Mesenchymal stem cell therapy for osteoarthritis: how apoptotic cells modulate inflammation

Abstract

Purpose: Mesenchymal stem cells (MSCs) enhance regeneration when locally delivered in pre-clinical models of osteoarthritis (OA). Their mechanism of action, however, is still poorly understood. Preliminary data show low engraftment of GFP-expressing syngeneic MSCs in mouse knee joints, consistent with published data using molecular techniques. We hypothesise that implanted cells undergo apoptosis or cell death. There is growing evidence that apoptotic MSCs exert an antiinflammatory effect, by release of immunosuppressive cytokines and, indirectly, through phagocytes. This study aims to investigate the immunomodulatory potential of apoptotic MSCs in vitro, to correlate with in vitro results in a mouse model of OA. Methods: Collagenase-induced OA (CIOA) was performed on C57BL/6 mice and 2x10 5 GFP-expressing MSCs were intra-articularly (IA)-delivered in the animals. Knee joints were harvested 3 days post-IA-injection and digested into a single cell suspension before sorting implanted GFPþ MSCs using the BD FACS Aria II. Retrieved cells were expanded in culture and characterised by cell surface markers and differentiation. Their immunosuppressive capacity was tested in vitro on murine lymphocytes and bone marrow-derived macrophages (BMDM). Apoptosis of MSCs was induced in vitro by treatment with Staurosporine (STS), while necrosis was produced by heat treatment (90 C, 5 min). Apoptosis was validated by pre-treating the cells with the pancaspase inhibitor Q-VD-OPH. All treatments were evaluated by flow cytometry using anti-Annexin V/Sytox Blue staining; activation of caspases was measured by FLICA assays. Apoptotic MSCs were co-cultured with activated murine splenocytes for 3 days. Suppression of proliferation was measured by quantification of Cell Trace Violet and expression of CD25. Conditioned media from apoptotic and necrotic MSCs were tested on LPS-activated BMDM and expression of pro-and anti-inflammatory molecules was assessed by ELISA (IL-6, IL-10, TGF-b, PGE2) and Griess assay (NO). Results: On average, 1.63% of the injected cells were successfully retrieved by cell sorting, with 82.1% sample purity. Sorted cells proliferated in culture retaining MSC-like morphology, expression of cell surface markers and differentiation potential. Retrieved cells have an increased immunosuppressive potential as observed on activated BMDMs and lymphocytes. STS successfully induced apoptosis, with activation of Caspase 3/7; Q-VD-OPH completely inhibited apoptosis, confirming the involvement of caspases. Necrotic, but most strikingly apoptotic MSCs, significantly prevented the proliferation and activation of murine splenocytes (Fig. 1). BMDM were also induced towards an anti-inflammatory phenotype, as shown by their secretory profile. Conclusions: MSCs can be administered and retrieved from murine knee joints and remain viable for characterisation. Retrieval yield is low, consistent with previous studies. IA-injected MSCs may contribute to local modulation of joint inflammation. Engrafted MSCs retrieved from the OA joint were ...

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“…One can consider inhibiting NO to prolong MSC survival, noting that it also will restrict the immunosuppression capacity of MSCs on the lymphocytes. Interestingly, Mancuso et al's study of knee osteoarthritis using an in vitro model revealed that apoptotic MSCs were more immunosuppressive than healthy MSCs [63]. Moreover, Chang et al SDF-1: stromal cell-derived factor 1; SCF: stem cell factor; IL-6: interleukin-6; VEGF: vascular endothelial growth factor; bFGF: basic fibroblast growth factor; PDGF: platelet-derived growth factor; ROS: reactive oxygen species; RNS: reactive nitrogen species; EGF: epidermal growth factor; HGF: hepatocyte growth factor; NGF: nerve growth factor; TGF-β: transforming growth factor-beta; LIF: leukemia inhibitory factor; NO: nitric oxide; CCL: C-C motif chemokine ligand (created with BioRender.com).…”

Magnetic Microcarriers with Accurate Localization and Proliferation of Mesenchymal Stem Cell for Cartilage Defects Repairing

“…BMSCs secrete various bioactive factors and stromal molecules to regulate the damaged tissue environment and direct subsequent regenerative processes, including cell migration, proliferation, and differentiation . Research has shown that MSCs transplantation can boost the microenvironment of the regenerated injured site, accelerating tissue repair and regeneration. − Ando et al generated a MSC-based tissue-engineered construct (TEC) to facilitate in vivo repair in a porcine chondral defect model; repaired tissue after TEC implantation exhibited levels of chondrogenic differentiation and integration similar to those in immature porcine experiments here. Zhang et al studied that BMSCs enhanced articular chondrocyte proliferation and migration, anabolic gene expression in the extracellular matrix, especially chondrocytes, and articular cartilage generation in vivo .…”