Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts

Abstract

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.

Mechanisms of stem cell based cardiac repair-gap junctional signaling promotes the cardiac lineage specification of mesenchymal stem cells

“…The ability of MSCs to participate in the gap junctional transfer of miRNA was previously reported for other co-culture systems, e.g. cancer cells 48 – 50 . Moreover, cardiogenic miRNAs, like miR-499 and miR-133 have been demonstrated to stimulate the cardiac differentiation of MSCs, including up-regulation of NKX2.5 and GATA-4 51 , 52 .…”

Communication of cAMP by connexin43 gap junctions regulates osteoblast signaling and gene expression

“…Whole cell extracts were prepared using modified RIPA buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 10 mM β-glycerophosphate, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 1X HALT protease and phosphatase inhibitor cocktail (Thermo Scientific) as described (31,32). Equal amounts of cell lysates were electrophoresed on SDS-PAGE gels and transferred to PVDF membranes.…”

Novel multi-functional fluid flow device for studying cellular mechanotransduction

“…Blots were probed for phospho- or total ERK1/2 (Cell Signaling Technologies), phospho- or total- pan-PKC (Cell Signaling Technologies), and GAPDH (Millipore), as described (Niger et al, 2012; Niger et al, 2010). Western blots were quantitated using ImageJ, as described (Liu et al, 2015). …”