Osteoarthritic Milieu Affects Adipose‐Derived Mesenchymal Stromal Cells

Abstract

The objective of this study was to define the effects of osteoarthritic (OA) milieu on good manufactured practice-adipose-derived mesenchymal stromal cells (GMP-ASC) that are commonly utilized in cell therapies. Two different OA milieu: OA synovial fluid (SF) and OAconditioned medium (CM) from synoviocytes were used to treat GMP-ASC both in normoxia or hypoxia. GMP-ASC were tested for cell migration, proliferation, cytokine receptors expression (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, CCR1, CCR2, CCR3, CCR5, IL6R), and cytokines (CXCL8/IL8, CXCL10/IP10, CXCL12/SDF-1, CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, IL6) release. Healthy SF was used as controls. We demonstrated that GMP-ASC show an increase in proliferation, migration, and modulation of CXCR1, CXCR3, CCR1, and CCR5 receptors in hypoxic condition. Moreover, GMP-ASC migration increased 15-fold when treated either with OA-SF or OA-CM compared with healthy SF both in normoxia and hypoxia. GMP-ASC treated in both OA milieu showed an increase in CXCR3, CCR3, and IL6R and a decrease in CCR1 and CCR2 receptors. In OA-SF, we detected higher amount of CXCL10/IP10 than in OA-CM, while CCL2/MCP1 and CCL4/MIP1β were higher in OA-CM compared with OA-SF. CXCL10/IP10 was the only chemokine of the OA milieu, which was downmodulated after treatment with GMP-ASC. In conclusion, we demonstrated specific effects of OA milieu on both GMP-ASC proliferation, migration, and cytokine receptor expression that were strictly dependent on the inflammatory and hypoxic environment. The use of characterized OA milieu is crucial to define the therapeutic effect of GMP-ASC and indicates that CXCL10/IP10-CXCR3 axis is partially involved in the GMP-ASC effect on synovial macrophages.

The Exposure to Osteoarthritic Synovial Fluid Enhances the Immunomodulatory Profile of Adipose Mesenchymal Stem Cell Secretome

“…Indeed, our data demonstrated and confirmed [22,45] the presence of proinflammatory molecules in osteoarthritic synovial fluid. As already reported [29][30][31], osteoarthritic synovial fluid influences the expression of molecules involved in immunomodulation. Furthermore, Sayegh et al recently demonstrated that coculture of AMSCs exposed to SF of patients affected by rheumatoid arthritis (RA), with activated monocyte or CD4 + cells, can inhibit the expression of CD40 and CD80 (monocyte's proinflammatory markers) and promote T reg expansion [46].…”

“…Although it is well known that the use of stem cells contributes to better clinical outcomes in OA patients, the molecular mechanisms responsible for the clinical findings have not been clarified. Recently, it has been reported that the exposure of MSCs to the articular microenvironment, represented by the osteoarthritic synovial fluid, could modulate some of the stem cells' properties such as proliferation, migration, cytokine receptor expression, cytokine secretion, and inhibition of lymphocyte proliferation [29][30][31].…”

Cartilage Protective and Immunomodulatory Features of Osteoarthritis Synovial Fluid-Treated Adipose-Derived Mesenchymal Stem Cells Secreted Factors and Extracellular Vesicles-Embedded miRNAs

“…In fact, most of the OA model was created by single cytokine supplementation in the ng/mL range in spite of several factors in the pg/mL range that are known to characterize OA-SF [ 20 ]. To date, only a few reports have described the ASC response after direct interaction with OA-SF, confirming their ability to inhibit T cell proliferation and promote T reg expansion, induce macrophage polarization into the M2-like phenotype and reduce monocyte differentiation into mature dendritic cells [ 21 , 22 , 23 , 24 ]. By preliminary analyses on a reduced number of cytokines, these effects were postulated to rely on specific ASC secreted factors (secretome) in the presence of OA-SF.…”

Impact of Isolation Procedures on the Development of a Preclinical Synovial Fibroblasts/Macrophages in an In Vitro Model of Osteoarthritis

“…Forward and reverse oligonucleotides for PCR amplification of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), CD80, CD86, CD163, CD206, IL6,TNFα, CCL3/MIP1α, CXCL10/IP10, IL10, TGFβ1, and CCL18 are described in Table S1 and real time PCR was carried out as previously described [ 36 ]. All primer efficiencies were confirmed to be high (>90%) and comparable ( Table S1 ).…”