Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Deliver Exogenous miR-105-5p via Small Extracellular Vesicles to Rejuvenate Senescent Nucleus Pulposus Cells and Attenuate Intervertebral Disc Degeneration

DOI: https://doi.org/10.21203/rs.3.rs-127320/v1

Abstract

Background: Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) have emerged as a promising new therapeutic strategy for intervertebral disc degeneration (IVDD). However, drawbacks of MSCs, including invasive access, donor age, and limited proliferative capacity, hinder the quantity and quality of MSC-sEVs. Induced pluripotent stem cell-derived MSCs (iMSCs) provide an indefinite source of MSCs with well-defined phenotype and function. This study aimed to investigate the therapeutic effect of sEVs derived from iMSC (iMSC-sEVs) on IVDD and explore the underlying molecular mechanisms.

Methods: IVDD models were established by puncturing tail disc in rats. Then iMSC-sEVs was injected into the punctured discs. The degeneration of punctured discs was assessed by MRI, HE and Immunofluorescence staining. In vitro, age-related phenotypes were used to determine the effects of iMSC-sEVs on senescent nucleus pulposus cells NPCs. Western blotting was used to detect the expression of Sirt6. miRNA sequencing analysis was used to find miRNAs that potentially mediate the activation of Sirt6.

Results: After intradiscally injecting iMSC-sEVs, NPC senescence and IVDD were significantly improved. In vitro, iMSC-sEVs could rejuvenate senescent NPCs and restore the age-related function by activating the Sirt6 pathway. Further microRNA sequence analysis showed that miR-105-5p was highly enriched in iMSC-sEVs and played a pivotal role in iMSC-sEVs-mediated therapeutic effect by downregulating the level of the cAMP-specific hydrolase PDE4D and could lead to Sirt6 activation.

Conclusion: iMSC-sEVs could rejuvenate the senescence of NPCs and attenuate the development of IVDD. iMSC-sEVs exerted their anti-aging effects by delivering miR-105-5p to senescent NPCs and activating the Sirt6 pathway. Our findings indicate that iMSCs are a promising candidate MSC for obtaining sEVs on a large scale while avoiding several defects related to the present applications of MSCs, and that iMSC-sEVs could be a novel cell-free therapeutic tool for the treatment of IVDD.

1. Introduction

Intervertebral disc degeneration (IVDD) is a widely recognized contributor to lower-back pain. IVDD increases both the burden on global health care systems and the risk of disability[1]. The IVD comprises an inner nucleus pulposus (NP) surrounded by the annulus fibrosus. NP cells (NPCs) located in the inner NP are responsible for producing a gelatinous extracellular matrix that includes collagen II and proteoglycan, which enable the disc to cope with diverse external mechanical stimuli[2]. Accumulating evidence indicates that the senescence of NPCs plays a key role in the pathological progression of IVDD[3]. These senescent NPCs show distinct catabolic features characterized by decreased proliferation capacity, loss of functional capability, and increased secretion of a senescence-associated secretory phenotype[4]. In addition, senescent NPCs also reinforce senescence in an autocrine manner or affect tissue homeostasis in a paracrine manner, leading to a vicious cycle of local catabolism and inflammation. Therapeutic strategies designed to ameliorate senescence of NPCs should effectively delay the progression of IVDD.

Recently, mesenchymal stem cell (MSC) transplantation has shown promising therapeutic potential in alleviating aging-associated phenotypes[5, 6]. Despite their potential therapeutic applications, the direct use of stem cell transplantation still faces several hurdles, such as the risk of tumorigenesis and undesirable immune response[7]. Recent evidence has indicated the therapeutic potential of small extracellular vesicles (sEVs) secreted by MSCs derived from different tissues in alleviating cellular senescence while avoiding the undesirable immune response and the risk of tumorigenesis[8, 9]. However, harvesting MSCs from different tissues, such as bone marrow and adipose tissue, is invasive. In addition, limitations such as the decreased proliferative potential and therapeutic efficacy of MSCs during expansion in vitro has impeded the industrial production of sEVs[10].

