Elsevier

Life Sciences

Volume 272, 1 May 2021, 119208
Life Sciences

Extracellular vesicles from GPNMB-modified bone marrow mesenchymal stem cells attenuate bone loss in an ovariectomized rat model

https://doi.org/10.1016/j.lfs.2021.119208Get rights and content

Abstract

Aims

The efficacy of anti-osteoporotic treatments is still limited. Our study aimed to investigate the effect of extracellular vesicles (EVs) derived from bone marrow-derived MSCs (BMSCs) overexpressing glycoprotein non-melanoma clone B (GPNMB) on osteoporosis (OP).

Main methods

Lentiviral vector for GPNMB overexpression or its negative control was generated and transfected into BMSCs. EVs enriched with GPNMB (GPNMB-EVs) were extracted from GPNMB-modified BMSC-conditioned medium and then identified. Cellular uptake and proliferation were analyzed using the Dil-labeled assay and CCK-8 assay, respectively. Cytochemical staining, western blot, and RT-qPCR analysis were performed to assess the effect of GPNMB-EVs on osteogenic differentiation of BMSCs in vitro. Dickkopf-1 (DKK1) as the inhibitor was applied to explore the Wnt/β-catenin signaling pathway involved in the GPNMB-EV-induced osteogenic differentiation. In vivo experiments were conducted using an ovariectomized (OVX) rat model of postmenopausal osteoporosis, and then assessed the effect of GPNMB-EVs by micro-CT, and histological and immunohistochemical assays.

Key findings

GPNMB-EVs were taken up by BMSCs, and they noticeably promoted the proliferation of BMSCs. Additionally, GPNMB-EVs activated the Wnt/β-catenin signaling to stimulate osteogenesis in BMSCs. In vivo examination showed that GPNMB-EVs remarkably improved trabecular bone regeneration and alleviated the osteoporotic phenotype in the OVX-induced rat model of OP.

Significance

EVs derived from GPNMB-modified BMSCs significantly stimulated the proliferation and osteogenic differentiation of BMSCs via the activation of Wnt/β-catenin signaling and attenuated the bone loss in the OVX-induced rat model of OP. Our findings suggest the promising potential of GPNMB-EVs as cell-free therapy for the treatment of OP.

Introduction

Osteoporosis (OP) is a chronic and disabling disease that presents with low bone mass and high risk of fragility fractures, which is highly detrimental to both society and individuals [1]. The pathogenesis of OP is mainly thought to be the enhanced osteoclast-mediated bone resorption relative to bone formation [2]. Although antiresorptive modulators and anabolic agents are utilized for anti-osteoporotic treatment, these drugs are often constrained by their unwarranted side effects and limited efficacy, which impede the long-term medication management and compliance [3,4]. Therefore, it is highly necessary to develop safer and more effective strategies for the prevention and treatment of OP.

Mesenchymal stem cells (MSCs) have gained broader attention in treating various diseases, including OP, owing to their extraordinary regenerative potential [5,6]. In recent years, an increasing evidence has suggested that the reparative effects of MSCs are potentially achieved by their paracrine actions, especially the secretion of extracellular vesicles (EVs) [[7], [8], [9]]. EVs are a group of heterogenous nanovesicles secreted by the majority of cells, among which exosomes (40–120 nm in diameter), as the most appealing subtype, have been shown to be involved in cell-to-cell communication by conveying a series of cargos (lipids, RNAs, and proteins) to target cells, thereby regulating their biological functions [10,11]. Indeed, MSC-derived EVs (MSC-EVs) exert similar therapeutic effects in tissue repair and regeneration with their originating MSCs, such as decreasing cardiac fibrosis [12],and promoting wound healing [13] and cartilage regeneration [14]. Furthermore, MSC-EVs are reported to induce new bone formation by enhancing the activity of osteoblasts and facilitating angiogenesis [[15], [16], [17]]. Several studies have revealed that systemic administration of MSC-EVs alleviates the osteoporotic phenotype and stimulates bone regeneration [[18], [19], [20]]. This highlights the tremendous potential of MSC-EV application for the treatment of skeletal disorders, especially for OP. However, unmodified MSC-EVs only display moderate therapeutic efficiency [21]. Genetically engineered MSCs with desired RNAs or proteins to obtain EVs might be expected to augment the osteogenic capability of MSC-EVs.

