Wharton's Jelly mesenchymal stromal cell-derived extracellular vesicles promote nucleus pulposus cell anabolism in an in vitro 3D alginate-bead culture model

Abstract

Background: Intradiscal transplantation of mesenchymal stromal cells (MSCs) has emerged as a promising therapy for intervertebral disc degeneration (IDD). However, the hostile microenvironment of the intervertebral disc (IVD) may compromise the survival of implanted cells. Interestingly, studies reported that paracrine factors, such as extracellular vesicles (EVs) released by MSCs, may regenerate the IVD. The aim of this study was to investigate the therapeutic effects of Wharton's Jelly MSC (WJ-MSC)-derived EVs on human nucleus pulposus cells (hNPCs) using an in vitro 3D alginate-bead culture model.

Methods: After EV isolation and characterization, hNPCs isolated from surgical specimens were encapsulated in alginate beads and treated with 10, 50, and 100 μg/mL WJ-MSC-EVs. Cell proliferation and viability were assessed by flow cytometry and live/dead staining. Nitrite and glycosaminoglycan (GAG) content was evaluated through Griess and 1,9-dimethylmethylene blue assays. hNPCs in alginate beads were paraffin-embedded and stained for histological analysis (hematoxylin-eosin and Alcian blue) to assess extracellular matrix (ECM) composition. Gene expression levels of catabolic (MMP1, MMP13, ADAMTS5, IL6, NOS2), anabolic (ACAN), and hNPC marker (SOX9, KRT19) genes were analyzed through qPCR. Collagen type I and type II content was assessed with Western blot analysis.

Results: Treatment with WJ-MSC-EVs resulted in an increase in cell content and a decrease in cell death in degenerated hNPCs. Nitrite production was drastically reduced by EV treatment compared to the control. Furthermore, proteoglycan content was enhanced and confirmed by Alcian blue histological staining. EV stimulation attenuated ECM degradation and inflammation by suppressing catabolic and inflammatory gene expression levels. Additionally, NPC phenotypic marker genes were also maintained by the EV treatment.

Conclusions: WJ-MSC-derived EVs ameliorated hNPC growth and viability, and attenuated ECM degradation and oxidative stress, offering new opportunities for IVD regeneration as an attractive alternative strategy to cell therapy, which may be jeopardized by the harsh microenvironment of the IVD.

Keywords: exosome; extracellular vesicles; intervertebral disc; intervertebral disc degeneration; intervertebral disc regeneration; low back pain; mesenchymal stromal cells.

FIGURE 1

Gating strategy to evaluate hNPC viability. Cells were stained with Fixable Viability Dye conjugated with eFluor780 fluorochrome to exclude dead cells (FVD+). Gating strategy based on Forward and Side scatter is shown. FVD, fixable viability dye; hNPC, human nucleus pulposus cells.

FIGURE 2

Characterization of WJ‐MSCs. (A) Cell surface markers (CD90, CD105, CD73, and SSEA3) detected by flow cytometric analysis. (B) Multilineage differentation of WJ‐MSCs towards the osteogenic, adipogenic, and chondrogenic lineages was confirmed by Alizarin Red, Oil red O, and Alcian Blue staining of WJ‐MSC pellets (left and center, scale bar: 50 μm; right, scale bar: 100 μm). WJ‐MSCs, Wharton's Jelly mesenchymal stromal cells.

FIGURE 3

Identification and characterization of WJ‐MSC‐EVs. (A) Particle size distribution of WJ‐MSC‐EVs measured by NTA. (B) TEM imaging showed the typical EV morphology in WJ‐MSC‐EV samples (left, scale bar: 200 nm; right box, scale bar: 20 nm). (C) WB analysis of EV protein markers CD63, CD9, and TSG101. (D) Representative images of hNPCs incubated with PBS or PKH26‐labeled WJ‐MSC‐EVs (red). hNPC nuclei were stained with DAPI (blue). Magnification: 20× scale bar: 100 μm. DAPI, 4′,6‐diamidino‐2‐phenylindole, EVs, extracellular vesicles, hNPCs, human nucleus pulposus cells, NTA, nanoparticle trafficking analysis, PBS, phosphate‐buffered saline, TEM, transmission electron microscopy, WB, Western blot, WJ‐MSCs, Wharton's Jelly mesenchymal stromal cells.

