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"INTRODUCTION Therapeutic application of mesenchymal stromal cells (MSCs) has been suggested as a promising regenerative treatment for osteoarthritis (OA) by virtue of their paracrine-mediated chondroprotective and immunomodulatory activity. Nonetheless, collection of MSCs is an invasive procedure and the therapeutic efficacy of autologous MSCs are subject to large variability due to the varying potency of individual MSCs.1 In the current study we therefore set out to explore human induced pluripotent stem cell (hiPSC) derived MSCs (hiMSCs) as sustainable cell sources for OA stem-cell treatment by identifying and characterizing the therapeutic OA-secretome of hiMSC that contribute to a beneficial chondrocyte state.
METHODOLOGY An established protocol was applied to generate hiMSCs with high similarity to hBMSCs.2 To mimic the inflammatory environment, generated hiMSCs and hBMSCs were exposed to different cytokines (IL6 or a combination of TNFα and IFNγ). RNA sequencing was performed to determine the secretome of licensed hiMSCs (N=6). Data were analyzed in R using DESeq2 package while considering significant differentially expressed genes (DEGs) in comparison to unlicensed controls when FDR<0.05. Luminex was applied to quantify cytokine levels in cell culture media. Effects of licensed hiMSCs on human primary articular chondrocytes (hPACs; N=3, RAAK study) were determined by RT-qPCR following 3 days of co-culture in transwells.
RESULTS Cell licensing with IL6 resulted in modest changes in gene expression effect sizes and 5 FDR-significant genes. Of note among these genes was SOCS3 encoding suppressor of cytokine signaling 3 (FC=2.0, FDR=2.2x10-2). In contrast, TNFα+IFNγ showed substantial differences as compared to unlicensed cells. Particularly, with respect to well-known immunomodulatory genes such as IDO1 (over 1000-fold induction), MCP1, and HLA-DRA (both around 100-fold induction). Likewise, we demonstrated significant increase of secreted cytokines in the culture media of TNFα+IFNγ exposed hiMSCs. The changes in licensed secretome of hiMSCs were highly comparable to those in hBMSCs, independent of the licensing factor applied, suggesting that the therapeutic mode of action is the same.
Effects of hiMSCs licensed with either IL6 or TNFα+IFNγ were explored in co-cultures with hPACs. The inverse effects of both licensing methods on chondrocytes with respect to expression of COL2A1 and OA markers MMP13, ADAMTS5, CD55 and IL11 suggested that IL6-licensed hiMSCs exerted direct pro-chondrogenic activity.
CONCLUSION Our results demonstrate that hiPSC-derived MSCs should be considered promising candidates as stable source for application in cell therapy. Particularly, IL6 licensed hiMSCs showed direct chondroprotective effects as reflected by decrease in OA markers. Transcriptome wide analyses revealed SOCS3 as a candidate effector. Further studies will address whether, in vivo, this may result in additional beneficial effects by virtue of its immune-suppressive activity.
In line with previously determined similarities between characteristics of our hiMSCs and hBMSCs, the current study demonstrates potential of iPSC-derived MSCs in response to inflammatory environment. Findings pave the way to further explore their application in the clinic as off-the-shelve cell source to treat osteoarthritis.
References
1. Barry et al, J Orthop Res 37, 1229-1235 (2019)
2. Rodriguez-Ruiz et al, Cell Tissue Res 386, 309-320 (2021)"
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