Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation

Abstract

Background: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF).

Methods: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups.

Results: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR.

Conclusion: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.

Keywords: BMDSCs; Decidualization; Exosomes; eSF; miRNAs.

Fig. 1

Characterization of extracellular vesicles (EVs). (a) Morphology of EVs showing a typical cup-shaped structure with a bilayer membrane and a diameter of approximately 100 nm, measured by transmission electron microscopy (TEM). (b i & ii) Shows the main peak and the median particle size respectively, in the range of exosomes determined by the nanoparticle tracking analysis (NTA)

Fig. 2

Analysis of RNA-seq. RNA seq analysis showing the top 25 miRNAs that are highly expressed in extracellular vesicles from BMDSCs. MiR-21-5p is most highly expressed followed by miR-100-5p, miR-143-3p, miR-let-7i, and miR-let-7b, with a minimum of 5000 copies

Fig. 3

Increased cell proliferation by hsa-miR-100-5p and miR-143-3p. (a) Shows the significant increase in cell counts and (b) shows significant increase in Ki67 mRNA levels compared to mimic-negative control (CTL) for 72 hr. Each bar represents the data from six individual experiments, and each experiment was performed in duplicate. Data are shown as mean ± standard error (SEM) CTL vs. mimic (** p < 0.01, *p < 0.05)

Fig. 4

miR-100-5p upregulates regenerative markers in eSFs. Transfection of miR-100-5p in eSFs significantly increased the mRNA levels of regeneration markers (a) TGF-ß3, (b) VEGFA, (c) MMP7, and (d) HGF compared to controls. Each bar represents the data from four individual experiments, and each experiment was performed in triplicate. Data are shown as mean ± standard error (SEM) CTL vs. mimic (*p < 0.05), ***p < 0.001, **** p < 0.0001)

Fig. 5

Inhibition of decidualization by miR-100-5p and miR-143-5p. eSFs with and without transfection of miR-100-5p and miR-143-5p were treated with cAMP + MPA. Prolactin was estimated in conditioned medium by ELISA. (a) Shows significant increase in PRL levels after 7 days in normal cells treated with cAMP + MPA compared to no treatment. (b) Shows no significant increase in PRL levels in cells transfected with miRNAs compared to cells without transfection. Each bar represents the data from two individual experiments, and each experiment was performed in duplicate. Data are shown as mean ± standard error (SEM) CTL vs. Decidualization (*** p < 0.001)

Fig. 6

Effect of miR-100-5p on decidualization markers. mRNA levels of decidualization markers were determined by qRT-PCR. (a) Shows the significant upregulation of mRNA levels of decidualization markers HOXA10, IGFBP-1, PRL, PR-B, and PR in normal eSFs treated with cAMP + MPA for 7 days (DEC), compared to control group (CTL). (b) Shows that miRNA miR-100-5p significantly downregulates mRNA levels of decidualization markers HOXA10, IGFBP-1, PRL, PR-B and PR significantly in eSFs compared to non-transfected cells with the mimic control. Each bar represents the data from four individual experiments, and each experiment was performed in triplicate. Data are shown as mean ± standard error (SEM) CTL vs. DEC and mimic negative control vs. miR-100-5p transfection. (*p < 0.05), **p < 0.01, ***p < 0.001)

Fig. 7

Inhibition of decidualization of eSFs in vitro by miR-100-5p and miR-143-3p. (a) Normal eSFs (HESCs), (b) eSFs transfected with miRNA mimic control. Cells gradually changed to typical decidual cells after 7 days of cAMP + MPA treatment. (c & d) eSFs transfected with miR-100-5p and miR-143-3p respectively. No typical or partial decidual changes were observed after 7 days treatment of cAMP + MPA