Magnetic Ion Channel Activation (MICA)-Enabled Screening Assay:A Dynamic Platform for Remote Activation of Mechanosensitive Ion Channels

This study reports results of a mechanical platform-based screening assay (MICA) to evaluate the remote activation of mechanosensitive ion channels. Here, we studied ERK pathway activation and the elevation in intracellular Ca2+ levels in response to the MICA application using the Luciferase assay and Fluo-8AM assay, respectively. Functionalised magnetic nanoparticles (MNPs) targeting membrane-bound integrins and mechanosensitive TREK1 ion channels were studied with HEK293 cell lines under MICA application. The study demonstrated that active targeting of mechanosensitive integrins via RGD (Arginylglycylaspartic acid) motifs or TREK1 (KCNK2, potassium channel subfamily K member 2) ion channels can stimulate the ERK pathway and intracellular calcium levels compared to non-MICA controls. This screening assay offers a powerful tool, which aligns with existing high-throughput drug screening platforms for use in the assessment of drugs that interact with ion channels and influence ion channel-modulated diseases

Therapeutic benefit for late, but not early, passage mesenchymal stem cells on pain behaviour in an animal model of osteoarthritis

Background: Mesenchymal stem cells (MSCs) have a therapeutic potential for the treatment of osteoarthritic (OA) joint pathology and pain. The aims of this study were to determine the influence of a passage number on the effects of MSCs on pain behaviour and cartilage and bone features in a rodent model of OA. Methods: Rats underwent either medial meniscal transection (MNX) or sham surgery under anaesthesia. Rats received intra-articular injection of either 1.5×106 late passage MSCs labelled with 10 μg/ml SiMAG, 1.5×106 late passage mesenchymal stem cells, the steroid Kenalog (200 μg/20 μL), 1.5×106 early passage MSCs, or serum-free media (SFM). Sham-operated rats received intra-articular injection of SFM. Pain behaviour was quantified until day 42 postmodel induction. Magnetic resonance imaging (MRI) was used to localise the labelled cells within the knee joint. Results: Late passage MSCs and Kenalog attenuated established pain behaviour in MNX rats, but did not alter MNX-induced joint pathology at the end of the study period. Early passage MSCs exacerbated MNX-induced pain behaviour for up to one week postinjection and did not alter joint pathology. Conclusion: Our data demonstrate for the first time the role of a passage number in influencing the therapeutic effects of MSCs in a model of OA pain

Author Correction: Ultra Short Echo Time MRI of Iron-Labelled Mesenchymal Stem Cells in an Ovine Osteochondral Defect Model

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Author Correction: Ultra Short Echo Time MRI of Iron-Labelled Mesenchymal Stem Cells in an Ovine Osteochondral Defect Model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Highly efficient delivery of functional cargoes by the synergistic effect of GAG binding motifs and cell-penetrating peptides

Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application

The use of MRI and MNP to image and track cells in vivo for arthritic cell-based therapies

Cell-based therapies have been proposed as novel approaches to treating Osteoarthritis and Rheumatoid Arthritis. A non-invasive means of monitoring cell populations, post implantation could prove valuable in the clinical translation of such therapies. We propose the use of superparamagnetic iron oxide nanoparticles (SPIONs; internalised by cell populations) with magnetic resonance imaging (MRI) to image and track cell populations in vivo. We investigate the potential of commercially available SPIONs (SiMAG, Lumirem, Nanomag and P904) as potential labelling/tracking agents for in vivo investigations. Human mesenchymal stem cells (hMSC) and porcine chondrocytes were labelled with SPIONs under passive incubation conditions in either serum free media or serum containing media (24 hrs). SiMAG (10 μgFe/ml) demonstrated greatest potential with highest comparative internalised Fe content (labelled in serum free media) in vitro. SPION-labelled cell population maintained viability and proliferative capacity apart from SiMAG-labelled chondrocytes (10 μgFe/ml). Furthermore, SiMAG-labelled hMSC populations demonstrated successful differentiation down mesodermal lineages and retained key cell surface markers. MRI visibility thresholds were investigated (in vitro and ex vivo). Dose dependant contrast was generated only by SiMAG-labelled populations in vitro when MR imaged. In vitro minimum visibility of SiMAG-labelled populations was influenced by various ex vivo tissues with similar contrast developing in muscle and fat tissue samples but not for ligament. Finally, an ex vivo model of articular cartilage damage confirmed the potential clinical application of SPIONS as cell tracking agents at optimised conditions (cell dosage and SiMAG concentration) using a clinical system. An AIA murine model (Rheumatoid Arthritis) and a MNX rat model (Osteoarthritis) were implemented to image and track implanted SiMAG-labelled MSCs (murine) in vivo for 7 and 29 days, respectively. Additionally, clinically relevant functional outcomes were also monitored. Relevant in vitro assessment was performed where mMSCs efficiently internalised SiMAG with no impairment on cell activity. Good contrast was generated in both studies with SiMAG-labelled cell population located within the synovial cavity after 7 days (mouse study) and 29 days (rat study) by MRI. Administration of MSCs significantly reduced joint swelling in the mouse study without influence from the presence of SiMAG. mMSCs significantly influenced weight bearing asymmetry with little influence on paw withdraw threshold indicating potential antinocieotive properties of MSCs. In summary, SiMAG has demonstrated great potential as a labelling and tracking agent to be implemented for imaging and tracking cell populations

Autonomous magnetic labelling of functional mesenchymal stem cells for improved traceability and spatial control in cell therapy applications

Mesenchymal stem cells (MSCs) represent a valuable resource for regenerative medicine treatments for orthopaedic repair and beyond. Following developments in isolation, expansion and differentiation protocols, efforts to promote clinical translation of emerging cellular strategies now seek to improve cell delivery and targeting. This study shows efficient live MSC labelling using silica-coated magnetic particles (MPs), which enables 3D tracking and guidance of stem cells. A procedure developed for the efficient and unassisted particle uptake was shown to support MSC viability and integrity, while surface marker expression and MSC differentiation capability were also maintained. In vitro, MSCs showed a progressive decrease in labelling over increasing culture time, which appeared to be linked to the dilution effect of cell division, rather than to particle release, and did not lead to detectable secondary particle uptake. Labelled MSC populations demonstrated magnetic responsiveness in vitro through directed migration in culture and, when seeded onto a scaffold, supporting MP-based approaches to cell targeting. The potential of these silica-coated MPs for MRI cell tracking of MSC populations was validated in 2D and in a cartilage repair model following cell delivery. These results highlight silica-coated magnetic particles as a simple, safe and effective resource to enhance MSC targeting for therapeutic applications and improve patient outcomes