Characterization of the Secretome from Spheroids of Adipose-Derived Stem Cells (SASCs) and Its Potential for Tissue Regeneration

Abstract

Introduction: Spheroids are spherical aggregates of cells that mimic the three-dimensional (3D) architecture of tissues more closely than traditional two dimensional (2D) cultures. Spheroids of adipose stem cells (SASCs) show special features such as high multilineage differentiation potential and immunomodulatory activity. These properties have been attributed to their secreted factors, such as cytokines and growth factors. Moreover, a key role is played by the extracellular vesicles (EVs), which lead a heterogeneous cargo of proteins, mRNAs, and small RNAs that interfere with the pathways of the recipient cells.

Purpose: The aim of this work was to characterize the composition of the secretome and exosome from SASCs and evaluate their regenerative potential.

Materials and methods: SASCs were extracted from adipose samples of healthy individuals after signing informed consent. The exosomes were isolated and characterized by Dinamic Light Scattering (DLS), Scanning Electron Microscopy (SEM), and Western blotting analyses. The expression of mRNAs and miRNAs were evaluated through real-time PCR. Lastly, a wound-healing assay was performed to investigate their regenerative potential on different cell cultures.

Results: The SASCs' exosomes showed an up-regulation of NANOG and SOX2 mRNAs, typical of stemness maintenance, as well as miR126 and miR146a, related to angiogenic and osteogenic processes. Moreover, the exosomes showed a regenerative effect.

Conclusions: The SASCs' secretome carried paracrine signals involved in stemness maintenance, pro-angiogenic and pro-osteogenic differentiation, immune system regulation, and regeneration.

Keywords: adipose tissue; extracellular vesicles; secretome; spheroids of adipose stem cells; stemness and mesenchymal differentiation.

Figure 1

Exosomes characterization. (A) DLS analysis for physical characterization, (B) SEM analysis for exosomal size and shape characterization, and (C) CD63, typical exosomal expression marker, by Western blotting analysis.

Figure 2

Exosomal internal cargo: analysis of 12 mRNAs. (A) Stemness-related mRNAs (Sox2, Nanog, Pou5f1 and Prom1); (B) angiogenesis-related mRNAs (Vegfa, Vegfr2, Hif1a, Igf1 and Cd31); (C) mesenchymal differentiation-related mRNAs (Sox9, Runx2 and Pparg).

Figure 3

Exosomal internal cargo: analysis of 11 miRNAs. (A) More-expressed miRNAs (miR126 and miR146a); (B) less-expressed miRNAs (miR100, miR221, miR140, miR30c, miR143, miR451, miR182, miR142-3p, and miR495).

Figure 4

Wound-healing assay on (A) endothelial cells, (B) fibroblasts, and (C) osteoblasts (20× magnification). In the first two columns, the controls (secretome and exosomes) are shown. In the second two columns, total secretome and isolated exosomes added to the cell specific culture media are shown. Percentage of the wounded area on (D) endothelial cells, (E) fibroblasts, and (F) osteoblasts at 0, 1, and 2 days.

Figure 4

Wound-healing assay on (A) endothelial cells, (B) fibroblasts, and (C) osteoblasts (20× magnification). In the first two columns, the controls (secretome and exosomes) are shown. In the second two columns, total secretome and isolated exosomes added to the cell specific culture media are shown. Percentage of the wounded area on (D) endothelial cells, (E) fibroblasts, and (F) osteoblasts at 0, 1, and 2 days.