Induced pluripotent stem cells (iPSCs) are a subpopulation of stem cells that can be reprogrammed from any tissue type in the body. iPSCs have a unique ability to proliferate indefinitely and display totipotency in vitro[11, 12]. MSCs derived from iPSCs (iMSCs) could expand over 40 passages with high efficiency in vitro[13]. Furthermore, iMSCs possess MSC-like therapeutic effects in the treatment of tissue regeneration[14, 15]. Along with the advantages in acquisition and proliferation of iMSCs compared with those of MSCs, sEVs can be abundantly obtained from iMSCs, which is convenient for industrial production.

The therapeutic effects of iMSC-derived sEVs (iMSC-sEVs) on cellular senescence are unclear. We speculated that like MSC-sEVs, iMSC-sEVs might also have anti-senescence functions. Since iMSC-sEVs can be harvested infinitely from iMSCs, the demonstration of an anti-senescence function was explored in this study, with the goal of informing the development of an improved treatment of IVDD.

2. Materials And Methods

2.1 Cell culture

A human iPSC line purchased from the Cell Bank of the Chinese Academy of Sciences was cultured in mTESR1 medium (STEMCELL Technologies). The iMSCs were transduced as previously described[16]. Briefly, mTeSR1 medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM, Hyclone) supplemented with 10% foetal bovine serum (FBS; Sigma-Aldrich), 1% penicillin/streptomycin, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (Gibco). These cells were continuously passaged in the MSC medium until they developed a homogeneous fibroblastic morphology. Human dermal fibroblasts (FBs) purchased from the Cell Bank of the Chinese Academy of Sciences were cultured in high-glucose DMEM (Hyclone) with 10% FBS (Sigma-Aldrich) and 1% penicillin/streptomycin. Human NPCs were isolated from normal NP tissue derived from lumbar trauma patients who underwent spinal fusion with no degenerative signal on magnetic resonance imaging (MRI). The method of isolation has been previously described[17]. NPCs were cultured in DMEM/F-12 medium (Hyclone) with 10% FBS (Gibco) and 1% penicillin/streptomycin. These cells were incubated at 37 °C in a humidified atmosphere of 5% CO2.

2.2 Characterization of iMSCs and sEVs

Surface antigens of iMSCs were detected using flow cytometry analysis. Cells were collected and incubated with 3% bovine serum albumin (Sigma-Aldrich) for 30 min to block non-specific antigen binding. The cell suspensions were incubated with antibodies for iMSC-specific surface markers that included CD29, CD34, CD44, CD45, CD73, or HLA-DR (all from BD Biosciences) at 4 °C for 30 min. The Guava easyCyte™ flow cytometer (Millipore) was used to analyze surface antigens.

iMSC-sEVs or FB-sEVs were isolated from iMSC or FB culture supernatants as previously described[18]. Briefly, after reaching 80% confluency, iMSCs and FBs were washed with PBS. The iMSCs were cultured with serum-free MSC medium (StemRD), and FBs were cultured with high-glucose containing extracellular vesicle-depleted FBS (10%) for 48 h at 37 °C in an atmosphere of 5% CO2. The medium were collected and centrifuged at 300 g for 10 min at 4 °C to remove remaining cells, followed by 2000 g for 10 min at 4 °C to remove dead cells, followed by 10000 g for 30 min at 4 °C. The supernatant was ultra-centrifuged at 100000 g for 70 min at 4 °C. This step was repeated once. The sEVs were resuspended in PBS for use in the experiments. The morphology of sEVs was observed by transmission electron microscopy (TEM) by using a model H-7650 device (Hitachi) operating at an accelerating voltage of 80.0 kV. The concentration and size distribution of the sEVs were measured by nanoparticle analysis (NTA) by using a ZetaView PMX 110 (Particle Metrix). Expressions of characteristic markers of sEVs (CD9, CD63, Tsg101, GM130, and Actin) were tested by western blotting.

