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Hijacking cell signalling pathways with magnetic nanoactuators for remote-controlled stem cell therapies of neurodegenerative disorders

Periodic Reporting for period 3 - MAGNEURON (Hijacking cell signalling pathways with magnetic nanoactuators for remote-controlled stemcell therapies of neurodegenerative disorders)

Reporting period: 2018-07-01 to 2019-12-31

Neurodegenerative disorders (ND) are expected to surpass cancer as the most common group of medical conditions by 2040. A prominent example of ND is Parkinson’s disease (PD), a medical condition that already affects 6.5 million people worldwide, a number expected to more than double by 2030. Curative treatment of PD and ND is considered as one of the great challenges to medical science and alternative medical routes needs to be devised since current therapies are not yet satisfactory.
Among these, cell replacement therapy (CT) is considered the most promising approach. It consists of transplanting cells into the brain to replace degenerating neurons. The transplantation of DA neurons derived from developing embryos into PD patients has already been tested in clinical trials. Despite promising results, practical, medical and ethical concerns restrict the use of human fetal tissue. With the advent of stem cell reprogramming technologies, induced pluripotent stem cells (iPS) generated from the patient’s own cells have thus emerged as a promising alternative source of DA. Yet, there are still serious obstacles for replacement therapy using iPS-derived neurons. Furthermore, precursor neurons transplanted into the brain region where neuronal cell bodies degenerate have to grow long distances to achieve re-innervation and functional repair.
The MAGNEURON project aims at developing a novel technology for magnetic actuation of cellular functions to treat ND. The innovative concept of our technology is to remote-control cellular signaling pathways by means of biofunctionalized magnetic nanoparticles (bMNPs) engineered to function as intracellular signaling nanoplatforms. Once delivered into the cytoplasm of target cells, bMNPs can activate signaling pathways in response to external magnetic fields in either a spatial and/or a temporal mode.
With this novel technology, our long-term goal is to fundamentally advance cell therapies and regenerative medicine in the brain by remote-controlling the differentiation and oriented growth of transplanted cells. In the context of PD, we will focus on the control of signaling pathways known to promote the survival, differentiation and growth of transplanted DA precursor neurons. The key objectives of our project are: (i) the development of the chemical, biochemical and physical tools for the magnetic control of signaling in stem cells and neuronal cells, (ii) the demonstration of magnetic actuation of differentiation of DA precursor neurons and guidance of DA neurites in single-cell in vitro assays and (iii) the application of the technology with cells transplanted into organotypic brain slices and rodents.
"Over the time course of the project, we have generated two generations of synthetic and protein-based magnetic nanoparticles (MNPs) with well-controlled magnetic and colloidal properties that meet the trade-off between their magnetic response and their intracellular mobility required for spatial manipulation in cells. Proof-of-concept experiments were achieved using the “space mode” to demonstrate magnetic activation of Rac1 and Cdc42 signaling in different cell lines. We also achieved spatial control of the HRAS signaling. We have generated human iPS derived dopaminergic neurons and successfully demonstrated delivery and manipulation of MNPs in the cytoplasm of the cell soma all the way into the nerve endings using a magnetic tip.
To increase the throughput of our manipulations, we established Micro-Magnetic Array (MMA) technology that allows massively parallelized magnetic manipulation under highly reproducible conditions. Using a direct ""endosomal approach"", we demonstrated our ability to trap adherent migrating cells and we performed proof of concept induction of directed neurite outgrowth using primary neurons.
We have demonstrated the differentiation of stem cells into neuronal cells via activating Wnt signaling by means of mechanical actuation of MNPs (“temp mode”) that are targeted to endogenous Frizzled at the cell surface by means of Wnt-mimicking peptides.
These developments have been flanked by our efforts to develop organotypic slice culture model for PD. A slice culture model that mimics the degeneration of dopaminergic neurons was successfully developed during this project and significant dopaminergic outgrowth of transplanted cells (tissue and dissociated cells) was observed and quantified in slices. Dopaminergic neurons were successfully loaded with MNPs and magnets exerted effects on these cells in wells and microfluidic devices. Exposure of MNP-loaded transplanted dopaminergic neurons to a powerful magnet near the striatum increased neuronal outgrowth from the transplantation site in the SN towards the striatum.
Our work has been disseminated in more than 10 publications in peer-review journals, 70 conferences and workshops, on a website and in several media such as general audience meetings. We organized two international workshops over the time course of the project. We applied for one patent, and we planned to apply for one more. We contacted two companies to evaluate the commercial exploitation of the know-how generated in the MAGNUERON project."
Manipulation of magnetic nanoparticles inside cells is still in its infancy and the MAGNEURON consortium is pioneering this methodology towards its application in regenerative neuromedicine. Particular innovations so far are therefore mostly related to method development and fundamental insights into the capabilities and limitations of this methodology. Important developments achieved include the design of different types of MNPs that are compatible with sustained intracellular control. In particular, we have designed semi-synthetic ferritin-based protein cages, a new class of magnetic nanoparticles, which will find use well beyond the framework of our project. This development was made possible by a thorough understanding of the behaviour of MNPs inside cells that was achieved by the collaboration of the MAGNEURON project. Another key innovation is the Micro-Magnetic Array (MMA) technology. In conjunction with tailored protocols for intracellular MNP delivery that were established within the consortium, the MMA technology substantially simplifies the application of high magnetic fields and therefore will be key for further spreading of the methodology.
With the “temp mode” approach, we have performed proof-of-concept experiments of magnetically controlled cell differentiation in organotypic brain slice cultures. For the “space mode”, we encountered limitations in the transfer of the technology from single cell assays to in vivo model systems. We thus utilized all the knowhow of the project participants to setup a parallel approach that relies on endosomal uptake of magnetic nanoparticles and their further manipulation by magnetic forces to control cell behavior.
We expect the MAGNEURON project to have a great impact in the field of neurodegenerative medicine and in fundamental science by opening a new route for innovative therapies. We have identified technical limitations, both in terms of magnetic apparatus and remote control of intracellular signaling, but we have proposed alternative solutions and the knowledge acquired will be instrumental for the future developments of the magnetic approach.