Endometrial epithelial cells with high ALDH activity control uterine development and regeneration

Reviewer #1 (Public review):

The manuscript by Tang et al. characterizes the expression dynamics and functional roles of aldehyde dehydrogenase 1 activity in uterine physiology. Using a combination of in vivo lineage tracing and cell ablation coupled with organoid culture, the authors propose that Aldh1a1 lineage-marked cells contribute to uterine gland development and epithelial regeneration. The descriptive data will be of interest to reproductive biologists and clinicians and will build on established hypotheses in the field. The manuscript is well written and scientifically sound; however, several experimental limitations and interpretation caveats should be addressed.

The methods surrounding the passage number and duration of culture following sorting prior to transcriptomic profiling should be clarified in the figure legends. Related to this, the representative images in Figures 1D and 1E do not appear consistent with the quantification presented in Figures 1F-H and should be reconciled.

The conclusion that ALDH1A1+ cells are enriched in populations with stem cell characteristics relies primarily on transcriptomic analysis. Protein-level co-localization should be performed to strengthen this claim.

The overlap of 19 genes between the data set here and AXIN2 HI data is presented as evidence of shared stemness identity, but no statistical assessment of this overlap is provided. A hypergeometric test should be performed to determine whether this overlap is greater than expected by chance.

The impact of tamoxifen injection on Aldh1a1 expression should be characterized in the neonatal uterus, as tamoxifen itself has known estrogenic activity that could confound interpretation of the lineage tracing results at early postnatal timepoints. Related to this, while low-dose tamoxifen is shown to label individual cells within 24 hours of injection, the translation dynamics of the label following Cre-mediated recombination can require up to 72 hours. The presence of only a few labeled clones at PND8 but multiple separate clones per cross-section at later timepoints warrants discussion and may reflect labeling kinetics rather than clonal expansion.

It would strengthen the in vivo ablation data to validate the degree of cell death following diphtheria toxin treatment directly. It is possible that a general decrease in cell number rather than specific loss of a stem cell population is responsible for the observed reduction in gland number and FOXA2 expression (Tongtong et al 2017).

The lineage tracing data in the postpartum endometrium demonstrate that Aldh1a1-marked cells are present during regeneration, but it remains unclear whether these cells are preferentially activated or expanded in response to tissue injury. Coupling these studies with diphtheria toxin-mediated ablation during active regeneration would more directly test the proposed regenerative role of this population.

The contribution of stromal Aldh1a1 lineage-positive cells is underexplored in the discussion, given the lineage tracing data showing stromal labeling across multiple timepoints and its potential relevance to mesenchymal-to-epithelial transition.

Finally, the word 'control' may overstate the functional evidence presented. 'Contribute' may be more accurate given the partial and context-dependent nature of the phenotypes observed.

Reviewer #2 (Public review):

Tang et al. investigated the contribution of Aldh1a1+ cells, as putative stem/progenitor cells, to endometrial development, maintenance during the estrous cycle, and postpartum repair in mouse models. They employed in vitro organoid formation and in vivo lineage tracing models coupled with RNA-seq to test the stem-ness of Aldh1a1+ cells. They found that mouse endometrial cells with high ALDH activity (using the ALDEFLUOR assay) formed more and larger organoids and were enriched for stem/progenitor cell gene signatures. Similar results were shown using endometrial cells from a human patient sample. Epithelial ALDH1A1 expression was shown to be hormonally regulated, becoming more restricted to the glands, a putative epithelial stem cell niche, under estrogen stimulation. Using lineage-tracing initiated postnatally/prepubertally, Aldh1a1+ epithelial cells were shown to expand, contributing to both the luminal and glandular epithelium into adulthood, whereas adult initiation of labeling showed expansion of stromal Aldh1a1+ cells but not epithelial. Postnatal ablation of single-labeled Aldh1a1+ epithelial cells resulted in impaired gland development. Lastly, Aldh1a1-lineage traced cells (adult labeled) were present during postpartum endometrial repair as were epithelial/mesenchymal transitional cells.