2.3 Establishment of IVDD model and treatment in rats

Twelve-week-old Sprague–Dawley rats were used. All experimental procedures were approved by the Animal Research Committee of the First Affiliated Hospital of the University of Science and Technology of China. The rat model of IVDD was established as previously described[19]. Briefly, the experimental level rat tail disc (Co5/6) was punctured using a 20-gauge needle (IVDD group). The puncture was made through the center of the disc to the opposite side, and the needle was rotated 180° and held for 10 s. Rats that were not treated were the negative control. One week after the initial surgery, 2 uL of sterile saline containing 1 × 1010 iMSC-sEVs/mL was injected into the punctured discs by using a 33-gauge needle, in the IVDD group. The negative group received an injection of FB-sEVs. The injections were repeated every 2 weeks. At week 8, MRI was performed on all rats, and the rats were euthanized for further analysis.

2.4 MRI

After 8 weeks of the puncture procedure, all rats were examined by MRI examination to evaluate the degenerative changes in sagittal T2-weighted images by using a 3.0 T clinical magnet (Siemens). T2-weighted sections in the median sagittal plane were obtained using the following settings: fast-spin echo sequence with a time to repetition of 5400 ms and time to echo of 920 ms; 320 (h) 256 (v) matrix; field of view of 260; and four excitations. The section was 2 mm thick with a 0-mm gap. The degree of IVDD in the MR images was evaluated by the Pfirrmann grading system[20].

2.5 Histological analysis

Rats were sacrificed and tail samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, dehydrated in a gradient alcohol, and embedded in paraffin. The specimens were cut into 5 um-thick sections. Hematoxylin-eosin (H&E) staining was performed to observe IVDD. Briefly, all sections were deparaffinized, rehydrated, and stained in hematoxylin solution for 5 min. After differentiation in 1% acid alcohol for 30 s, the sections were stained in eosin solution for 30 s to 1 min. Sections were observed by optical microscopy. Immunofluorescence (IF) staining was used to detect age-related P16 protein. The sections were deparaffinized, rehydrated, antigen retrieved, and blocked and incubated with primary antibody against P16 (1:200; Invitrogen) overnight at 4 °C. The sections were incubated with Alexa Fluor 594-conjugated secondary antibody (1:400) for 1 h, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI).

2.6 Senescence-associated 𝛽 -galactosidase (SA-𝛽-gal) staining

SA-𝛽-Gal activity of NPCs was detected using a cellular senescence staining kit (Beyotime Biotechnology) according to the manufacturer’s instructions. Senescent cells were identified as blue-stained cells by phase-contrast microscopy. The proportion of positive cells was determined by counting the number of blue cells and dividing by the number of cells observed.

2.7 Proliferation assay

Cell proliferation was measured using a cell counting kit-8 (CCK-8, Dojindo Molecular Technologies). NPCs from different treatment groups were seeded into 96-well plates at a density of 3000 cells per well. Ten microliters of CCK8-solution was added to 100 uL of medium and incubated for 2 h at 37 °C. The absorbance at 450 nm was measured using a model 680 microplate reader (Bio-Rad).

2.8 Western blotting analysis

Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The resolved proteins were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 for 2 h at room temperature. The membranes were then incubated with primary antibodies against the following proteins: P16 (1:1000; Abcam), collagen II (1:1000; Abcam), aggrecan (1:1000; Abcam), matrix metalloproteinase 3 (MMP3, 1:1000; Abcam), A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4, 1:1000; Abcam), cAMP-specific 3',5'-cyclic phosphodiesterase 4D (PDE4D, 1:1000; Abcam), Sirt6 (1:1000; Abcam), and Actin (1:3000; Abcam) at 4 °C overnight. Subsequently, membranes were incubated with peroxidase-conjugated anti-rabbit IgG (1:3000; Abcam) or anti-mouse IgG (1:3000; Abcam) for 1 h at room temperature. Finally, the proteins were visualized by ECL (Thermo Fisher Scientific).

