Amyotrophic Lateral Sclerosis (ALS) is a fatal neuromuscular disorder caused by degeneration of motor neurons (MNs) in the spinal cord, brainstem, and cortex. It belongs to a group of heterogeneous disorders called “motor neuron diseases”, in which ALS is the most common form in adults. The progressive MN degeneration leads to a gradual muscle atrophy and paralysis. Patients affected by ALS usually die 3-5 years after the onset of symptoms due to respiratory failure. Up to now, no effective cure is available for ALS beyond supportive care and Riluzole, which only modestly prolongs Survival. In the early stage of the disease, MN loss and consequent muscle denervation are compensated by axonal sprouting and reinnervation by the remaining MNs, but this mechanism is insufficient in the long term. Thanks to their multiple beneficial mechanisms, stem cell transplantation represents a promising therapeutic strategy for ALS and other neurodegenerative disorders. In fact, transplanted stem cells can provide therapeutic effect by modulating the micro-environment through the production of neurotrophic factors, eliminating toxic molecules, reducing neuroinflammation and generating auxiliary neural networks. Moreover, stem cells can eventually replace degenerating cells. A novel source for stem cell transplantation consists in the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs). Since iPSCs are directly derived from adult tissues, they bypass ethical issue of the embryo manipulation and are patient-specific, potentially reproducing ALS features in vitro. This means that they are a promising tool for stem cells transplantation and to model human pathologies in vitro. In this study, we isolated a specific subpopulation of neural stem cells (NSCs) derived from differentiated iPSCs. Compared to other types of stem cell, NSCs are particularly appropriate for ALS treatment due to their peculiar ability to differentiate into neurons, astrocytes and oligodendrocytes. The rationale of this study consists in the selection of a subpopulation of NSCs able to engraft and migrate through the nervous system parenchyma, to protect degenerating MNs and to improve ALS phenotype. We selected NSCs for the presence of three markers: Lewis X (or LeX), CXCR4 e β1 integrin. Lewis X is a glycoprotein marker of stem cells with a relevant role in cell adhesion and migration. CXCR4 is a chemokine receptor, which increases the sensitivity of the cells to be recruited by the host spinal cord that produces chemoattractant cytokines. β1 integrin is a subunit of VLA4, a receptor that allows cells to cross the blood-brain barrier, particularly in the presence of inflammation as in ALS animal models and human patients. In order to evaluate the ability of LeX+CXCR4+β1+ NSCs to engraft into the nervous system and to improve ALS phenotype, we performed intrathecal injection of these cells in the SOD1G93A mouse model. Transplantation resulted in an efficient engraftment of the cells, which reached central nervous system bypassing blood brain barrier, and in the protection of MNs and their axons from degeneration. This determined a preservation of neuromuscular junction (NMJ) innervations by maintaining their integrity and inducing axonal sprouting. These beneficial effects on neuropathological phenotype correlated with a significant increased survival and improved neuromuscular function of transplanted SOD1G93A mice. We also demonstrated the beneficial effects of LeX+CXCR4+β1+ NSCs in a human in vitro model of ALS. When co-cultured with these cells, iPSC-derived MNs from ALS patients showed an improvement in terms of survival and axonal growth. We then analyzed the molecular mechanisms underlying NSC protection demonstrating that our NSC subpopulation exerted positive effects through neurotrophic factors production, inhibition of the GSK3β activity, and limiting astrocytes proliferation through activation of vanilloid receptor. The results of this study suggest that effective protection of MNs and NMJs can be achieved targeting multiple deregulated cellular and molecular mechanisms in both MNs and glial cells in ALS models. This is particularly relevant for ALS because different pathological mechanisms likely contribute to its onset, making NSC transplantation a promising therapeutic approach for ALS.

TRANSPLANTATION OF A SPECIFIC NEURAL STEM CELL SUBPOPULATION AS A THERAPEUTIC APPROACH FOR AMYOTROPHIC LATERAL SCLEROSIS / M. Bucchia ; tutor: G. P. Comi ; co-tutore: S. P. Corti ; coordinatore: M. Clerici. - : . DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2017 Feb 10. ((29. ciclo, Anno Accademico 2016. [10.13130/bucchia-monica_phd2017-02-10].

