(165ac) Human Mesenchymal Stem Cell-Derived Exosomes for Biologically Active Annulus Fibrosus Repair

Intervertebral disc (IVD) herniation is a common condition with an incidence rate of approximately 5 to 20 cases per 1000 adults.¹ The IVD is the largest avascular organ and is made up of an inner jelly-like core called the nucleus pulposus (NP), which is surrounded by collagen and elastin fibers making up the annulus fibrosus (AF).² In a herniated IVD, NP material protrudes through defects present in the AF, causing compression on nerve roots. Surgical interventions include discectomy procedures to remove protruding NP tissue to alleviate compression and symptoms. However, this treatment strategy does not repair AF defects or address the IVDs limited healing capacity, resulting in up to 24% of patients undergoing re-operation to address reherniation or progressive IVD degeneration after their initial procedure.³Therefore, there is an unmet clinical need to develop therapeutic strategies that promote endogenous repair of AF tissue and maintain IVD function.

Next generation treatment strategies are focused on biologically active molecules that can slow degenerative processes and enhance repair. Mesenchymal stem cells (MSCs) have emerged for treating human diseases, with much of their regenerative qualities attributed to the factors that they naturally release called extracellular vesicles (EVs).⁴ EVs often serve as mediators of cellular communication since they can transfer their molecular cargo across cells and affect cell function.⁵ Exosomes, the smallest class of EVs, range in size from 40 – 150 nm, and are enriched in bioactive factors including proteins, signaling molecules, mRNAs, and micro-RNAs (miRs), which allows them to control biological processes by activating receptors of recipient cells.^5,6 Exosomes have been shown to elicit therapeutic effect in a number of different organ systems, including kidneys, liver, cardiovascular applications, and neurological applications.⁶However, their ability to serve as potential therapeutic factors to promote regenerative AF repair is not well established. The purpose of this study was to examine the therapeutic potential of MSC-derived exosomes on bovine AF cells following a biochemical challenge. We isolated and characterized MSC-derived exosomes and then studied their ability to induce cell migratory behavior and proliferation, and to rescue AF cell phenotypic markers from a pro-inflammatory challenge with TNFα.

Methods

Cell Culture and EV Isolation

Human bone marrow-derived MSCs (hBM-MSCs) from three biological donors (MSC00115, MSC00175, MSC00179) were cultured according to manufacturer’s instructions in either hypoxic (Hx) conditions (18.6% O₂, 5% CO₂, and 70.2% N₂) or normoxic (Nx) conditions (5% O₂, 5% CO₂, and 83.8% N₂) until cells reached 90% confluency. The cell culture media was then replaced with serum-deprived media and EV production supplement and cultured for 24 h. MSC-derived exosomes were isolated by differential centrifugation of conditioned media over a 30% sucrose cushion.

EV Characterization

Transmission Electron Microscopy (TEM) was performed to visualize exosome structure. Dynamic Light Scattering (DLS) was performed to determine exosome particle size distribution. After extracting proteins from MSC-derived exosomes, western blot was performed using exosomal marker TSG101 and Calnexin as a control.

Cell Proliferation Assay

The CellTiter-Glo® 2.0 Assay was used to measure AF cell proliferation according to manufacturer’s instructions. Bovine AF cells from three biological donors were treated with either Nx or Hx exosomes treatment groups. (N = 3/group). Luminescence was measured at pre-determined assay timepoints.

Transwell Migration Assay

Bovine AF cell migration was evaluated using the Transwell Migration Assay. Media was prepared in the following conditions: serum-free media (negative control), serum free media supplemented with CCL5/RANTES, and serum-free media supplemented with 50 µg/mL of MSC-derived exosomes in both Nx and Hx conditions. Membranes were imaged on a widefield microscope.

Cell Rescue Experiment and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Bovine AF cells from ten biological donors were cultured to 85% confluency. Cells were divided into the following treatment groups: (1) serum-free media (untreated control), (2) serum-free media supplemented with TNFα, serum-free media treated with Hx MSC-derived exosomes from donors (3) MSC00115, (4) MSC00175, (5) MSC00179 for 1 hour and then supplemented with TNFα. Cells were then incubated for 24 hours. Cell RNAs were extracted by guanidinium thiocyanate-phenol-chloroform extraction and then used to synthesize cDNA for the qRT-PCR reaction.

Results and Discussion

This study examines the regenerative potential of hBM-MSC-derived exosomes on AF cells. EVs consist of a heterogeneous population of nanoparticles that are generally classified into different groups based on size and biogenesis. Given the overlaps in size between the different groups, it is necessary to employ both biophysical and biochemical characterization methods as a quality control measure to confirm the exosome identity of isolated EVs. TEM imaging showed the cup-like morphology of EVs with a lipid bilayer (Figure 1). DLS results showed the mean diameter of isolated EVs fell within the typical range reported for exosomes (Figure 2). Diameter size did not vary significantly across Nx or Hx culture conditions, which had mean diameters of 113.26 ± 30.56 nm (N = 3) and 88.62 ± 34.49 nm (N = 3), respectively. Furthermore, western blot analysis showed all EV groups were positive for TSG101, an established positive exosome marker, and negative for Calnexin, an established negative exosome marker. The combination of these biophysical and biochemical characterization techniques confirm that isolated EVs were exosomes.

Results of the cell proliferation assay showed that 72 h treatment with Hx exosomes showed significant increases in normalized luminescence for 2 of the 3 donors (Figure 3). Additionally, normalized luminescence was significantly greater for Hx than Nx exosomes at 72 h for the same two donors derived from MSC00115 and MSC00175. Furthermore, results of the transwell migration assay showed that treatment with Hx exosomes across all groups were significantly increased for all Hx treatment groups and for Nx exosomes derived from MSC00175 and MSC00179 (Table 1). Furthermore, exosomes induced substantially greater migration than CCL5. These results indicate that MSC-derived exosomes induce proliferative and migratory responses in AF cells, which is an important finding since cellular senescence is a pathological hallmark of IVD degeneration. Cell proliferation and migration serve to promote mitotic activity within the IVD, and we suggest that exosomes may offer potential to mitigate the effects of IVD degeneration and support endogenous tissue repair.

Hx exosomes were selected for downstream use to treat TNFα-challenged AF cells since they demonstrated the most significant cell proliferation and migration responses compared to Nx exosomes. Results from qRT-PCR showed exosome treatment exerted a protective effect against damage induced by TNFα by restoring gene expression levels back to untreated control levels for genes associated with a normal AF phenotype (COL1A1, COL2A1, ACAN, SCX, and MKX).⁷ Furthermore, exosome treatment was shown to modulate catabolic and inflammatory responses by attenuating MMP1, IL6, and NLRP3, which are implicated in IVD degeneration.⁸

Overall, these results show that exosomes were successfully isolated and characterized from MSCs and that they can induce regenerative effects in bovine AF cells. Exosomes were shown to promote chemotactic and proliferative AF cell responses, and rescue a healthy AF cell phenotype from a TNFα challenge. Taken together, these findings suggest that exosomes may have therapeutic potential as a treatment strategy for AF repair by enhancing the poor IVD healing capacity and promoting regenerative repair.

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