2.9 qRT-PCR analysis

Extraction of sEV RNA was performed using Exoquick (QIAGEN). qRT-PCR was performed using the QuantiTect® SYBR Green PCR Master Mix. The default PCR setting was 40 cycles of 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. Specific amplicons were identified by melting curve analysis.

2.10 Uptake of iMSC-sEVs and FB-sEVs

The uptake of sEVs by NPCs was observed using green fluorescent dye (DiO; Life Technologies) to label iMSCs and FBs according to the manufacturer’s instructions. The sEVs released by the labeled iMSCs or FBs were also labeled with DiO. The NPCs were cultured with the conditioned medium containing DiO-labeled sEVs, for 12 h.

2.11 Transport of miRNAs inhibitors into iMSC-sEVs

The miR-105-5p inhibitor and control miRNA inhibitor were purchased from QIAGEN. The miR-105-5p inhibitor and control miRNA inhibitor were transferred to iMSC-sEVs by using the Exo-Fect siRNA/miRNA Transfection Reagent Kit (SBI) according to the manufacturer’s instructions.

2.12 Statistical analysis

The data were presented as means ± standard deviation. Statistical significance (P values) was determined by One-way analysis of variance (ANOVA) or Student’s t-test. Statistical significance was determined to be P < 0.05.

3. Results

3.1 Characterization of iMSCs and sEVs derived from iMSCs or FBs

iMSCs were successfully derived from iPSCs. As shown in previous studies[21], iMSCs expressed CD29, CD44, and CD73 and were negative for CD34, CD45, and HLA-DR (Fig. 1a). Next, we characterized sEVs derived from iMSCs or FBs. Western blotting analysis indicated that sEVs expressed sEV markers, such as CD9, CD63, and TSG101 and were negative for GM130 and Actin (Fig. 1b). TEM revealed that sEVs from iMSCs or FBs displayed a cup-shaped morphology (Fig. 1c). NTA showed that the sizes of the iMSC-sEVs and FB-sEVs ranged from 80 to 200 nm (Fig. 1d).

3.2 Intradiscal injection of iMSC-sEVs ameliorate progression of IVDD and senescence of the NPCs in a rat model

A rat model of IVDD was successfully established 8 weeks after needle puncture. One week after the initial surgery, iMSC-sEVs were injected into the punctured disc for treatment. FB-sEVs injection was used as a negative control. MRI indicated that T2-weighted signal intensities in the IVDD group were weaker than those in the Control group. This change was ameliorated by iMSC-sEVs, while no significant improvement was observed using FB-sEVs (Fig. 2a). Pfirrmann MRI grade scores, which indicate the degree of disc degeneration, demonstrated significantly higher scores in the IVDD group compared to the Control group, and scores decreased markedly after the application of iMSC-sEVs (Fig. 2b). Next, we observed the histological changes of the IVDs in each group by HE staining. The treatment with iMSC-sEVs remarkably reduced the loss of NPCs and restored intervertebral height in a rat model of IVDD, while no significant difference was observed between the IVDD and FB-sEV injection groups (Fig. 2c). Previous studies found that the senescence of NPCs was closely related to the pathological progression of IVDD. Therefore, we further explored whether the therapeutic effects of iMSC-sEVs on IVDD involved the altered senescence of NPCs. IF staining for the age-related P16 protein indicated that iMSC-sEVs, but not FB-sEVs, significantly alleviated the senescence of NPCs 4 weeks after needle puncture (Fig. 2d). These results suggested that iMSC-sEVs significantly delayed the pathological progression of IVDD and ameliorated the senescence of NPCs in the rat model.