TRANSPLANTATION OF A SPECIFIC NEURAL STEM CELL SUBPOPULATION AS A THERAPEUTIC APPROACH FOR AMYOTROPHIC LATERAL SCLEROSIS

M. Bucchia
2017-02-10

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a fatal neuromuscular disorder caused by degeneration of motor neurons (MNs) in the spinal cord, brainstem, and cortex. It belongs to a group of heterogeneous disorders called “motor neuron diseases”, in which ALS is the most common form in adults. The progressive MN degeneration leads to a gradual muscle atrophy and paralysis. Patients affected by ALS usually die 3-5 years after the onset of symptoms due to respiratory failure. Up to now, no effective cure is available for ALS beyond supportive care and Riluzole, which only modestly prolongs Survival. In the early stage of the disease, MN loss and consequent muscle denervation are compensated by axonal sprouting and reinnervation by the remaining MNs, but this mechanism is insufficient in the long term. Thanks to their multiple beneficial mechanisms, stem cell transplantation represents a promising therapeutic strategy for ALS and other neurodegenerative disorders. In fact, transplanted stem cells can provide therapeutic effect by modulating the micro-environment through the production of neurotrophic factors, eliminating toxic molecules, reducing neuroinflammation and generating auxiliary neural networks. Moreover, stem cells can eventually replace degenerating cells. A novel source for stem cell transplantation consists in the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs). Since iPSCs are directly derived from adult tissues, they bypass ethical issue of the embryo manipulation and are patient-specific, potentially reproducing ALS features in vitro. This means that they are a promising tool for stem cells transplantation and to model human pathologies in vitro. In this study, we isolated a specific subpopulation of neural stem cells (NSCs) derived from differentiated iPSCs. Compared to other types of stem cell, NSCs are particularly appropriate for ALS treatment due to their peculiar ability to differentiate into neurons, astrocytes and oligodendrocytes. The rationale of this study consists in the selection of a subpopulation of NSCs able to engraft and migrate through the nervous system parenchyma, to protect degenerating MNs and to improve ALS phenotype. We selected NSCs for the presence of three markers: Lewis X (or LeX), CXCR4 e β1 integrin. Lewis X is a glycoprotein marker of stem cells with a relevant role in cell adhesion and migration. CXCR4 is a chemokine receptor, which increases the sensitivity of the cells to be recruited by the host spinal cord that produces chemoattractant cytokines. β1 integrin is a subunit of VLA4, a receptor that allows cells to cross the blood-brain barrier, particularly in the presence of inflammation as in ALS animal models and human patients. In order to evaluate the ability of LeX+CXCR4+β1+ NSCs to engraft into the nervous system and to improve ALS phenotype, we performed intrathecal injection of these cells in the SOD1G93A mouse model. Transplantation resulted in an efficient engraftment of the cells, which reached central nervous system bypassing blood brain barrier, and in the protection of MNs and their axons from degeneration. This determined a preservation of neuromuscular junction (NMJ) innervations by maintaining their integrity and inducing axonal sprouting. These beneficial effects on neuropathological phenotype correlated with a significant increased survival and improved neuromuscular function of transplanted SOD1G93A mice. We also demonstrated the beneficial effects of LeX+CXCR4+β1+ NSCs in a human in vitro model of ALS. When co-cultured with these cells, iPSC-derived MNs from ALS patients showed an improvement in terms of survival and axonal growth. We then analyzed the molecular mechanisms underlying NSC protection demonstrating that our NSC subpopulation exerted positive effects through neurotrophic factors production, inhibition of the GSK3β activity, and limiting astrocytes proliferation through activation of vanilloid receptor. The results of this study suggest that effective protection of MNs and NMJs can be achieved targeting multiple deregulated cellular and molecular mechanisms in both MNs and glial cells in ALS models. This is particularly relevant for ALS because different pathological mechanisms likely contribute to its onset, making NSC transplantation a promising therapeutic approach for ALS.
COMI, GIACOMO PIETRO
CLERICI, MARIO SALVATORE
Settore MED/26 - Neurologia
TRANSPLANTATION OF A SPECIFIC NEURAL STEM CELL SUBPOPULATION AS A THERAPEUTIC APPROACH FOR AMYOTROPHIC LATERAL SCLEROSIS / M. Bucchia ; tutor: G. P. Comi ; co-tutore: S. P. Corti ; coordinatore: M. Clerici. - : . DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2017 Feb 10. ((29. ciclo, Anno Accademico 2016. [10.13130/bucchia-monica_phd2017-02-10].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/470092
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