Exosomal miR-335 derived from mature dendritic cells enhanced mesenchymal stem cell-mediated bone regeneration of bone defects in athymic rats

Abstract

Background: Transplantation of bone marrow-derived mesenchymal stem cells (BM-MSCs) embedded in a bio-compatible matrix has been demonstrated as a promising strategy for the treatment of bone defects. This study was designed to explore the effect and mechanism of exosomes derived from mature dendritic cells (mDC-Exo) on the BM-MSCs-mediated bone regeneration using the matrix support in an athymic rat model of femoral bone defect.

Methods: The BM-MSCs were isolated from rats and incubated with osteoblast induction medium, exosomes derived from immature DCs (imDC-Exo), mDC-Exo, and miR-335-deficient mDC-Exo. BM-MSCs treated without or with mDC-Exo were embedded in a bio-compatible matrix (Orthoss^®) and then implanted into the femoral bone defect of athymic rats.

Results: mDC-Exo promoted the proliferation and osteogenic differentiation of BM-MSCs by transferring miR-335. Mechanistically, exosomal miR-335 inhibited Hippo signaling by targeting large tongue suppressor kinase 1 (LATS1) and thus promoted the proliferation and osteogenic differentiation of BM-MSCs. Animal experiments showed that mDC-Exo enhanced BM-MSCs-mediated bone regeneration after bone defect, and this effect was abrogated when miR-335 expression was inhibited in mDC-Exo.

Conclusion: mDC-Exo promoted osteogenic differentiation of BM-MSCs and enhanced BM-MSCs-mediated bone regeneration after femoral bone defect in athymic rats by transferring miR-335.

Keywords: Bone defect; Dendritic cell; Exosome; Mesenchymal stem cells; miR-335.

Fig. 1

Identification of exosomes and BM-MSCs. The exosomes isolated from imDCs and mDCs were referred to as imDCs-Exo and mDCs-Exo, respectively. a The morphological characterization of imDCs-Exo and mDCs-Exo by TEM. b The particle size of imDCs-Exo and mDCs-Exo measured using nanoparticle tracking analysis. c Expression of the exosomal markers (CD63, and Alix) and the exosome negative marker (Calnexin) confirmed by western blot. d The surface antigens including CD29, CD44, CD90, CD34, and CD45 of rat BM-MSCs was detected by flow cytometry. The positive rate of each surface marker was presented. e Osteogenic differentiation of BM-MSCs was confirmed by Alizarin Red staining. f Adipogenic differentiation of BM-MSCs was identified using Oil Red O staining

Fig. 2

mDC-Exo promoted proliferation and osteogenic differentiation of BM-MSCs. a Immunofluorocytochemical staining for BrdU (green) and DNA (DAPI, blue) in BM-MSCs and b quantitation of the number of BrdU-positive cells in BM-MSCs in the groups of control, imDC-Exo, and mDC-Exo. c ALP activities expressed in optical density (OD) values at 520 nm, d–f protein levels of ALP and Runx2 determined by western blot, g, h representative Alizarin red staining assessing calcium deposits and quantification of the percentage of Alizarin red dyeing area in BM-MSCs in the groups of control, induction, imDC-Exo, and mDC-Exo on the 7th and 14th day of culture. **P < 0.01, vs. Control

Fig. 3

mDC-Exo promoted osteogenic differentiation of BM-MSCs by transferring miR-335. a Higher expression of miR-672, miR-335, miR-124, and miR-125a-5p in mouse mDC-Exo relative to imDC-Exo (GSE33179). *P < 0.05, vs. imDC-Exo. b Expression of miR-672, miR-335, miR-124, and miR-125a-5p determined by qRT-PCR analysis in imDC-Exo and mDC-Exo separated according to the method mentioned above. *P < 0.05, vs. imDC-Exo. c miR-335 expression determined by qRT-PCR analysis in mDC-Exo-NC and mDC-Exo-miR-335I. **P < 0.01, vs. mDC-Exo-NC. d ALP activities expressed in optical density (OD) values at 520 nm, e, f Runx2 mRNA and protein levels, and g miR-335 expression in BM-MSCs in the groups of control, induction, mDC-Exo, and mDC-Exo-NC, and mDC-Exo-miR-335I on the 7th day of culture. *P < 0.05, **P < 0.01, vs. Control; ^#P < 0.05, ^##P < 0.01, vs. mDC-Exo-NC. h The exosomes isolated from Cy3 (red)-miR-335-transfected mDCs were labeled with the lipophilic fluorescent dye Dio (green) and incubated with BM-MSCs (nuclei stained with Hoechst 33,342, blue). The uptake of exosomes by BM-MSCs was observed by confocal laser microscopy. (I) ALP activities expressed in OD values at 520 nm, (J-K) Runx2 mRNA and protein levels in BM-MSCs transfected with miR-335 mimics and mimic NC. **P < 0.01, vs. mimic NC

Fig. 4

Exosomal miR-335 promoted osteogenic differentiation of BM-MSCs by targeting LATS1. LATS1 mRNA (a) and protein (b, c) levels in BM-MSCs in the groups of control, induction, mDC-Exo, and mDC-Exo-NC, and mDC-Exo-miR-335I on the 7th day of culture. **P < 0.01, vs. Control; ^#P < 0.05, vs. mDC-Exo-NC. d Predicted binding sites for miR-335 in the 3′-UTR of LATS1 (Targetscan). e The interaction between miR-335 and LAST1 3′-UTR was analyzed by luciferase activity assay. **P < 0.01, vs. mimic NC. LAST1 mRNA (f) and protein (g) levels in BM-MSCs transfected with miR-335 mimics, mimic NC, miR-335 inhibitors, or inhibitor NC. *P < 0.05, **P < 0.01, vs. mimic NC; ^#P < 0.05, vs. inhibitor NC. h, i The protein levels of LATS1, p-LAST1, YAP, p-YAP, TAZ, and p-TAZ, j ALP activities expressed in optical density (OD) values at 520 nm, k–m Runx2 mRNA and protein levels in BM-MSCs transfected with LAST1 overexpression vector/empty vector and treated with mDC-Exo or PBS. *P < 0.05, **P < 0.01, vs. vector + PBS; ^#P < 0.05, ^##P < 0.01, vs. vector + mDC-Exo

Fig. 5

mDC-Exo enhanced BM-MSCs-mediated bone regeneration by transferring miR-335. Rats were randomly divided into five groups: Matrix, Matrix + BM-MSCs, Matrix + BM-MSCs + mDC-Exo, Matrix + BM-MSCs + mDC-Exo-NC, and Matrix + BM-MSCs + mDC-Exo-miR-335I. a Bone regeneration was monitored via micro-CT scanning at 4, 8 and 12 weeks post-surgery. b Quantification of new bone volume from micro-CT scanning. c Representative H&E staining showing bone defect repair of posterior limbs of rats. d Quantification of new bone area from H&E staining. The protein levels of e ALP, Runx2, and f, g LAST1 in the bone examined by western blot. *P < 0.05, **P < 0.01, vs. Matrix; ^#P < 0.05, ^##P < 0.01, vs. Matrix + BM-MSCs; ^$P < 0.05, ^$$P < 0.01, vs. Matrix + BM-MSCs + mDC-Exo-NC