Culture enhanced mesenchymal stromal cells with increased potency

Abstract

Background & Aim Mesenchymal stromal cells (MSCs) have been pursued for treatment of many inflammatory and fibrotic diseases due to their capacity to modulate immune cell (such as monocyte/macrophage, MΦ) phenotype and to dampen fibrosis. With the goal of developing an MSC product with increased potency, the Viswanathan lab has pioneered a strategy to generate 3D MSC aggregates with enhanced immunomodulatory functions (US62/397,572). The objective of this work is to assess the ability of culture enhanced MSCs to reduce inflammation and fibrosis, and to investigate mechanism of action by probing extracellular vesicle (EV) release and microRNA (miR) expression in 3D versus 2D MSCs. In parallel, hypoxic MSC cultures will also be investigated. Methods, Results & Conclusion MSCs were isolated from human bone marrow and subject to a serum-free 3D cell aggregation protocol (US62/397,572) with conventional 2D culture in serum-free medium or with 10% fetal bovine serum serving as controls. To evaluate MSC potency, gene expression analysis (quantitative PCR) was performed on MSCs licensed with tumour necrosis factor-α (TNFα) or interferon-γ (IFNγ) for 24 h. Results showed that TNFα-licensed 3D MSCs displayed highest expression of the anti-inflammatory marker TNFα-stimulated gene 6 ( TSG6 ) (Fig. 1) which has been correlated to MSC anti-inflammatory function. IFNγ-licensed 3D MSCs displayed reduced transcript levels of pro-inflammatory markers chemokine (C-X-C motif) ligand 8 ( CXCL8 ) and cyclooxygenase-2 ( COX2 ), as well as the pro-fibrotic marker transforming growth factor-β ( TGFβ ). Co-culture of MSCs with human peripheral blood-derived monocytes demonstrated that MSCs promote a shift toward a more homeostatic “M2-like” MΦ phenotype with higher expression of CD163 and CD206 relative to M1 markers CD86 and HLA-DR measured by flow cytometry (Fig. 2). A trend toward increased expression of CD163 was observed in 3D MSC co-cultures relative to serum 2D MSCs (p=0.0802) but no other statistical differences were noted. Taken together, these results demonstrate that 3D MSCs may have improved immunomodulatory capacity and are less fibrotic and inflammatory relative to MSCs cultured in 2D. In ongoing work, we are evaluating EV release and miR expression in enhanced MSCs (3D and hypoxic cultures) and will examine the role of these factors in mediating MΦ polarization and fibroblast phenotype in vitro . The results will aid in developing MSC therapies with increased potency for treatment of inflammatory and fibrotic conditions.