Introduction

Mesenchymal stromal cells (MSCs) are an emerging therapeutic tool for treatment of osteoarthritis (OA) with recent systematic reviews of early stage clinical trials for knee OA with data from over 500 patients indicating safety and some efficacy with modulation of pain^1,2. Historically, the initial rationale for MSC use in osteoarthritis (OA) was based on their ability to undergo chondrogenic differentiation. However, we first demonstrated that MSCs do not engraft to the degenerating cartilage of the caprine knee joint post medial meniscectomy and anterior cruciate ligament transection but were detected in regenerated meniscal tissue and synovium³ with associated cartilage protection. This and other studies suggested that the engrafted cells might act to secrete factors that contribute to tissue repair. However, the underlying mechanism of action following intra-articular (IA) MSC injection has not been elucidated.

Content

In an effort to address this lack and define how MSCs act to modulate development or treatment of knee OA, MSCs were retrieved from the joint after IA delivery in a murine collagenase induced osteoarthritis (CIOA) model. The retrieval was performed at times representing early stage or acute OA as well as at a later time with established cartilage degradation in joints. Briefly, CIOA was induced in C57BL/6 mice and syngeneic mouse GFP+ bone marrow derived MSCs (BM-MSCs) were injected into knee joints at days 14 and 56, representing the early acute and established OA joint with cartilage degradation confirmed respectively. For cell retrieval, mice were sacrificed three days following IA-injection and knee joints digested in collagenase type I for six hours before undergoing FACS-coupled cell sorting. Each sample was compared to GFP+ BM-MSCs retrieved from SHAM joints, where the knee joints were injected with saline. Retrieved cells were pooled for analysis of their molecular profile by RNA sequencing. BM-MSCs, licensed in vitro with a single dose of IL-6 (50ng/ml) and a combination of interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and interferon gamma (IFN- γ) each at 50ng/ml were also included as in vitro controls for this analysis. After treatment for 72 hours, these cells were also processed for RNA sequencing. In silico analysis was performed to identify the transcriptome profile of retrieved MSCs in relation to the disease stage. Major canonical pathways and the predicted secretome were also analysed to determine relevant biological processes and functions that can contribute to the ability of MSC to modulate development of OA.

Differentially upregulated genes (DEGs) associated with early retrieval or the initial response of MSCs to joint injury (Day 14) were associated with cell survival, cell proliferation and regulation of apoptosis and autophagy, whereas in late OA-retrieved cells the gene pattern indicated a strong immunomodulatory profile with an increase of genes involved in the complement cascade, secretion of cytokines and M2 macrophage markers. In terms of the predicted secretome, 157 elements were identified as associated with early OA with 278 differentially regulated in response to the established OA environment. The largest cluster for 14D retrieved cells was enriched for metabolism and extracellular matrix organization, whereas immune response and cytokine-cytokine interactions-related genes were dominant in 56D retrieved cells. The Wnt Signalling Pathway was enriched at both time points, with Wnt antagonists SFRP2 and SFRP4 common elements.

In summary, this data characterized the transcriptome profile of therapeutically licensed MSCs exposed to an in vivo OA environment. As a higher number of cells were retrieved from CIOA induced joints compared to SHAM controls, indicating that the inflammatory environment seems to promote cell survival. The surviving cells also responded differently to the disease stage with suggesting that the therapeutic response of MSCs is strongly influenced by the environment to which the cells are delivered. Early stage OA is characterized by an imbalance of the extracellular matrix between anabolic and catabolic responses, therefore it is most likely that the initial response driven by MSCs will be targeting to counteract these processes. In the later disease stage where OA has been established with associated changes to the immune environment, MSCs are driven to express genes mostly involved in immune processes, suggesting a strong immunomodulatory action. However, the molecular response of the surviving MSCs at this time point also suggests potential for cartilage protection and other disease-modifying responses.

References

1. Song et al., J Orthop Translat. 2020 Sep; 24: 121–130 (https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7452318/)

2. Yubo et al., PLoS One. 2017 Apr 27;12(4):e0175449 (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175449

3. Murphy et al., Arthritis Rheum 2003 48:3464-74. (https://pubmed.ncbi.nlm.nih.gov/14673997/)

Acknowledgments

Science Foundation Ireland (SFI), the European Regional Development Fund and Abbvie (Grant Number 13/RC/2073)