Boosting the Biogenesis and Secretion of Mesenchymal Stem Cell-Derived Exosomes

Abstract

A limitation of using exosomes to their fullest potential is their limited secretion from cells, a major bottleneck to efficient exosome production and application. This is especially true for mesenchymal stem cells (MSCs), which can self-renew but have a limited expansion capacity, undergoing senescence after only a few passages, with exosomes derived from senescent stem cells showing impaired regenerative capacity compared to young cells. Here, we examined the effects of small molecule modulators capable of enhancing exosome secretion from MSCs. The treatment of MSCs with a combination of N-methyldopamine and norepinephrine robustly increased exosome production by three-fold without altering the ability of the MSC exosomes to induce angiogenesis, polarize macrophages to an anti-inflammatory phenotype, or downregulate collagen expression. These small molecule modulators provide a promising means to increase exosome production by MSCs.

Keywords: exosome secretion; proteomics; small molecules.

Figure 1

Effects of small molecules on MSC cell viability and exosome production efficiency. (A) Cell proliferation rate. (B) The cell viability and metabolism of mesenchymal stem cells (MSCs) treated with different doses of compounds measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. (C) The production efficiency of exosomes derived from MSCs treated with different doses of compounds. (D) The production efficiency of exosomes derived from different MSCs treated with combinations of compounds. (E) The cell proliferation rate of MSCs in response to treatment with small molecule modulators. (F) The cell viability and metabolism of MSCs after treatment with small molecule modulators measured by the MTT assay. (G) The size distribution of exosomes by nanoparticle tracking analysis (NTA) in response to the treatment of MSCs with small molecule modulators. (H) Transmission electron microscopy (TEM) images of exosomes, scale bar 200 = nm, Exo: Exosomes. (I) CD63 and CD9 exosomal surface marker protein expression by Western Blotting. One-way ANOVA with Dunnett’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 3–6 each group. FT: fenoterol; NE: norepinephrine; FK: forskolin; MeDA: N-methyldopamine; Mepn: mephenesin.

Figure 2

Effects of exosomes derived from MSCs treated with small molecule modulators on collagen expression. (A) COL1A1 expression in human cardiac fibroblasts (HCF) incubated with exosomes isolated from MSCs treated with small molecule modulators as determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). (B) MTT assay of MSC-derived exosomes on HCFs. (C) COL1A1 expression in human cardiac fibroblasts (HCF) incubated with small molecule modulators. (D) MTT assay of compound treatment on HCFs. One-way ANOVA with Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3–4.

Figure 3

Polarization of macrophages with compound-treated MSC-derived exosomes. (A) Inflammatory macrophage (M1) markers iNOS and IL6. (B) Anti-inflammatory macrophage (M2) markers Arg1 and CD206. (C) M2/M1 ratios. One-way ANOVA with Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3.

Figure 4

Tube formation assay. (A) Representative images of the angiogenesis effect of compound-treated MSC-derived exosomes. (B–G) Quantification of angiogenesis using ImageJ. (B) Total length. (C) Total branching length. (D) The number of nodes. (E) The number of junctions. (F) The number of meshes. (G) The mesh index. One-way ANOVA with Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 5.

Figure 5

The effect of small molecule compounds on exosomal production-related pathways after 48 h treatment. RT-PCR quantification of (A) nSMase2 (SMPD3), (B) Hrs, (C) Tsg101, (D) Stam1, (E) Alix, (F) microphthalmia-associated transcription factor (MITF), (G) Rab27a, and (H) Rab27b. One-way ANOVA with Dunnett’s multiple comparisons test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n = 3–5.

Figure 6

Proteomic profile of MSC exosomes 48h after norepinephrine and N-methyldopamine (NE + MeDA) treatment. (A) Raw abundance values from the NT and NE + MeDa groups showed no statistical difference using a paired t-test across samples. (B) Linear regression analysis comparing non-treated MSCs (NT) and MSCs treated with NE + MeDA. (C) Data visualized using a volcano plot with red dots indicating statistically significantly different proteins with an abundance change of 2-fold or higher. (D) Cytoscape 3.2.1 visualization of Kegg, Reactome, and Panther Pathway hits of differentially expressed proteins using ClueGo v.2.5.4. The font size is mapped to the p value with Bonferroni step down correction, and the node size is mapped to number of statistically significant gene hits.