Glycoprotein non-melanoma clone B (GPNMB), also called osteoactivin (OA), is a multi-functional transmembrane glycoprotein expressed in numerous tissues, including bone [22]. Previous studies have shown that GPNMB is capable of regulating cell proliferation, adhesion, differentiation, and extracellular matrix synthesis [23]. Abdelmagid et al. indicated that GPNMB depletion impaired osteoblast differentiation while overexpression of GPNMB facilitated osteoblast differentiation [[24], [25], [26], [27]]. They also found that GPNMB deficiency resulted in a reduction in the number of differentiated osteoblasts and the impairment of osteogenesis in DBA/2J mice [28]. Other researchers have found that overexpression of GPNMB could induce transdifferentiation of C2C12 myoblasts into osteoblasts [29]. Moreover, GPNMB strongly contributes to rescuing the decreased osteoblast differentiation of MSCs caused by dexamethasone [30]. Hence, GPNMB plays a critical role in osteoblast differentiation and bone homeostasis. Nevertheless, none of the studies have reported the role of GPNMB as a cargo of MSC-EVs in the regulation of osteogenesis in OP.

In this study, we innovatively generated EVs with improved version of GPNMB (GPNMB-EVs) through overexpressing GPNMB in MSCs, and investigated the effects of GPNMB-EVs on an ovariectomized (OVX)-induced rat model of OP (Fig. 1). We found that GPNMB could augment the therapeutic efficacy of EVs for OP and provided support for the potential application of EVs as a gene delivery carrier to transfer the therapeutic molecules for cell-free therapy.

Section snippets

Culture and identification of bone marrow-derived MSCs (BMSCs)

Bone marrow-derived MSCs (BMSCs) were harvested from the femurs and tibias of healthy 2-week-old (weighing 30–40 g) SD rats under aseptic conditions. Cell suspension was centrifuged and grown in α-MEM (Hyclone, USA) with 10% FBS (Gibco, USA) and 1% (v/v) penicillin /streptomycin (Invitrogen, USA), and maintained with 5% CO2 at 37 °C. Half volume of the medium was replaced every 48 h. Cells were trypsinized and passaged until they reached about 80%–90%. BMSCs at early passages (passage numbers

Characterization of BMSCs and transfection efficiency

The typical ‘spindle-shaped’ BMSCs were visualized by an inverted microscope (Fig. 2a), and they could differentiate into osteoblasts, adipocytes, as well as chondrocytes in response to different induction media (Fig. 2b). Additionally, FCA showed positive expression of stemness markers CD29 and CD90 in BMSCs, but negative expression of hematopoietic cell markers CD34 and CD45 (Fig. 2c). The GPNMB-modified BMSCs were generated by transduction with GPNMB overexpressing lentivirus followed by

Discussion

The senescence of MSCs and impaired capacity of osteogenic differentiation are strongly associated with the pathological process of OP [33,35]. Although systemic transplantation of MSCs has been a promising tool for the treatment of OP, the low efficiency of transplanted MSCs in the recipient bone tissues is still a serious barrier for cell transplantation therapy [36]. In the current study, we found that EVs derived from BMSCs overexpressing GPNMB (GPNMB-EVs) presented promotion effects on the

Conclusion

Our results show that GPNMB-EVs can effectively promote the proliferation and osteogenic differentiation of BMSCs and attenuate OVX-induced bone loss in a rat model. The possible mechanism is via the Wnt/β-catenin signaling pathway. Although other molecular mechanisms regarding the protective effects of GPNMB-EVs remains obscure, our findings provide evidence that GPNMB-EVs may be a novel therapeutic strategy for the clinical therapy in OP. Future studies are still needed to assess the