This study addresses an important area of research in the field of endometrial stem/progenitor cell biology. The authors are commended for their use of multiple complementary methods, including lineage tracing, DTR-mediated cell ablation, organoid assays, and RNA-seq in mouse and human models to assess the stem-like nature of Aldh1a1+ cells. The data support the stem/progenitor phenotype of Aldh1a1+ epithelial cells during endometrial development; however, there are noted discrepancies between organoid formation assays and lineage tracing experiments regarding the stemness of Aldh1a1+ epithelial cells in adults. Specifically, organoids were generated from adult cells and demonstrated in vitro stem cell activity; however, in vivo lineage-tracing of adult cells either during the estrous cycle or postpartum repair does not show expansion of Aldh1a1+ cells, suggesting they do not have stem/progenitor activity. Additionally, the stem-ness of epithelial vs stromal Aldh1a1+ cells is confounded in the study because epithelial cells were not purified for organoid experiments, epithelial cells were not exclusively lineage-traced as stromal cells were also labeled, and mesenchymal-epithelial transition was suggested to occur during postpartum repair. The following specific comments are presented to detail these concerns:

(1) The statement in the brief summary, "...critical for lifelong endometrial regeneration," is not supported by the data provided.

(2) AlDH1A1 is not restricted to the endometrial epithelium, and epithelial cells were not purified by flow cytometry for experiments in Figure 1. Figure 2 clearly shows the presence of mesenchymal cells, even using the described method for enriching for epithelial cells. Therefore, contaminating mesenchymal cells with high ALDH activity may confound the experimental results in Figure 1, either through promoting epithelial cell growth or through MET. The authors should provide clear evidence of epithelial purity in organoid experiments or that mesenchymal cells are not contained in the ALDHhi population. These comments also apply to the human organoid experiments in Figure 7.

(3) Lines 186-187: Susd2 was increased in EpSC clusters, yet this is a mesenchymal stem/progenitor marker in humans. The authors should discuss the implications of this.

(4) In Figure 5, RFP+ epithelial cells should be quantified as in previous figures to substantiate the statement in lines 279-280, "At PPD5, the proportion of RFP+ epithelial cells had expanded relative to PPD1 and PPD3 (Figure 5E-E')." Especially because in the low mag images (C-E), RFP+ epithelial cells appear to be most abundant at PPD1 and decrease at PPD3 and PPD5, suggesting that they may not be involved in endometrial regeneration/repair (contradicting the interpretation in line 285). Further, if there is in fact a decrease over postpartum repair, then regeneration should be removed from the title of the manuscript. RFP+ stromal cells should also be quantified.

(5) For Figure 7F, it should be clearly stated in the main text that the results are from one patient sample and the data presented are experimental replicates, so as not to be confused with biological replicates (the same for Supplementary Figure S4). Were B and G in Figure 7 also from one patient?

(6) Lines 425-427: "Ovariectomized mice treated with 90-day E2 pellets, on the other hand, showed a complete restriction of ALDH1A1 to the glandular crypts." In Figure 2 S' ALDH1A1+ cells are visible in the LE (the staining is lighter than in the GE but looks real), contradicting this statement.

(7) Lines 466-467: "In cycling mice, we found sporadic cells that expressed both stromal and epithelial markers in the ALDHA1+ cells." These data are not presented.

(8) These data support the role of Aldh1a1+ cells in endometrial epithelial development, but conclusions about their role in repair/regeneration should be tempered as the data are much weaker here.

Reviewer #3 (Public review):

Summary:

Tan et al demonstrated the importance of ALDH-high cells in the epithelial development in the mouse endometrium, and these cells displayed properties of stem cells.

Strengths:

The findings are solid, supported and validated through a combination of technical methods. I appreciated this combined use of mouse and human endometrial cells to strengthen the findings. Genomic results from a single-cell sequencing dataset were informative as they depicted the different stages of the estrus cycle during the regeneration process. Verification with immunostainings with various markers made it convincing for readers to visualize the cell's location, progression, and status at different timepoints. Utilizing human endometrial cells further demonstrated that the phenomenon observed in mice can be translated to humans.

This work will greatly advance the understanding of endometrial regeneration for reproductive biologists.

Weaknesses:

No major weaknesses were identified by this reviewer.