Human umbilical cord blood-derived MSCs trans-differentiate into endometrial cells and regulate Th17/Treg balance through NF-κB signaling in rabbit intrauterine adhesions endometrium

Abstract

Purpose: The fundamental cause of intrauterine adhesions (IUAs) is the destruction and reduction in stem cells in endometrial basal layer, resulting in endometrial reconstruction very difficult. The purpose of this study was to investigate the effects and underlying mechanism of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on the endometrial reconstruction after transplantation.

Methods: hUCB-MSCs were isolated and identified by flow cytometry, osteogenic, adipogenic and chondrogenic differentiation assays. The rabbit IUA models were established and set five groups (control, 14/28th day after surgery, estrogen and hUCB-MSCs treatment). The number of endometrial glands and the fibrosis rate were evaluated using HE and Masson staining, respectively. Endometrial proliferation, angiogenesis and inflammation were evaluated by immunohistochemical staining of ER, Ki-67and TGF-β1, respectively. Single-cell RNA sequencing (scRNA-seq) was applied to explore the cell differentiation trajectory after hUCB-MSCs transplanted into IUA endometrium. Finally, molecular mechanism of hUCB-MSCs repairing damaged endometrium was investigated by RNA sequencing, qRT-PCR and Western blot assays.

Results: After transplantation of the hUCB-MSCs, the increase in endometrial gland number, estrogen receptor (ER) and Ki-67 expression, and the decrease in fibrosis rate and TGF-β expression (P < 0.05), suggested the endometrial repair, angiogenesis and inflammatory suppression. The therapeutic effect of hUCB-MSCs was significantly improved compared with 28th day after surgery and estrogen group. ScRNA-seq demonstrated that the transplanted hUCB-MSCs can trans-differentiate into endometrial cells: epithelial, fibroblast and macrophage. RNA sequencing of six IUA samples combined with qRT-PCR and Western blot assays further revealed that hUCB-MSCs may regulate Th17/Treg balance through NF-κB signaling, thus inhibiting the immune response of damaged endometrium.

Conclusions: Our study demonstrated that hUCB-MSCs can repair damaged endometrium through trans-differentiation, immunomodulatory capacities and NF-κB signaling, suggesting the treatment value of hUCB-MSCs in IUA.

Keywords: Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs); Intrauterine adhesion (IUA); NF-κB signaling; Trans-differentiation; scRNA-seq.

Fig. 1

Establishment of IUA model and characterization of hUCB-MSCs. A Normal uterus after laparotomy without treatment. B Uterus after IUA surgery. An LPS surgical suture placed in the uterine cavity after curettage. C The abdomen after suture. D Schematic diagram of hUCB-MSCs injected into rabbit uterus wall. E Representative image of hUCB-MSCs. Scale bar: 100 μm. F, G Representative image of hUCB-MSCs adipogenic (F) and osteogenic (G) differentiation. Scale bar: 200 μm. H Representative image of hUCB-MSCs chondrogenic differentiation. Scale bar: 100 μm. I–L Surface antigens of hUCB-MSCs detected by flow cytometry assay. Cells were positive for CD73, CD90 and CD105, but negative for CD34, CD45, CD11b, CD19 and HLA-DR

Fig. 2

Histological analysis of uterine transverse section. A Micrographs representing HE-stained endometrial tissue from 5 groups, i.e., rabbits without surgery (control), 14/28th day after surgery, and treated with estrogen/hUCB-MSCs. Original magnification at ×400. B Endometrial gland number in 5 groups. C Micrographs representing Masson-stained endometrial tissue from 5 groups. Original magnification at ×400. D Endometrial fibrosis in 5 groups. Each group has three independent experiments

Fig. 3

Immunohistochemistry of ER, Ki-67 and TGF-β1 (A, C, E). Micrographs of ER (A), Ki-67 (C) and TGF-β1 (E) expression from 5 groups, i.e., rabbits without surgery (control), 14/28th day after surgery, and treated with estrogen/hUCB-MSCs. (B, D, F). The expression of ER (B), Ki-67 (D) and TGF-β1 (F) in endometrium in 5 groups. All original magnification at ×400. Each group has three independent experiments

Fig. 4

Identification of endometrium populations with single-cell transcriptomic analysis. A The workflow shows the collection and processing of obtained endometrium sample for scRNA-seq. B t-distributed stochastic neighbor (t-SNE) visualization of all human-derived cells displayed with different colors for clusters. C A heatmap revealed the expression levels of the indicated genes for human-derived cells. D The heatmap represents the corresponding relationship of 8 cell clusters and human-derived cell types; red (or blue) color indicates a high (or low) proportion. E t-SNE visualization of four main human-derived cell types after re-annotation

Fig. 5

Cell differentiation trajectory of hUCB-MSCs and the cell type proportion of rabbit-derived cells. A The trajectory with 3 branches identified by pseudotime analysis. B The specific cell distribution of four cell types in differentiation trajectory. C Four mainly cell types and three rare cell types identified in rabbit-derived cells. D The proportion of four mainly cell types in IUA rabbit-derived cells, merged (human and rabbit) endometrial cells and normal human endometrial cells from a reference literature

Fig. 6

RNA sequencing and bioinformatics analysis reveal biological pathways involved in IUA treated with hUCB-MSCs. A The volcano plot of differential expressed genes in IUA before and after hUCB-MSCs transplantation. Red (or blue) color indicates up-regulated (or down-regulated) genes. B The heatmap of representative differential expressed genes. C The enriched GO terms in GO enrichment analysis. D The enriched KEGG pathways in pathway enrichment analysis. E The NFKB1 mRNA expression (FPKM) measured by RNA sequencing in two groups, each group has three samples. F The NFKB1 mRNA expression level detected by qRT-PCR assay in 5 groups, each group has three samples. G The representative WB image of NF-κB p65 protein expression. H The NF-κB p65 protein expression level measured by WB in 5 groups, i.e., rabbits without surgery (control), 14/28th day after surgery, and treated with estrogen/hUCB-MSCs