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. 2017 May 10;8(1):108.
doi: 10.1186/s13287-017-0563-9.

Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells

Kang Lu  1 , Hai-Yin Li  1 , Kuang Yang  1 , Jun-Long Wu  1 , Xiao-Wei Cai  2 , Yue Zhou  1 , Chang-Qing Li  3
Affiliations

Exosomes as potential alternatives to stem cell therapy for intervertebral disc degeneration: in-vitro study on exosomes in interaction of nucleus pulposus cells and bone marrow mesenchymal stem cells

Kang Lu et al. Stem Cell Res Ther. .

Abstract

Background: The stem cell-based therapies for intervertebral disc degeneration have been widely studied. However, the mechanisms of mesenchymal stem cells interacting with intervertebral disc cells, such as nucleus pulposus cells (NPCs), remain unknown. Exosomes as a vital paracrine mechanism in cell-cell communication have been highly focused on. The purpose of this study was to detect the role of exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) and NPCs in their interaction with corresponding cells.

Methods: The exosomes secreted by BM-MSCs and NPCs were purified by differential centrifugation and identified by transmission electron microscope and immunoblot analysis of exosomal marker proteins. Fluorescence confocal microscopy was used to examine the uptake of exosomes by recipient cells. The effects of NPC exosomes on the migration and differentiation of BM-MSCs were determined by transwell migration assays and quantitative RT-PCR analysis of NPC phenotypic genes. Western blot analysis was performed to examine proteins such as aggrecan, sox-9, collagen II and hif-1α in the induced BM-MSCs. Proliferation and the gene expression profile of NPCs induced by BM-MSC exosomes were measured by Cell Counting Kit-8 and qRT-PCR analysis, respectively.

Results: Both the NPCs and BM-MSCs secreted exosomes, and these exosomes underwent uptake by the corresponding cells. NPC-derived exosomes promoted BM-MSC migration and induced BM-MSC differentiation to a nucleus pulposus-like phenotype. BM-MSC-derived exosomes promoted NPC proliferation and healthier extracellular matrix production in the degenerate NPCs.

Conclusion: Our study indicates that the exosomes act as an important vehicle in information exchange between BM-MSCs and NPCs. Given a variety of functions and multiple advantages, exosomes alone or loaded with specific genes and drugs would be an appropriate option in a cell-free therapy strategy for intervertebral disc degeneration.

Keywords: Differentiation; Exosomes; Intervertebral disc degeneration; Mesenchymal stem cell; Migration; Nucleus pulposus cell; Proliferation.

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Figures

Fig. 1
Fig. 1
Characterization of BM-MSC and NPC-derived exosomes by TEM. a PBS used as a control group; b exosomes derived from BM-MSCs; c exosomes derived from NPCs. Exosomes were stained with phosphotungstic acid and observed by TEM (×100,000). The two kinds of exosomes were roughly identical, ranging from 30 to 100 nm. Scale bar = 200 nm. BM-MSC bone marrow mesenchymal stem cell, Exo exosomes, NPC nucleus pulposus cell, PBS phosphate-buffered saline
Fig. 2
Fig. 2
Western blot analyses to test the proteins in exosomes. Western blot analysis was performed on the vesicles to validate exosomal markers CD63, TSG101 and negative protein Calnexin. NPC nucleus pulposus cell, BM-MSC bone marrow mesenchymal stem cell, CL cell lysate, Exo exosomes
Fig. 3
Fig. 3
Reciprocal uptake of exosomes by BM-MSCs and NPCs. BM-MSCs and NPCs were respectively incubated with PKH67-labeled exosomes for 4 hours and were observed with a fluorescence confocal microscope. Green, PKH67 fluorescence; red, phalloidine blue; DAPI-stained nuclei. Exosomes were absorbed by responding cells. BM-MSC bone marrow mesenchymal stem cell, Exo exosomes, NPC nucleus pulposus cell
Fig. 4
Fig. 4
NPC exosome chemotactic assay to BM-MSCs. a Migration activity of BM-MSCs treated by NPC exosomes at concentrations of 0, 1, 10 and 100 μg/ml. b Number of transmitted cells in the transwell migration assay in (a). Values are the mean ± SD. n =9. *P < 0.05. BM-MSC bone marrow mesenchymal stem cell, Exo exosomes, NPC nucleus pulposus cell
Fig. 5
Fig. 5
Effects of NPC exosomes on NPC-specific protein expression in BM-MSCs. (a) Gene expression of ACAN (a), SOX-9 (b), COL2A1 (c), HIF-1α (d), CA12 (e) and KRT19 (f) in BM-MSCs was significantly upregulated by NPC exosomes with time. Expression of these genes was normalized to β-actin and control (BM-MSCs alone). Values are the mean ± SD. n = 3.*P < 0.05, **P < 0.01, ***P < 0.001. (b) NPC-specific protein expression in BM-MSCs described in (a) analyzed by western blot analysis. (c) TGF-β in NPC exosomes. NPC nucleus pulposus cell
Fig. 6
Fig. 6
Comparison of gene expression in BM-MSCs after coculture with NPCs and after stimulation by NPC exosomes. a Schematic diagram of experimental groups. NPCs and BM-MSCs were cocultured by an indirect method using a transwell system. NPCs were cultured in the upper chamber and BM-MSCs in the lower chamber to coculture for 14 days. NPC exosomes alone were added to BM-MSCs in the Exo group. BM-MSCs without treatment were taken as the control group. b After 14 days of treatment, qRT-PCR detected relative mRNA expression in BM-MSCs. Expression levels of ACAN (P < 0.01), SOX-9 (P < 0.05), COL2A1 (P < 0.01), HIF-1α (P < 0.01) and CA12 (P < 0.05), but not KRT19 (P > 0.05), mRNA were significantly higher in the Exo group than the coculture group. *P < 0.05, **P < 0.01, ***P < 0.001. BM-MSC bone marrow mesenchymal stem cell, Exo exosomes, NPC nucleus pulposus cell
Fig. 7
Fig. 7
NPC proliferation rate increased with the time for which NPCs were stimulated by BM-MSC exosomes. The group of day 12 has the greatest relative proliferation rate (49% compared with control). The day 6, day 9 and day 12 groups had significant difference from the control group (*P < 0.05, ***P < 0.001). The day 3 group had no significant statistical difference (P > 0.05). Results shown as the relative proliferation rate to the control group
Fig. 8
Fig. 8
Extracellular matrix expression of degenerate NPCs after NPCs were stimulated by BM-MSC exosomes. a qRT-PCR analysis showed an increase in ACAN (a), COL2A1 (b), SOX-9 (c) and TIMP-1 (f) and a decrease in MMP-1 (d) and MMP-3 (e) mRNA expression with the time of stimulation by BM-MSC exosomes. *P < 0.05, **P < 0.01, ***P < 0.001. b Western blot analysis showed that the effect of BM-MSC exosomes on NPCs in the protein profile was almost the same as gene expression
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