Secretion of WNT7A by UC-MSCs assist in promoting the endometrial epithelial regeneration

Abstract

Stem cell therapy for intrauterine adhesions (IUAs) has been widely used in clinical treatment. However, intravenous injection lacks sufficient targeting capabilities, while in situ injection poses challenges in ensuring the effective survival of stem cells. Furthermore, the mechanism underlying the interaction between stem cells and endometrial cells in vivo remains poorly understood, and there is a lack of suitable in vitro models for studying these problems. Here, we designed an extracellular matrix (ECM)-adhesion mimic hydrogel for intrauterine administration, which was more effective than direct injection in treating IUAs. Additionally, we analyzed the epithelial-mesenchymal transition (EMT) and confirmed that the activation of endometrial epithelial stem cells is pivotal. Our findings demonstrated that umbilical cord mesenchymal stem cells (UC-MSCs) secrete WNT7A to activate endometrial epithelial stem cells, thereby accelerating regeneration of the endometrial epithelium. Concurrently, under transforming growth factor alpha (TGFA) stimulation secreted by the EMT epithelium, UC-MSCs upregulate E-cadherin while partially implanting into the endometrial epithelium.

Keywords: Cell biology; Female reproductive endocrinology; Stem cells research.

Graphical abstract

Figure 1

Development of ECM-adhesion mimic hydrogels and treatment for intrauterine adhesions (A) Heatmap of the normalized spectral abundance factor (NSAF) for matrisome proteins in rat uterine tissues (n = 4). (B) Proportion of matrisome proteins in rat uterine tissues. (C) Schematic representation of hydrogel synthesis and UC-MSC encapsulation. (D) Brightfield image of hydrogel-loaded UC-MSCs (scale bar: 100 μm). (E) Electron microscopy image of the hydrogel (scale bar: 200 μm). (F) Schematic representation of the treatment for IUA rats.

Figure 2

UC-MSC promote endometrial epithelial cell proliferation and reverse the EMT process (A) Organoid formation efficiency of EEO cells cocultured with UC-MSCs (scale bar: 100 μm, n = 3, ∗p < 0.05). (B) Brightfield images of normal EEO, EMT-induced EEO, and UC-MSCs+EMT-induced EEO (scale bar: 100 μm). (C) Time-lapse recording of the EMT process (scale bar: 100 μm). (E) Time-lapse recording of the MET process after coculture of UC-MSCs and EMT-induced EEO (scale bar: 100 μm). (D) and (F) Immunofluorescence staining of E-cadherin and vimentin (scale bar: 100 μm).

Figure 3

Single-cell landscapes revealed the trajectory of EEOs and UC-MSCs (A) UMAP plot of scRNA-seq data from a total of 4 individual samples, including EEO and UC-MSC samples. (B) Changes in total cell proportions across 4 samples. (C) Expression of marker genes of each cell type in the EEO and UC-MSC samples. (D) The cell trajectory presented by pseudotime analysis.

Figure 4

Immunofluorescence staining to validate markers of cell populations among EEOs and UC-MSCs (A) Immunofluorescence images of markers for luminal epithelial cells (luminal epi), glandular epithelial cells (glandular epi), EMT epithelial cells (EMT epi), proliferating EMT epithelial cells (proliferating epi), mesenchymal cells (Mes), UC-MSCs, and UC-derived mesenchymal cells (UC-derived mes) (scale bar: 100 μm). (B) Schematic representation of the trajectory analysis of the EMT and coculture processes.

Figure 5

Epithelial cells with stemness characteristics are crucial to the epithelial regeneration process (A) Expression of PROM1 in epithelial cells after coculture with UC-MSCs. (B) Histogram plot showing the PROM1-and PROM1+ populations in EEO cells and UC-MSCs (n = 3; replicates are shown in Figures S5A–S5C). (C) Dot plot showing CFSE+ and E-cadherin+ populations in EEO_EMT and normal EEO cells cocultured with UC-MSCs (n = 3; replicates are shown in Figure S5D). (D) Immunofluorescence staining to validate the presence of the E-cadherin and PanCK proteins in the cell populations among the EEO_EMT and UC-MSCs (scale bar: 100 μm). (E) Immunofluorescence staining to validate the presence of the E-cadherin and PanCK proteins in the cell populations among the normal EEO and UC-MSC (scale bar: 100 μm). (F) Schematic representation of cell sorting of LGR5⁺ and LGR5⁻ cell populations. (G) Different behaviors were observed between the LGR5⁺ and LGR5⁻ cell populations. Bright field images of LGR5⁺ cells, LGR5⁻ cells and LGR5⁻ cells+UC-MSCs are shown (scale bar: 100 μm). (H) Organoid formation efficiency of LGR5+ cells, LGR5⁻ cells and LGR5⁻ cells+UC-MSCs (n = 3, ∗p < 0.05).

Figure 6

Ratios of epithelial cells with stemness characteristics and up-regulated pathways after EMT induction and co-culture (A) Time-lapse recording of EMT 120 and 144 h groups (scale bar: 50 μm). (B) Bright field images of normal EEO and EMT induced EEO (scale bar: 100 μm). Histogram plot showing LGR5⁻ and LGR5+ populations in EEO cells (n = 3; replicates are shown in Figures S5E–S5H). (C) KEGG analysis shows upregulated pathways in EMT 96 h and coculture 96 h samples.

Figure 7

Wnt7A and TGFA are signaling molecules in the interaction between EEOs and UC-MSCs (A) Cell communication connections between EMT epithelial cells and UC-derived mesenchymal cells. (B) Schematic representation of Wnt7A and TGFA detection. (C) Concentration statistics of Wnt7A and TGFA (n = 6, ∗p < 0.05). (D) Schematic representation of cell sorting in LGR5+ cell populations. (E) Time-lapse recording of the group supplemented with WNT7A (112 ng/mL) and the group without WNT7A (scale bar: 50 μm). (F) Cell Viability of WNT7A-supplemented groups (112 ng/mL and 93 ng/mL), and group without WNT7A (n = 3, ∗p < 0.05). (G) Schematic representation of p-Erk1/2 detection and immunofluorescence validation of E-cadherin. (H) Detection of p-Erk1/2 expression level (n = 3, ∗p < 0.05). (I) and (J) Immunofluorescent staining to detect E-cadherin of UC-MSCs in groups supplemented with TGFA (52 ng/mL and 26 ng/mL; scale bar: 100 μm).