Advanced technologies for neural transplantation: new approaches for neural repair and modelling diseases

© 2019 Stefano FrausinMany neurological diseases affecting the central nervous system are still incurable because of the scarce and incomplete knowledge of disease mechanisms and the lack of effective treatments. Despite the effort, these disorders represent a challenge for medical science. The poorly representative disease models and technical limitation in deriving and characterising neural cells after transplantation into animal model hindered the development of new therapies. Cell therapy has become a promising therapeutic approach for diseases characterised by cell loss, although significant hurdles need to be overcome before it can be progressed into mainstream therapies. Today, brain tumours remain incurable and there are no therapies able to cure these diseases. Well-defined GBM in vivo models and cellular technologies might be useful for the investigation of pathophysiological mechanisms regulating brain tumours, helping the development of new therapeutic strategies. This thesis explores the potential and the limitations of using human gene manipulated cell lines into different disease contexts: cell therapy for motor neuron disease (MND) and brain tumours. Using a differentiation protocol, we investigated the possibility to derive and characterise spinal motor neurons (spMNs) derived from human pluripotent stem cells (hPSCs). We demonstrated that the specification of different spMN subtypes can be regulated using a combination of small molecules for defined timing. These results contribute to clarify important human developmental aspects and to propose an in vitro source of spMNs useful to study disease mechanisms and propose a cell-based therapy for MND. Accordingly, we conducted a systematic evaluation of the abilities and properties of spMNs derived from different reporter hPSC lines after transplantation into the rat spinal cord. This research highlight that grafted cells require a period of 6 months to mature and integrate into the host spinal motor circuitry. This study demonstrates that stem cell therapy might be a promising approach for treating diseases affecting the central nervous system and characterised by cell loss. Two reporter tumour cell lines derived from patient biopsies were grafted into the brain of athymic mice to establish, study and characterise an animal model of GBM. The results highlight the utility of tagged cells in brain tumour research, which allow us to better understand not only the tumour generation and progression but also evaluate the efficacy of new treatments. Furthermore, we presented a new tool for studying the role of ions in GBM, the chemogenic platform DREADD. GBM cells were engineered to express Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and used to chronically manipulate calcium and potassium homeostasis in order to evaluate their effect on cell metabolism and cell behaviour in vivo. Our results show that these ions are involved in key tumour mechanisms such as cell proliferation and migration. This work contributes to a mixed literature where there has been conjecture as to whether the dysregulation of ions homeostasis is responsible of key tumour mechanisms. Taken together, these studies offer new tools that might be useful for the understanding and comprehension of biological events related to a huge number of diseases affecting the central nervous system and will have implications in clinical approaches for treating MND and GBM

DIAnet International School Proceedings 2013. Interdisciplinary Methods for the Sustainable Development of the Danube Region. Gorizia, 13rd - 22nd April 2013

Sustainable Development (SD) in the Danube River Basin needs an Interdisciplinary approach. Researchers must be able to identify, analyse and take into account the legacies of the past and the long history of human interactions with the environment in this densely inhabited region. This introductory contribution to DIANET 2013 maps the 7 pillars of the EU strategy for the Danube onto the ‘magic triangle’ of SD. Finding solutions requires understanding the present situation through the study of socio-natural sites. Such sites form by interaction of humans and the river system. Practices result in material arrangements which are created and kept in working condition by human actions. Benign or wicked, short- or longstanding legacies can be distinguished, their fate determines the potential for sustainable futures

Introduction [Interdisciplinary Methods for the sustainable development of Danube Region, Gorizia, 13rd - 22nd April 2013]

The objective of this publication is to collect and disseminate the proceedings of the first edition of the DIAnet International School which was held in Gorizia, Italy, between 13th and 22nd April 2013. The core is made up by the works produced by the participants in the School that are basically project proposals along the lines of the Danube:Future project, which is closely related to the DIAnet International School as the following paragraphs will try to explain. Before and around the School participants’ efforts and results, are the contribution and involvement of many people and institutions, on which this introduction wants to shed a light, thus defining the perspective and context within which the DIAnet International School has been conceived and shaped, and the scope and objectives that drive and direct its developmen

Wharton\u2019s jelly derived mesenchymal stromal cells: Biological properties, induction of neuronal phenotype and current applications in neurodegeneration research

Multipotent mesenchymal stromal cells, also known as mesenchymal stem cells (MSC), can be isolated from bone marrow or other tissues, including fat, muscle and umbilical cord. It has been shown that MSC behave in vitro as stem cells: they self-renew and are able to differentiate into mature cells typical of several mesenchymal tissues. Moreover, the differentiation toward non-mesenchymal cell lineages (e.g. neurons) has been reported as well. The clinical relevance of these cells is mainly related to their ability to spontaneously migrate to the site of inflammation/damage, to their safety profile thanks to their low immunogenicity and to their immunomodulation capacities. To date, MSCs isolated from the post- natal bone marrow have represented the most extensively studied population of adult MSCs, in view of their possible use in various therapeutical applications. However, the bone marrow-derived MSCs exhibit a series of limitations, mainly related to their problematic isolation, culturing and use. In recent years, umbilical cord (UC) matrix (i.e. Wharton\u2019s jelly, WJ) stromal cells have therefore emerged as a more suitable alternative source of MSCs, thanks to their primitive nature and the easy isolation without relevant ethical concerns. This review seeks to provide an overview of the main biological properties of WJ-derived MSCs. Moreover, the potential application of these cells for the treatment of some known dysfunctions in the central and peripheral nervous system will also be discussed

A combined cell and gene therapy approach for homotopic reconstruction of midbrain dopamine pathways using human pluripotent stem cells

Midbrain dopamine (mDA) neurons can be replaced in patients with Parkinson's disease (PD) in order to provide long-term improvement in motor functions. The limited capacity for long-distance axonal growth in the adult brain means that cells are transplanted ectopically, into the striatal target. As a consequence, several mDA pathways are not re-instated, which may underlie the incomplete restoration of motor function in patients. Here, we show that viral delivery of GDNF to the striatum, in conjunction with homotopic transplantation of human pluripotent stem-cell-derived mDA neurons, recapitulates brain-wide mDA target innervation. The grafts provided re-instatement of striatal dopamine levels and correction of motor function and also connectivity with additional mDA target nuclei not well innervated by ectopic grafts. These results demonstrate the remarkable capacity for achieving functional and anatomically precise reconstruction of long-distance circuitry in the adult brain by matching appropriate growth-factor signaling to grafting of specific cell types

In vivo survival and differentiation of Friedreich ataxia iPSC-derived sensory neurons transplanted in the adult dorsal root ganglia

Friedreich ataxia (FRDA) is an autosomal recessive disease characterized by degeneration of dorsal root ganglia (DRG) sensory neurons, which is due to low levels of the mitochondrial protein Frataxin. To explore cell replacement therapies as a possible approach to treat FRDA, we examined transplantation of sensory neural progenitors derived from human embryonic stem cells (hESC) and FRDA induced pluripotent stem cells (iPSC) into adult rodent DRG regions. Our data showed survival and differentiation of hESC and FRDA iPSC-derived progenitors in the DRG 2 and 8 weeks post-transplantation, respectively. Donor cells expressed neuronal markers, including sensory and glial markers, demonstrating differentiation to these lineages. These results are novel and a highly significant first step in showing the possibility of using stem cells as a cell replacement therapy to treat DRG neurodegeneration in FRDA as well as other peripheral neuropathies