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

Abstract

Aim: Mesenchymal stromal cells (MSCs) emerged as a promising therapeutic option for osteoarthritis (OA) management, in particular those isolated from adipose tissue (hASCs) and amniotic membrane (hAMSCs). The cartilage protective and immunomodulatory features of hASCs and hAMSCs are ascribed to secreted factors, including extracellular vesicles (EVs) and embedded miRNAs. The purpose of this study was to compare EVs and shuttled miRNAs from both MSC types and discuss them in the frame of OA pathological tissues.Methods: Human hASCs and hAMSCs were analyzed by flow cytometry. EVs were analyzed by flow cytometry, nanoparticle tracking analysis, and electron microscopy. High-throughput qRT-PCR miRNA data available in the literature were compared. Abundant miRNAs and their experimentally validated targets were associated with those reported to drive OA pathology at cartilage, synovia, and macrophage levels. Four tools (Genorm, Normfinder, BestKeeper, and Delta Ct) were used to identify EVs stable reference genes.

Endogenous Controls for the Evaluation of Osteoarthritis-Related miRNAs in Extracellular Vesicles from Bone-Marrow-Derived Mesenchymal Stromal Cells and the Impact of Osteoarthritis Synovial Fluid

“…CD44, CD73 and CD90, all defining BMSC-EVs [ 54 ] and present on parental cells, were strongly positive ( Figure 1 D). As in MSC-EVs from adipose tissue [ 41 ], amniotic membrane [ 41 ], cord blood [ 53 ] and previously shown in bone marrow as well [ 53 ], BMSC-EVs strongly expressed both CD63 and CD81 EV markers, while CD9 staining gave an extremely weak signal ( Figure 1 E).…”

“…Moreover, customized drug-loaded EVs are gradually applied in the field of OA therapy by either loading miRNA into the secreting cells or directly mixing EV with purified particles [ 61 , 62 , 63 ], although this technology has to be improved in terms of efficiency [ 64 ]. Eventually, differential tissue regenerative and inflammatory modulating properties related to EV-miRNAs emerged comparing ASCs and hAMSCs [ 41 ]. It should be noted that all these approaches and studies relied on the quantification and comparison of molecules between different donors, tissue sources or conditions.…”

“…According to the literature, 14 miRNAs and 1 small RNA (U6 snRNA) were selected for stability analysis and optimal reference gene (RG) identification ( Table 1 ) [ 30 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]: has-let-7d-5p 002283; hsa-miR-16a-5p 000391; has-miR-22-5p 002301; hsa-miR-23a-3p 000399; has-miR-24-3p 000402; hsa-miR-26a-5p 000405; has-miR-29a-5p 002447; hsa-miR-34a-5p 000426; hsa-miR-101-3p 002253; hsa-miR-103a-3p 000439; hsa-miR-221-3p 000524; hsa-miR-423-5p 002340; hsa-miR-425-5p 001516; has-miR-660-5p 001515; U6 snRNA 001973. The following assay was selected for OA-related miRNA analysis ( Table 1 ) [ 44 ]: has-miR-193b-5p 002366.…”

Donor Sites and Harvesting Techniques Affect miRNA Cargos of Extracellular Vesicles Released by Human Adipose-Derived Mesenchymal Stromal Cells

“…Collection of abdominal adipose tissue by lipoaspiration and peri-trochanteric adipose tissue by surgical excision were previously described [ 14 , 15 ]. In these two patient groups, adipose tissue was collected as waste material during regenerative medicine procedures based on fat-derived products and during total hip arthroplasty, respectively.…”

“…The present study aimed to analyze the composition of miRNA cargos of EVs released by different human ASCs to investigate the possible impact of the site and the methodology of adipose tissue-harvesting. The miRNA content of ASC-EVs isolated from adipose tissue was obtained through direct excision of peri-trochanteric (hip) adipose tissue (H-ASC) or from abdominal adipose tissue, either excised surgically (A-ASC) or by lipoaspiration (LipoA-ASC), using identical experimental approaches and platforms [ 14 , 15 ]. MiRNA expression profiles were analyzed by bioinformatic tools for miRNA–mRNA interactions using expression data from OA-affected tissues and cells.…”