3.3 iMSC-sEVs can ameliorate the senescent phenotype of NPCs and age-related dysfunction in vitro

To further investigate the effect of iMSC-sEVs on senescent NPCs, we first established an in vitro model of cellular senescence by treating NPCs with 10 ng/mL tumor necrosis factor-alpha (TNF-α). The proportion of senescent NPCs and the expression of age-related P16 protein increased, while the proliferation of NPCs decreased (Fig. 3b-f). Before examining the therapeutic effects of iMSC-sEVs on senescent NPCs, we first determined whether iMSC-sEVs or FB-sEVs could be endocytosed into senescent NPCs. DiO-labeled iMSC-sEVs or FB-sEVs were present in the perinuclear region after incubation for 12 h, suggesting iMSC-sEVs or FB-sEVs uptake by senescent NPCs (Fig. 3a). To observe the effects of iMSC-sEVs on senescent NPCs, senescent NPCs were incubated with 1 × 1010 iMSC-sEVs/mL for 7 days. The iMSC-sEVs significantly reduced the proportion of senescent NPCs (Fig. 3b and c). The CCK-8 assay demonstrated that iMSC-sEVs treatment restored the proliferation of TNF-α-induced senescent NPCs (Fig. 3d). Western blot analysis also suggested that the expression of age-related P16 protein was significantly downregulated after iMSC-sEVs treatment (Fig. 3e and f). Accordingly, the decreases in the levels of the Collagen II and Aggrecan anabolism markers of the extracellular matrix (ECM) and the increases in the MMP-3 and ADAMTS-4 catabolism markers of ECM were suppressed after senescent NPCs were treated with iMSC-sEVs but not FB-sEVs (Fig. 3g and h). These results revealed that iMSC-sEVs can ameliorate the senescent phenotype of NPCs and age-related dysfunction in vitro.

3.4 iMSC-sEVs alleviate senescence of NPCs by up-regulating Sirt6 in vitro

Sirt6 is a key protein that protects cells from senescence by more efficient repairing of DNA double-strand breaks[22]. The expression level of Sirt6 can decrease with cellular senescence, and the reactivation of Sirt6 in senescent cells can alleviate cellular senescence and age-related dysfunction[23–25]. Therefore, we investigated whether Sirt6 is involved in the effects of iMSC-sEVs on senescent NPCs. Western blotting analysis showed that the expression of Sirt6 declined in senescent NPCs. iMSC-sEVs treatment recovered the expression of Sirt6 but not FB-sEVs (Fig. 4a and b). To explore whether the upregulation of Sirt6 resulted in the therapeutic effects of iMSC-sEVs, we treated senescent NPCs with iMSC-sEVs and the Sirt6 inhibitor (OSS_128167). iMSC-sEVs failed to decrease the SA-𝛽-gal activity in senescent NPCs when co-treated with the Sirt6 inhibitor (Fig. 4c and d), as well as the expression of P16 (Fig. 4f and g). The CCK-8 assay demonstrated that the Sirt6 inhibitor also abolished iMSC-sEVs mediated restoration of proliferation ability in senescent NPCs (Fig. 4e). Accordingly, iMSC-sEVs mediated the upregulation of anabolism markers of ECM (Collagen Ⅱ and Aggrecan), and the downregulation of catabolism markers of ECM (MMP-3 and ADAMTS-4) were obviously blocked by the Sirt6 inhibitor treatment (Fig. 4h and i). These results suggested that iMSC-sEVs can ameliorate the senescent phenotype of NPCs and age-related dysfunction in vitro through Sirt6 activation.

