G. Kaur (Ann Arbor, US)

University of Michigan Biomedical Engineering
PhD student in cartilage healing and regeneration (CHaR) lab in Biomedical Engineering; University of Michigan. I am interested in utilizing the principles of tissue engineering; synthetic biology and immunology to develop mesenchymal stem cell based cartilage regeneration therapies under inflammation.

Presenter Of 1 Presentation

Stem Cells

P-12.2.8 - Utilizing Auto-Regulated RUNX2 Suppression in Mesenchymal Stem Cell Derived Chondrocytes to Attenuate Knee Joint Inflammation

Abstract

Purpose

Articular cartilage has limited healing capacity and human mesenchymal stem cell (hMSC)-based cartilage injury/defect repair is impaired by the inflammatory knee microenvironment, enacted predominantly by M1 macrophages. Matrix metalloproteinases (MMPs) and inflammatory cytokines released by M1 macrophages induce cartilage degradation, suppress hMSC chondrogenesis and induce RUNX2 expression in hMSC-derived chondrocytes (hMdChs), which further upregulates MMP expression. hMdChs are also induced to produce additional inflammatory cytokines, establishing pro-inflammatory crosstalk between M1 macrophages and hMdChs. We hypothesized that RUNX2 suppression in hMdChs can prevent inflammation induced cartilage degradation and disrupt hMdCh-M1 Macrophage crosstalk. We utilized a synthetic gene circuit inducing tunable autonomous suppression of RUNX2 in hMdChs and a co-culture model mimicking the joint inflammatory environment, to test our hypothesis.

Methods and Materials

Conditioned media(M1CM) was collected from THP-1 derived M1 macrophages. Unmodified hMSCs (WT) and hMSCs containing gene circuits expressing short-hairpin RNA for RUNX2 (sRX2) or a scrambled sequence (Scr) were placed into chondrogenic pellet culture (10ng/ml TGF-β) for 21 days. 21D pellets were treated with M1CM for 72 hours and their double conditioned media (DCM) was used to treat M1 macrophages for an additional 72 hours. RUNX2 activity and matrix turnover in MdCh pellets were analyzed using luciferase reporter and DMMB assays. Control and DCM treated M1 macrophages were evaluated for inflammatory and anti-inflammatory markers using immunofluorescence and RNAseq.

Results

sRX2-hMdChs suppressed RUNX2, which reduced inflammation-induced cartilage matrix loss upon M1CM treatment (Fig.1). sRX2-hMdCh-DCM treatment of M1 macrophages reduced inflammatory marker expression and increased anti-inflammatory marker expression, indicating a phenotype shift (Fig.2).

p-12.2.8.png

Conclusion

We established a novel role of RUNX2 in mediating pro-inflammatory hMdCh-M1 macrophage crosstalk and showed that RUNX2 suppression in hMdChs prevents inflammation induced matrix loss and disrupts this crosstalk to attenuate macrophage inflammation. We demonstrate that auto-regulated gene circuits can modulate MdCh-macrophage interactions for creating a conducive environment for MSC-based cartilage repair.
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