TY - JOUR
T1 - 3D-MSCs A151 ODN-loaded exosomes are immunomodulatory and reveal a proteomic cargo that sustains wound resolution
AU - Camões, Sérgio P.
AU - Bulut, Ozlem
AU - Yazar, Volkan
AU - Gaspar, Maria M.
AU - Simões, Sandra
AU - Ferreira, Rita
AU - Vitorino, Rui
AU - Santos, Jorge M.
AU - Gursel, Ihsan
AU - Miranda, Joana P.
N1 - Funding Information:
This study was partially supported by FCT (PTDC/MED-TOX/29183/2017, PTDC/MED-QUI/31721/2017 and PTDC/SAU-SER/30197/2017), by strategic funding for iMed.ULisboa (UIDB/04138/2020 and UIDP/04138/2020), for UnIC (UID/00051/2020) and for iBiMED (UID/BIM/04501/2020), and by European Union, Quadro de Referência Estratégico Nacional (QREN), Fundo Europeu de Desenvolvimento Regional (FEDER) and Programa Operacional Fatores de Competitividade (COMPETE) (POCI-01-0145-FEDER-007628 and LISBOA-01-0145- FEDER-030197). VIB Proteomics Core and COST Actions (EU Framework Programme Horizon 2020) CA16113 and CA16119 are also acknowledged. The authors thank Dr. Tânia Carvalho for her valuable assistance on histological analysis. SPC also acknowledges EFIS-IL short-term fellowship.
Publisher Copyright:
© 2022
PY - 2022/11
Y1 - 2022/11
N2 - Introduction: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. Objectives: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. Methods: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. Results: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-β, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-β, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. Conclusion: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines.
AB - Introduction: Non-healing wounds remain a major burden due to the lack of effective treatments. Mesenchymal stem cell-derived exosomes (MSC-Exo) have emerged as therapeutic options given their pro-regenerative and immunomodulatory features. Still, little is known on the exact mechanisms mediated by MSC-Exo. Importantly, modulation of their efficacy through 3D-physiologic cultures together with loading strategies continues underexplored. Objectives: To uncover the MSC-Exo-mediated mechanism via proteomic analyses, and to use 3D-culture and loading technologies to expand MSC-Exo efficacy for cutaneous wound healing. Methods: MSC-Exo were produced in either 3D or 2D cultures (Exo3D/Exo2D) and loaded with an exogenous immunosuppressive oligodeoxynucleotide (A151 ODN). Both, loaded and naïve exosomes were characterised regarding size, morphology and the presence of specific protein markers; while IPA analyses enabled to correlate their protein content with the effects observed in vitro and in vivo. The Exo3D/Exo2D regenerative potential was evaluated in vitro by assessing keratinocyte and fibroblast mitogenicity, motogenicity, and cytokine secretion as well as using an in vivo wound splinting model. Accordingly, the modulation of inflammatory and immune responses by A151-loaded Exo3D/Exo2D was also assessed. Results: Exo3D stimulated mitogenically and motogenically keratinocytes and fibroblasts in vitro, with upregulation of IL-1α and VEGF-α or increased secretion of TGF-β, TNF-α and IL-10. In vivo, Exo3D reduced the granulation tissue area and promoted complete re-epithelization of the wound. These observations were sustained by the proteomic profiling of the Exo3D cargo that identified wound healing-related proteins, such as TGF-β, ITGA1-3/5, IL-6, CDC151, S100A10 and Wnt5α. Moreover, when loaded with A151 ODN, Exo3D differentially mediated wound healing-related trophic factors reducing the systemic levels of IL-6 and TNF-α at the late stage of wound healing in vivo. Conclusion: Our results support the potential of A151-loaded Exo3D for the treatment of chronic wounds by promoting skin regeneration, while modulating the systemic levels of the pro-inflammatory cytokines.
KW - 3D-cultured mesenchymal stem/stromal cells
KW - Exosomes
KW - Immunomodulation
KW - Immunosuppressive oligodeoxynucleotide loading
KW - Proteomics
KW - Wound healing
UR - http://www.scopus.com/inward/record.url?scp=85126097251&partnerID=8YFLogxK
U2 - 10.1016/j.jare.2022.01.013
DO - 10.1016/j.jare.2022.01.013
M3 - Article
C2 - 36328741
AN - SCOPUS:85126097251
SN - 2090-1232
VL - 41
SP - 113
EP - 128
JO - Journal of Advanced Research
JF - Journal of Advanced Research
ER -