3.5 iMSC-sEVs downregulate PDE4D expression and activate the Sirt6 signalling pathway by delivering miR-105-5p into senescent NPCs

Various miRNAs encapsulated in sEVs can be transported to the recipient cells and regulate gene expression post-transcriptionally by binding to the 3ʹ untranslated region (UTR) or amino acid coding sequences of the target gene[26, 27]. To identify the miRNAs with therapeutic activity in iMSC-sEVs, we first performed miRNA sequence analysis of sEVs secreted by iMSCs or FBs. Ninety-two differentially expressed miRNAs were evident in iMSCs-sEVs compared with FB-sEVs. Given that no therapeutic effect on senescent NPCs was observed using FB-sEVs, we focused on the upregulated miRNAs in iMSCs-sEVs compared to those in FB-sEVs. Ten miRNAs in iMSCs-sEVs were significantly upregulated compared to their levels in FB-sEVs (Fig. 5a). qRT-PCR validated the levels of the 10 upregulated miRNAs (Fig. 5b). Next, we predicted the potential target genes of the 10 upregulated miRNAs by using the TargetScan and miRbase websites. Bioinformatic analysis demonstrated that miR-105-5p could bind the 3ʹUTR of PDE4D, which inactivates the Sirt6 pathway by decreasing the cellular levels of the second messenger cAMP (Fig. 5c)[28]. A luciferase assay was used to validate whether miR-105-5p could directly target PDE4D. Luciferase activity was significantly decreased when transfected cells were incubated with miR-105-5p mimics. However, this effect was blocked by mutating the target sites in the 3ʹUTR of PDE4D (Fig. 5d). Moreover, western blotting analysis also showed that the expression level of PDE4D in senescent NPCs treated with iMSCs-sEVs was significantly downregulated (Fig. 5e and f). These results suggested that iMSC-sEVs could deliver miR-105-5p into senescent NPCs and downregulate the expression level of PDE4D, and subsequentyl activate the Sirt6 signalling pathway.

3.6 Inhibition of miR-105-5p attenuates the therapeutic effect of iMSC-sEVs on senescent NPCs

To further investigate whether miR-105-5p is crucial in iMSC-sEVs-mediated rejuvenation of senescent NPCs, miR-105-5p inhibitor was incorporated into iMSC-sEVs. Binding of the inhibitor to miR-105-5p sequences abolished the biological function of miR-105-5p. We first examined the expression levels of PDE4D and Sirt6 after incubation of senescent NPCs with miRNA inhibitor-treated iMSC-sEVs. Expression of PDE4D was increased in the presence of miRNA inhibitor-treated iMSC-sEVs, whereas the expression of Sirt6 was decreased (Fig. 6a and b). The findings indicated that the inhibition of miR-105-5p promoted the activation of the Sirt6 signalling pathway. SA-𝛽-gal staining and the CCK-8 assay revealed that the inhibition of miR-105-5p significantly abolished the iMSC-sEVs-mediated reduction of the proportion of senescent NPCs and restoration of proliferation capacity (Fig. 6c-e). The level of age-related P16 protein also increased when senescent NPCs were treated with miRNA inhibitor-treated iMSC-sEVs (Fig. 6f and g). Additionally, the levels of the Collagen Ⅱ and Aggrecan anabolism markers of ECM decreased, and the levels of the MMP-3 and ADAMTS-4 catabolism markers of ECM increased upon treatment with miRNA inhibitor-treated iMSC-sEVs (Fig. 6h and i). These results demonstrated that the inhibition of miR-105-5p attenuated the therapeutic effect of iMSC-sEVs on senescent NPCs in vitro.