Abbreviations

    BMSCs

    bone marrow-derived mesenchymal stem cells

    GPNMB

    glycoprotein nonmelanoma clone B

    Len-GFP-GPNMB

    lentiviral vectors encoding Gpnmb gene with green fluorescent protein (GFP)

    Len-GFP-NC

    lentiviral vectors encoding only GFP as negative control

    EVs

    extracellular vesicles

    GPNMB-EVs

    EVs derived from GPNMB-transfected BMSCs

    NC-EVs

    EVs derived from empty vector-transfected BMSCs

    i.v.

    intravenous injection via the caudal vein

    PBS

    phosphate buffered solution

    FBS

    fetal bovine serum

    α-MEM

    modified Eagle's medium alpha

CRediT authorship contribution statement

Ba Huang, Investigation, Writing-original draft; Yongwei Su, Data curation, Visualization; Enpu Shen, Software, Methodology; Meng Song, Visualization, Supervision; Danping Liu, Conceptualization, Supervision; Hui Qi, Supervision, Writing-review & editing.

Declaration of competing interest

The authors declare that they have no conflicting interests.

Acknowledgments

We are deeply grateful to Prof. Hui Qi for her encouragement and support.

Funding

This research was supported by the National Natural Science Foundation of China (grant number: 81572140), the Natural Science Foundation of Beijing Municipality (grant number: 7172036), and Beijing Municipal Health Commission (grant number: BMC2018-4; BMC2019-9).

Ethics approval

Our experiments were approved and conducted in accordance with the Ethical Guidelines of Jinzhou Medical University (approved number: 2019021).

References (50)

  • M. Maqsood et al.

    Adult mesenchymal stem cells and their exosomes: sources, characteristics, and application in regenerative medicine

    Life Sci.

    (2020)
  • C.Y. Chen et al.

    Extracellular vesicles from human urine-derived stem cells inhibit glucocorticoid-induced osteonecrosis of the femoral head by transporting and releasing pro-angiogenic DMBT1 and anti-apoptotic TIMP1

    Acta Biomater.

    (2020)
  • D. Ingato et al.

    Good things come in small packages: overcoming challenges to harness extracellular vesicles for therapeutic delivery

    J. Control. Release

    (2016)
  • L. Sun et al.

    Safety evaluation of exosomes derived from human umbilical cord mesenchymal stromal cell

    Cytotherapy

    (2016)
  • F. Cosman et al.

    Clinician’s guide to prevention and treatment of osteoporosis

    Osteoporos. Int.

    (2014)
  • S. Das et al.

    Osteoporosis - a current view of pharmacological prevention and treatment

    Drug Des Devel Ther.

    (2013)
  • R. Eastell et al.

    Pharmacological management of osteoporosis in postmenopausal women: an Endocrine Society* clinical practice guideline

    J. Clin. Endocrinol. Metab.

    (2019)
  • M.B. Murphy et al.

    Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine

    Exp. Mol. Med.

    (2013)
  • B. Antebi et al.

    Stem cell therapy for osteoporosis

    Curr Osteoporos Rep

    (2014)
  • Y. Li et al.

    Mesenchymal stem cells-derived exosomes: a possible therapeutic strategy for osteoporosis

    Curr Stem Cell Res Ther

    (2018)
  • E.L.A. S et al.

    Extracellular vesicles: biology and emerging therapeutic opportunities

    Nat. Rev. Drug Discov.

    (2013)
  • N. Jabbari et al.

    Breast cancer-derived exosomes: tumor progression and therapeutic agents

    J. Cell. Physiol.

    (2020)
  • Z. Zhang et al.

    Pretreatment of cardiac stem cells with exosomes derived from mesenchymal stem cells enhances myocardial repair

    J. Am. Heart Assoc.

    (2016)
  • S. Rani et al.

    The exosome - a naturally secreted nanoparticle and its application to wound healing

    Adv. Mater.

    (2016)
  • C.H. Woo et al.

    Small extracellular vesicles from human adipose-derived stem cells attenuate cartilage degeneration

    J Extracell Vesicles

    (2020)
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