A culture engineering strategy to enhance mesenchymal stromal cells for treatment of osteoarthritis

Abstract

Purpose: Osteoarthritis (OA) is a debilitating disease associated with chronic inflammation, metabolic dysregulation, and progressive cartilage degradation in the joint. Mesenchymal stromal cells (MSCs) have been investigated as a promising therapy for the disease; however, insufficient in vivo potency and MSC donor variability can limit their efficacy. To address this, the Viswanathan lab has pioneered a proprietary culture engineering strategy to culture MSCs in 3D aggregates to augment their anti-inflammatory and immunomodulatory functions (US62/397,572). The objective of this work is to assess the ability of 3D MSCs to reduce inflammation, fibrosis, and cartilage degradation relative to conventional 2D culture controls within in vitro and in vivo OA models. Methods: MSCs were isolated from human bone marrow (REB# 14-7909) and subject to a proprietary 3D cell aggregation protocol under serum-free conditions (US62/397,572) with conventional 2D culture in serum-free medium or with 10% fetal bovine serum serving as controls. As a standard assessment of MSC potency, licensing experiments were performed by stimulating the MSCs with the pro-inflammatory cytokines tumour necrosis factor-alpha (TNFa; 10 ng/mL for 24 h) or interferon-gamma (IFNg; 30 ng/mL for 24 h) and OA-related gene expression was evaluated by quantitative PCR. Lastly, co-culture experiments were performed using MSCs and human peripheral blood monocytes (REB#14-7483), and flow cytometry was used to assess the effects of MSCs on the expression of monocyte/macrophage phenotypic markers after 48 h of culture. In ongoing work, we are investigating extracellular vesicle release (isolated by ultracentrifugation) and microRNA expression (profiled through microRNA-sequencing) in 2D versus 3D MSCs and will examine the role of these factors in mediating monocyte polarization and synovial fibroblast proliferation/migration in vitro. Results: After licensing with TNFa, 3D MSCs (serum-free 3D culture group) displayed higher transcript levels of the anti-inflammatory marker TNFa-stimulated gene 6 (TSG6) versus serum-free 2D and serum 2D controls by 6.5-fold and 12-fold, respectively, despite somewhat lower basal levels observed in unstimulated serum-free 3D MSCs (N¼2 MSC donors, n¼3 technical replicates/condition; Fig. 1A). In IFNglicensing experiments, 3D MSCs displayed reduced transcript levels of the 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 beta (TGFb) (N¼2, n¼3; Fig. 1B). Highest expression of the anabolic joint-lubricating marker proteoglycan 4 (PRG4) was observed in the serum-free 2D condition but serum-free 3D MSCs displayed higher mRNA levels relative to serum 2D controls, while no differences in the expression of the anti-inflammatory genes indoleamine 2,3-dioxygenase 1 (IDO) and TSG6 were observed across groups. As expected, co-culture of MSCs with monocytes promoted a shift toward a more homeostatic "M2-like" monocyte/macrophage phenotype ...

From Vial to Vein: Crucial Gaps in Mesenchymal Stromal Cell Clinical Trial Reporting

“…MSCs are fundamentally responsive to subtle changes in their environment. MSCs respond to changes in atmospheric gases ( Lin et al, 2014 ; Gorgun et al, 2021 ; Roemeling-Van Rhijn et al, 2013 ; Ejtehadifar et al, 2015 ; Kang et al, 2019 ; von Bahr et al, 2019 ), temperature ( Stolzing et al, 2006 ; Kubrova et al, 2020 ; Shimoni et al, 2020 ), hydrostatic pressure ( Steward et al, 2012 ; Becquart et al, 2016 ; Pattappa et al, 2019 ) and aggregation ( Robb et al, 2019 ; Yuan et al, 2019 ; Burand et al, 2020 ; Xie et al, 2021 ). It is surprising then, that the steps and duration between dose preparation and delivery of MSC therapies are ill-defined and under-reported.…”

Research progress of engineered mesenchymal stem cells and their derived exosomes and their application in autoimmune/inflammatory diseases

“…To improve the efficacy of MSCs for the treatment of OA, the Viswanathan lab pioneered an engineering strategy for culturing MSCs in 3D aggregates. The results showed that 3D MSCs reduced inflammation, fibrosis and cartilage degradation in both in vitro and in vivo models of OA compared to MSCs cultured using conventional 2D methods [ 138 ]. Furthermore, Brian et al isolated MSCs from the knee joint synovial fluid of three OA patients, epigenetically reprogrammed them, and then induced differentiation to establish a reprogrammed MSC (Re-MSC) line.…”

Mesenchymal Stromal Cell-Based Therapy—An Alternative to Arthroplasty for the Treatment of Osteoarthritis? A State of the Art Review of Clinical Trials

“…Increased complexity and non-standardized expansion protocols, alongside higher costs should be taken into consideration, when applying 3-D expansion techniques. However, reports about increased immunomodulatory and chondrogenic potential of MSCs, cultured in 3-D compared to conventional 2-D cultures, point towards promising technical developments in the field of tissue engineering for OA treatment [ 61 , 62 ]. The stromal vascular fraction (SVF) is a combination of ATMSCs, endothelial cells, growth factors, precursor cells, macrophages, t-regulatory cells, lymphocytes and others.…”