8, green), Acetylated-a-tubulin (AcTub, red), and DAPI (white).

Analysis of DEG in ALDHHI vs. ALDHLO mouse organoids.

Clustering analysis of stemness-related (A) and keratin genes (C) in the mouse epithelial cells established from ALDHHI or ALDHLO organoids. B) Overlap of ALDHHI genes that are increased and decreased with AXIN2HI endometrial epithelial stem cells in mouse.

Identification and characterization of epithelial stem cells in the adult WT mouse uterus at estrus and diestrus.

A) Dot plots showing the expression of Aldh1a1, Lgr5, and Axin2 across the epithelial cell subclusters from the estrus and diestrus phases. B) Estrus and diestrus phase expression of genes identifying epithelial, luminal, and epithelial stem cell clusters across each cluster of epithelial cells. C) Heatmap (row for gene, column for individual cell) comparing epithelial cell subclusters from our dataset to DEGs identified from EpSC DEGs of cluster 12 (Padilla-Banks et al., 2023). D) UMAP of epithelial cell subclusters from mouse samples obtained during the estrus (E) or diestrus phases (F). F-L) Lineage trajectories using pseudotime analysis of the epithelial cell types in estrus (F,G) and diestrus (H-L).

Estrogen and progesterone impact the expression and localization of ALDH1A1 in the WT mouse uterus.

A) Description of the experimental scheme used to ovariectomize and administer hormonal treatments to 6-8-week-old WT mice. B-E’) ALDH1A1 immunohistochemistry in the uterine cross-sections of ovariectomized mice treated with vehicle (B-B’), 1mg 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 the ovariectomized mice treated with Vehicle, P4, E2 or E2 + P4. Experiments were repeated in more than three mice per group. Data in F displayed as mean ± SEM analyzed by a One-Way ANOVA test with a Tukey’s post-hoc test. *, p<0.05; **, P<0.01; ***, p<0.001.

Comparison of ALDHHI and ALDHLO formation in the organoids from human eutopic endometrial epithelium.

A-B) Phase contrast confocal images and quantification of an organoid formation assay comparing ALDHHI and ALDHLO cells. Data in C are displayed as mean ± SD analyzed by a two-tailed t-test. *, p<0.05; **, P<0.01; ***, p<0.001.

Gene expression pathways are differentially regulated between ALDHHI and ALDHLO cells in the eutopic endometrial organoids.

Clustering of the top 100 differentially expressed genes in ALDHHI eutopic vs ALDHLO human endometrial epithelial organoids.

Genotyping strategy for the various mouse lines used in the study.

A-C) Gel electrophoresis results showing the PCR results for the Aldh1a1cre/ERT2 allele (A), for the Ai9/Ai9tdTomato reporter (B) and for the DTR knock-in and WT alleles (C).