The development of cell-based therapies aimed to promote tissue repair in central nervous system (CNS) diseases, represents one of the most challenging areas of investigation in the field of regenerative medicine. Several cell-replacement strategies have been developed in the last few years. Recent evidence from our own and other laboratories indicates that undifferentiated neural stem/precursor cells (NPCs) might very efficiently protect the CNS from chronic degeneration induced by inflammation both in small rodents as well as in primates. However, before envisaging any potential human applications of such innovative therapies we need to confront with some preliminary and still unsolved questions:


1.The ideal stem cell source for transplantation, whether it has to be from pluripotent or multipotent sources;

2.The ideal route of cell administration, whether it has to be focal or systemic;

3.The ideal balance between differentiation and persistence of stem cells into the targeted tissue and – last but not least –

4.The ideal mechanism of tissue repair to foster, whether it has to be cell replacement or tissue protection (rescue).


The current projects in the PluchinoLab are further exploring the cellular and molecular mechanisms regulating the therapeutic plasticity of NPCs in complex CNS diseases such as multiple sclerosis, and spinal cord injury. While keeping an eye on next generation stem cells, either induced pluripotent stem (iPS) or induced neural stem (iNS) cells that are being tested via classical experimental cell therapy approaches, we are also devoting special attention to the study of the different modalities by which stem cells speak with the brain and with the immune system (neuro/immune interactions).


































As first and principal strand of investigation, we are focusing at a novel mechanism of intercellular communication that works through the cell-to-cell transfer of extracellular membrane vesicles (EMVs). We are attempting on defining whether this form of communication exists for stem cells of the brain, and on elucidating its molecular signature and potential therapeutic relevance. We are very excited about our current preliminary data, as they suggest that EMV-mediated transfer of small non-coding nucleic acids – including microRNAs – is very likely be a major mechanism responsible for the functional instruction of neighbouring (target) cells by transplanted stem cells. In our experiments we are using state-of-the-art technologies to (i) investigate the EMV small RNAome; and (ii) demonstrate that ncRNAs from stem cells affect the expression of several genes and protein, and ultimately the function, of target cells. Then, we aim at (iii) demonstrating the therapeutic potential of the transfer of individual EMV-carried small ncRNAs in vitro and in vivo in rodents with experimental neurological diseases. Understanding how this communication occurs will allow us to identify both the stem cell molecular makeup and their therapeutic benefits. Using computational analysis, bioinformatics techniques and rodent models, this project hopes to determine how stem cell behavior can be translated into treatments for neurological disorders that include multiple sclerosis and spinal cord injuries (http://erc.europa.eu/succes-stories/world-multiple-sclerosis-day-erc-funded-research).








As follow up (and integration) of this first strand of highly translational research, we are also pursuing a more basic (fundamental) approach that focuses at the discovery of the cellular machinery controlling the mobility of small nucleic acid sequences (known to regulate the translation into protein products, and ultimately functions) from within the cell to the extracellular space/compartment. We have started from some datasets that we have generated in the laboratory in the last couple of years and now attempting to define the genetic loci; verify the functions of potential cellular binders and/or identify the molecular carriers of these mobile small nucleic acids. This is a key and strategic strand of research that will provide evidence and mechanisms whose understanding has profound implications towards both the elucidation of the pathophysiology of major neurodegenerative diseases as well as the designing of next generation therapeutics mimicking (or interfering with) the sophisticated cellular intercourse that orchestrates the shift from homeostasis to disease.



A third strand of investigation aims at the design, development, synthesis and testing of next generation RNA nanotherapeutics that will deliver short hairpin RNA (shRNA), microRNAs (miRs), aptamers, rybozimes, or other small RNAs to specific neural and non-neural cells in vitro and in vivo, for the treatment of inflammation-driven chronic neurological damage, such as that accumulating in multiple sclerosis and spinal cord injuries. We have recently launched collaboration with Professor Peixuan Guo at the University of Kentucky (UK) in applying RNA nanotechnologies for brain repair in syndromes where persistent inflammation leads to irreversible neural damage, such as multiple sclerosis, cerebral stroke and spinal cord injury. Professor Peixuan Guo is the William S. Farish Fund Endowed Chair in Nanobiotechnology at the UK College of Pharmacy and Markey Cancer Center, has established himself as one of the lead’s premier nanobiotechnology experts for his work in cancer therapeutics and RNA nanotechnology. Our backgrounds come from different ends of the scientific spectrum, which was one of the major factors in the partnership. We are constantly searching for innovative investigative approaches, and it was after we read a review on the emerging field of RNA nanotechnology published by Guo in Nature Nanotechnology that we became interested in how we could apply Guo’s approach to cancer to our own vision of creating novel nanomedicines to test in experimental neuroscience. As part of the collaboration, Jayden A. Smith, a post-doctoral scholar supported by our laboratory at Cambridge is spending six months in Guo’s lab at UK. Jayden will be specifically working at the construction of RNA nanostructures (from the conception, to the refinement of the assembly, to the final characterization) and learning how to apply Guo’s novel methods to neuroscience. The first translational phase of this project will encompass the test of RNA-nanostructures laboratory animals suffering from experimental multiple sclerosis, or contusion spinal cord injuries. Successful attempts in rodents will provide a strong rationale towards the development of clinical-grade next generation nanomedicines for the treatment of inflammation-driven neural damage in humans

(See news releases at http://www.pluchinolab.org/PluchinoLab/News/Entries/2012/3/17_Entry_1.html; http://www.neuroscience.cam.ac.uk/news/article.php?permalink=61ea8fd398; http://pharmacy.mc.uky.edu/display.php?id=854).

 

Volocity-based 3D reconstruction showing structural junctional connexin43 pattern (red; arrowheads) between the process of one transplanted NPC (green) and one juxtaposed F4/80+ spinal cord macrophage (blue).

From Cusimano et al. Brain. 2012; 135- 447-60.pdf

Co-authorship network of the Neural Stem Cells field.

The nodes in the network represent all authors with at least 20 publications that match the search term "Neural Stem Cells" in Europe PubMed Central (http://europepmc.org, accessed 20-5-2014).

An edge linking two authors means that they are co-authors of at least one publication and the width of the edge is proportional to the number of co-authored publications. Similarly, the size of each node is proportional to the total number of publications matching the search term for each author. The nodes were clustered using MCL and color-coded according to cluster membership. Tommaso Leonardi (tl344@cam.ac.uk), 20/05/2014