3.7 PDE4D overexpression in NPCs blocks the therapeutic effect of iMSC-sEVs on senescent NPCs

To examine the key role of PDE4D in the therapeutic effect of miR-105-5p incorporated in iMSC-sEVs, a PDE4D overexpressing adenovirus was synthesised and transfected into NPCs. We first explored the optimal multiplicity of infection (MOI) value for adenovirus transfection of NPCs. When the MOI was 10, the efficiency of adenovirus transfection into NPCs was almost 100%, and the efficiency of adenovirus transfection was not significantly improved when MOI continued to increase (Fig. 7a). Therefore, an MOI of 10 was used in the following experiments. Western blot analysis showed that the expression level of PDE4D protein could be significantly upregulated when adenovirus was transfected into NPCs (Fig. 7b and c). Next, we treated NPCs with TNF-α and PDE4D-overexpressing adenovirus and observed aged-related phenotypes 7 days later. iMSC-sEVs-mediated reduction in the proportion of senescent NPCs, restoration of proliferation capacity, and downregulation of age-related P16 protein were significantly attenuated (Fig. 7d-h). Accordingly, iMSC-sEVs-mediated increases in the levels of anabolism markers of ECM and the decreases in the catabolism markers of ECM were suppressed after overexpressing PDE4D (Fig. 7i and j). The results indicated the therapeutic activity of miR-105-5p transmitted by iMSC-sEVs by targeting the downregulation of PDE4D expression

4. Discussion

During disc degeneration, disorders in the physiological behavior of NPC cells and ECM synthesis eventually lead to biomechanical impairment of the IVD. Previous studies have suggested that senescence of NPCs is involved in these pathological changes[29], indicating that targeting senescent NPCs would be an effective treatment strategy for IVDD[30, 31]. Recent evidence suggests that MSC transplantation into degenerated discs may be beneficial due to their paracrine function. Among these paracrine bioactive substances, sEVs are attracting increasing interest. sEVs are a class of natural nanoparticles enclosed by a lipid bilayer with diameter of 30–150 nm. sEVs can deliver their internal contents of parental cells, including proteins, nucleic acids, and lipids, into the target cell resulting in the metabolic changes in recipient cells. sEVs pose no risks of tumorigenesis and immune response. MSC-derived sEVs may be capable of alleviating cellular senescence. However, challenges remain in the large scale preparation of sEVs derived from MSCs. The quantity and quality of MSC-derived sEVs are hindered by drawbacks that include the limited proliferative capacity of MSCs in vitro, MSC donor age, and the invasive acquisition of MSCs. One solution to these problems may be the use of MSCs derived from iPSCs. Numerous studies have found that iMSCs resemble some conventional MSCs derived from bone marrow or adipose tissue in terms of both phenotype and function[13, 32]. The potential advantages of iMSCs have been described previously.[13]

Recent studies have shown the promising therapeutic effects of iMSC-sEVs in tissue repair, including skin wound healing, osteoarthritis, bone defects, and limb ischemia[21, 33–35]. Consistent with these results, we presently demonstrated that the intervertebral injection of iMSC-sEVs could significantly delay IVDD in rats as well as the senescence of NPCs. In addition, in vitro experiments also showed that iMSC-sEVs remarkably alleviated the senescent phenotype of NPCs and age-related dysfunction.

DNA damage has long been implicated in cellular senescence. Unresolved DNA damage can accelerate cellular senescence and promote disease development[36]. DNA repair is regulated by DNA damage response genes. Sirt6 is one gene that responds to DNA repair and promotes longevity[37]. The expression level of Sirt6 decreases with cellular senescence and the reactivation of Sirt6 in senescent cells can alleviate cellular senescence and age-related dysfunction due to the more efficient DNA double-strand break repair[22, 23]. Here, to investigate whether Sirt6-mediated signalling is involved in the effects of iMSCs-sEVs on NPC senescence, the level of Sirt6 was detected. The effects of iMSC-sEVs on senescent NPCs were evidenced by the activation of the Sirt6 signalling pathway.