FIGURE 4

MTT assay for hNPC metabolic activity assessment. WJ‐MSC‐EVs significantly improved metabolic activity in the experimental group treated with 10 μg/mL and 100 μg/mL. Data are expressed as percent variation between the control and experimental groups (n = 5). *p < 0.05, **p < 0.01 compared to the control group. hNPCs, human nucleus pulposus cells, WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.

FIGURE 5

WJ‐MSC‐EVs promoted hNPCs proliferation and viability. (A) Cell count significantly increased after treatment with 10 and 50 μg/mL WJ‐MSC‐EVs at day 10 and 14, as compared with the control group (n = 5). (B) After 1 day, WJ‐MSC‐EV treatment significantly reduced hNPC death at 10 and 100 μg/mL (n = 5). (C) Live/Dead staining showed reduced cell death in hNPCs treated with WJ‐MSC‐EVs compared to control (n = 5). Magnification: 20×, scale bar: 100 μm.*p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group. ^# p < 0.05 compared to the 100 μg/mL group. AM, acetoxymethylester; hNPCs, human nucleus pulposus cells; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.

FIGURE 6

(A) NOS2 mRNA levels were significantly reduced by 100 μg/mL WJ‐MSC‐EVs (n = 5). WJ‐MSC‐EVs led to a significant decrease of nitrite concentration (B) released in cell culture supernatant after treatment with 50 and 100 μg/mL WJ‐MSC‐EVs compared to the control group (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group. hNPCs, human nucleus pulposus cells; NOS2, nitric oxide synthase 2; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.

FIGURE 7

WJ‐MSC‐EVs enhanced ECM synthesis in treated hNPCs. (A) GAG/DNA ratio in hNPCs after WJ‐MSC‐EVs treatment demonstrated a significant dose‐dependent increase in all experimental groups (10, 50, and 100 μg/mL WJ‐MSC‐EVs). Data are expressed as GAG/DNA ratio percent variation between the control and experimental groups (n = 8). *p < 0.05, **p < 0.01 compared to the control group. (B) Alcian Blue staining of hNPC alginate beads. Representative images are shown. Blue color indicates proteoglycans. Arrowheads point to proteoglycan deposits. Upper rows, scale bar: 500 μm; bottom boxes, scale bar: 100 μm. hNPCs treated with 50 and 100 μg/mL WJ‐MSC‐EVs showed a significant increase of COL2 (C) and a decrease of COL1 (D) content, respectively (n = 3). COL, collagen; GAG, glycosaminoglycans; hNPCs, human nucleus pulposus cells; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles. **p < 0.01, compared to the control group. °°p < 0.01 compared to the 10 μg/mL group. ^§§ p < 0.01 compared to the 50 μg/mL group. ^# p < 0.05 compared to the 100 μg/mL group.

FIGURE 8

WJ‐MSC‐EVs maintained hNPC phenotype and reduced catabolic gene expression. WJ‐MSC‐EV treatment resulted in a significant decrease of ADAMTS5 (A), MMP1 (B), MMP13 (C), and IL6 (D) mRNA levels, while increasing ACAN (E), SOX9 (F) and KRT19 (G) gene expression. Results were normalized based on GAPDH expression and calculated as fold change compared to the controls. See Materials and Methods for sample size. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to the control group. ACAN, aggrecan; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; hNPCs, human nucleus pulposus cells; IL, interleukin; MMP, matrix metalloproteinase; NOS, nitric oxide synthase; SOX, SRY‐box transcription factor; WJ‐MSC‐EVs, Wharton's Jelly mesenchymal stromal cell‐derived extracellular vesicles.