Amniotic membrane-mesenchymal stromal cells secreted factors and extracellular vesicle-miRNAs: Anti-inflammatory and regenerative features for musculoskeletal tissues

Abstract

Human amniotic membrane-derived mesenchymal stromal cells (hAMSCs) are easily obtained in large quantities and free from ethical concerns. Promising therapeutic results for both hAMSCs and their secreted factors (secretome) were described by several in vitro and preclinical studies, often for treatment of orthopedic disorders such as osteoarthritis (OA) and tendinopathy. For clinical translation of the hAMSC secretome as cell-free therapy, a detailed characterization of hAMSC-secreted factors is mandatory. Herein, we tested the presence of 200 secreted factors and 754 miRNAs in extracellular vesicles (EVs). Thirty-seven cytokines/chemokines were identified at varying abundance, some of which involved in both chemotaxis and homeostasis of inflammatory cells and in positive remodeling of extracellular matrix, often damaged in tendinopathy and OA. We also found 336 EV-miRNAs, 51 of which accounted for more than 95% of the genetic message. A focused analysis based on miRNAs related to OA and tendinopathy showed that most abundant EV-miRNAs are teno-and chondro-protective, able to induce M2 macrophage polarization, inhibit inflammatory T cells, and promote Treg. Functional analysis on IL-1β treated tenocytes and chondrocytes resulted in downregulation of inflammation-associated genes. Overall, presence of key regulatory molecules and miRNAs explain the promising therapeutic results of hAMSCs and their secretome for treatment of musculoskeletal conditions and are a groundwork for similar studies in other pathologies. Furthermore, identified molecules will pave the way for future studies aimed at more Enrico Ragni and Andrea Papait contributed equally to the work.

Comparison of miRNA cargo in human adipose-tissue vs. amniotic-membrane derived mesenchymal stromal cells extracellular vesicles for osteoarthritis treatment

“…The discriminating factor was hAMSC-EVs enriched miR-146a-5p that in several studies reduced M1 and promoted M2 macrophage polarization [63][64][65] . Consistently, the only study comparing hASCs and hAMSCs activity on macrophages reported a higher capacity for M2 polarization in favor of hAMSCs [20] . Together with inflammatory factors, other OA-related and synovia specific cytokines are preferential hAMSC-EVs miRNA targeted.…”

“…Molecular data suggest stronger commitment in antiinflammatory macrophage modulation for hAMSC-EVs and a less defined picture for the definition of the best EV type in cartilage protection, where harmful growth factors are the preferential hAMSC-EV target, whereas ECM was more protected by hASC-EVs' inhibitory activity on proteases and the presence of miR-125b-5p. The observed in vitro increased chondro-protection and M1:M2 synovial macrophage ratio reduction for hAMSCs with respect to hASCs [20] might be due to the highest EV release of hAMSCs that can overcome the similar chondro-protective ability. Future clinical studies to address the issue regarding EVs dose will be necessary, especially in the frame of GMP clinical products.…”

“…Topoluk et al [19] showed that hAMSCs have a greater differentiation potential toward bone and cartilage compared with hASCs. In addition, in a sophisticated in vitro coculture model of patient-matched human OA cartilage and synovium, hAMSCs resulted more chondroprotective and more effective at reducing the OA pro-inflammatory: anti-inflammatory (M1:M2) synovial macrophage ratio [20] . These features might be ascribed to different secretory activity, including EVs and their miRNAs.…”

The Role of Extracellular Vesicles in the Pathogenesis, Diagnosis, and Treatment of Osteoarthritis

“…The analysis based on miRNAs related to OA and tendinopathy showed that the most abundant EV-miRNA can induce polarization of M2 macrophages, and then protect the tendon and cartilage. Overall, the presence of key regulatory molecules and miRNAs explains the promising therapeutic results of AMSCs and their secretion group in the treatment of musculoskeletal diseases [ 41 ].…”

Allogenic Use of Human Placenta-Derived Stromal Cells as a Highly Active Subtype of Mesenchymal Stromal Cells for Cell-Based Therapies

“…Preclinical studies comparing the efficacy of atMSCs vs PSCs to regenerate the sphincter muscle in an animal model for urinary incontinence are well under way. Interestingly, MSCs derived from the amniotic membrane produced a secretome suitable for regeneration of musculoskeletal tissues [260].…”