Accumulating evidence indicates that sEVs are rich in a variety of miRNAs. The miRNAs encapsulated in sEVs can be transferred to recipient cells and can regulate their function by regulating gene expression post-transcriptionally[38]. However, few studies have examined miRNAs in iMSCs-sEVs. Presently, we provide the first evidence of the differential expression of miRNAs in iMSCs-sEVs by miRNA sequence analysis. There was no evidence of an effect of FB-sEVs on senescent NPCs. Thus, it seems reasonable to speculate that the crucial miRNAs that show therapeutic effect should be upregulated in iMSCs-sEVs. Among these upregulated miRNAs, miR-105-5p was predicted to bind the 3ʹUTR of PDE4D as observed by TargetScan and miRDB gene prediction website’s scrutiny of high scoring target genes. PDE4D is a cAMP-specific hydrolase. Inhibition of PDE4D activity could increase the cAMP concentration, which leads to AMP-activated phosphate kinase (AMPK) and Sirt6 activation[39], the eventual alleviation of age-related phenotypes, and extended lifespan. PDE4 inhibitors and cAMP analogues may protect against aging-related diseases, such as Alzheimer's disease[40]. To confirm the crucial role of miR-105-5p on the PDE4D-Sirt6 axis, we inhibited the biological function of miR-105-5p in iMSCs-sEVs. This inhibition abolished the therapeutic effect of iMSC-sEVs on senescent NPCs, and increased the expression levels of PDE4D and reduced the expression levels of Sirt6. In addition, the overexpression of PDE4D in senescent NPCs also blocked the therapeutic effect of iMSC-sEVs on senescent NPCs. These data reveal that iMSC-sEVs-mediated miR-105-5p transfer leads to higher cAMP concentrations by targeting PDE4D and activation of Sirt6, a key cascade involved in IVDD.

5. Conclusions

This is the first study to evaluate the therapeutic effects of iMSC-sEVs on IVDD. iMSC-sEVs could rejuvenate the senescence of NPCs and attenuate the development of IVDD. iMSC-sEVs exerted their anti-aging effects by delivering miR-105-5p to senescent NPCs and activating the Sirt6 pathway, which is a pivotal pathway response to DNA repair and promotes longevity. Our findings also indicate that iMSCs are a promising candidate MSC for obtaining sEVs on a large scale while avoiding several defects related to the present applications of MSCs, and that iMSC-sEVs could be a novel cell-free therapeutic tool for the treatment of IVDD. Notably, the effects of iMSC-sEVs on Sirt6 activation and age-related dysfunction were not entirely blocked by miR-105-5p antagomir treatment (Fig. 6c-i). This suggests that other molecular mechanisms might also be involved and requires further exploration.

Abbreviations

IVDD: Intervertebral disc degeneration; NPCs: Nucleus pulposus cells; MSC: Mesenchymal stem cell; sEVs: Small extracellular vesicles; iPSCs: Induced pluripotent stem cells; iMSCs: MSCs derived from induced pluripotent stem cells; iMSC-sEVs: iMSC-derived small extracellular vesicles; DMEM: Dulbecco’s Modified Eagle Medium; FBS: Foetal bovine serum; FBs: Fibroblasts; MRI: Magnetic resonance imaging; TEM: Transmission electron microscopy; NTA: Nanoparticle analysis; H&E: Hematoxylin-eosin; IF: Immunofluorescence; SA-𝛽-gal: Senescence-associated 𝛽 –galactosidase; CCK-8: Cell counting kit-8; MOI: Multiplicity of infection

Declarations

Acknowledgements

The authors appreciated the help of all the professors at the First Affiliated Hospital of the University of Science and Technology of China.

Authors’ contributions

Xu Li conceived the idea, oversaw the experiments, and provided the funding for the study. Yongjin Sun and Wenzhi Zhang performed the in vivo and in vitro experiments, and drafted the manuscript

Funding

This research is supported by National Natural Science Foundation of China. (81201383)

Competing interests

There are no conflicts to declare.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

This study was performed in compliance with the principles of the Helsinki Declaration and the Guidelines for the Care and Use of Laboratory Animals of the Chinese Institute of Health. Sprague–Dawley rats were purchased from the central animal laboratory of The First Affiliated Hospital of the University of Science and Technology of China. All procedures were approved by Animal Research Committee of the First Affiliated Hospital of the University of Science and Technology of China.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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