Publicação
Exploring the secretome of mesenchymal like stem cells for central nervous system regenerative medicine: a focus on Parkinson's disease
| Resumo: | In recent years, mesenchymal stem cells (MSCs) have emerged as strong therapeutic candidates for Central nervous system (CNS) regenerative medicine. Over the last decade, neuroregulatory molecules secreted by different tissue derived MSCs have shown to hold a tremendous therapeutic potential towards CNS protection and recovery in animal models of distinct CNS disorders. More recently, it has been discovered that MSCs also secrete microvesicles and exosomes which have been reported to act as reparative agents. Nevertheless, despite these progresses, is still not known if the MSCs secretome alone, without any further cell transplantation, induces similar therapeutic benefits. Moreover, it is still not known if the secretome of different tissue derived MSCs have similar or differential therapeutic impact on a neurodegenerative disease, such as Parkinson’s disease (PD). Finally, an in-depth proteomic analysis to the secretome of MSCs is yet to be made. As a consequence of this, the scope of the present thesis was to explore the potential of the sole use of different tissue derived MSCs secretome, namely derived from bone marrow (BMSCs), adipose tissue (ASCs) and the Wharton jelly surrounding the vessels of the umbilical cord [(WJ-MSCs/human umbilical cord perivascular cells (HUCPVCs)] for CNS regenerative medicine, namely in PD. For this purpose, we first studied (Chapter 2) the effect of the BMSCs, ASCs and HUCPVCs secretome, in the form of conditioned media (CM) collected at different time points (24h,96h), on the survival and neuronal differentiation of a neuroblastoma cell line (SH-SY5Y cells). Results showed that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival, induce neurite outgrowth, as well as their differentiation into neuron-like cells. These experiences further indicated that the secretome of the two cell populations was inducing SH-SY5Y cells towards a different phenotype. In chapter 3 it was revealed that ASCs secretome induced a higher survival rate in ventral mesencephalic cells (VMCs). Moreover, when the secretome of the three MSC like cell populations was individually administrated into a 6-hydroxydopamine (6-OHDA) hemiparkinsonian rat model of PD, it was observed that BMSCs secretome induced a higher functional recovery, as assessed by the stair case test, as well as an increase on the number of tyrosine hydroxylase (TH) positive cells in the substantia nigra. Finally, in chapter 4, an exhaustive proteomics approach based on liquid chromatography coupled with tandem mass spectrometry following information dependent and SWATH (sequential windowed data independent acquisition of the total high-resolution mass spectra) acquisitions was performed in order to characterize the secretome of BMSCs, ASCs and HUCPVCs. Through this approach, we have been able to identify and quantify 121 proteins, from which 20 have been shown to be involved in events related with neural repair. The latter not only included neurotrophic, neurogenic, axon guidance, axon growth and neurodifferentiative proteins, but also proteins playing roles against distinct pathogenic processes, including oxidative stress, apoptosis, excitotoxicity, inflammation, glial scarring and toxic protein deposition, which have been shown to be involved in several CNS disorders/injuries. Importantly, the latter proteins were found to be differently expressed within the secretome of the MSCs populations in study. This result, not only demonstrates that effectively there are differences within the secretome of the MSCs populations in study, but also suggests that the secretome of different tissue derived MSCs may have a different impact in neuroprotection, neuroreparative and neurodifferentiation phenomena, which can explain the results obtained in the studies conducted throughout the present thesis. In summary, the work developed in the present thesis adds new knowledge on the biological and molecular relevance of the secretome differences of different tissue derived MSCs in the context of CNS neuroprotection and neuron repair. Moreover, it also demonstrates that, although it is important to select the appropriate cell type for application, the sole use of MSCs secretome may be a promising cell free therapeutic tool for future application in CNS regenerative medicine. |
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| Autores principais: | Marques, Ana de Oliveira Resende Pires |
| Assunto: | Ciências Médicas::Ciências da Saúde |
| Ano: | 2015 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | In recent years, mesenchymal stem cells (MSCs) have emerged as strong therapeutic candidates for Central nervous system (CNS) regenerative medicine. Over the last decade, neuroregulatory molecules secreted by different tissue derived MSCs have shown to hold a tremendous therapeutic potential towards CNS protection and recovery in animal models of distinct CNS disorders. More recently, it has been discovered that MSCs also secrete microvesicles and exosomes which have been reported to act as reparative agents. Nevertheless, despite these progresses, is still not known if the MSCs secretome alone, without any further cell transplantation, induces similar therapeutic benefits. Moreover, it is still not known if the secretome of different tissue derived MSCs have similar or differential therapeutic impact on a neurodegenerative disease, such as Parkinson’s disease (PD). Finally, an in-depth proteomic analysis to the secretome of MSCs is yet to be made. As a consequence of this, the scope of the present thesis was to explore the potential of the sole use of different tissue derived MSCs secretome, namely derived from bone marrow (BMSCs), adipose tissue (ASCs) and the Wharton jelly surrounding the vessels of the umbilical cord [(WJ-MSCs/human umbilical cord perivascular cells (HUCPVCs)] for CNS regenerative medicine, namely in PD. For this purpose, we first studied (Chapter 2) the effect of the BMSCs, ASCs and HUCPVCs secretome, in the form of conditioned media (CM) collected at different time points (24h,96h), on the survival and neuronal differentiation of a neuroblastoma cell line (SH-SY5Y cells). Results showed that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival, induce neurite outgrowth, as well as their differentiation into neuron-like cells. These experiences further indicated that the secretome of the two cell populations was inducing SH-SY5Y cells towards a different phenotype. In chapter 3 it was revealed that ASCs secretome induced a higher survival rate in ventral mesencephalic cells (VMCs). Moreover, when the secretome of the three MSC like cell populations was individually administrated into a 6-hydroxydopamine (6-OHDA) hemiparkinsonian rat model of PD, it was observed that BMSCs secretome induced a higher functional recovery, as assessed by the stair case test, as well as an increase on the number of tyrosine hydroxylase (TH) positive cells in the substantia nigra. Finally, in chapter 4, an exhaustive proteomics approach based on liquid chromatography coupled with tandem mass spectrometry following information dependent and SWATH (sequential windowed data independent acquisition of the total high-resolution mass spectra) acquisitions was performed in order to characterize the secretome of BMSCs, ASCs and HUCPVCs. Through this approach, we have been able to identify and quantify 121 proteins, from which 20 have been shown to be involved in events related with neural repair. The latter not only included neurotrophic, neurogenic, axon guidance, axon growth and neurodifferentiative proteins, but also proteins playing roles against distinct pathogenic processes, including oxidative stress, apoptosis, excitotoxicity, inflammation, glial scarring and toxic protein deposition, which have been shown to be involved in several CNS disorders/injuries. Importantly, the latter proteins were found to be differently expressed within the secretome of the MSCs populations in study. This result, not only demonstrates that effectively there are differences within the secretome of the MSCs populations in study, but also suggests that the secretome of different tissue derived MSCs may have a different impact in neuroprotection, neuroreparative and neurodifferentiation phenomena, which can explain the results obtained in the studies conducted throughout the present thesis. In summary, the work developed in the present thesis adds new knowledge on the biological and molecular relevance of the secretome differences of different tissue derived MSCs in the context of CNS neuroprotection and neuron repair. Moreover, it also demonstrates that, although it is important to select the appropriate cell type for application, the sole use of MSCs secretome may be a promising cell free therapeutic tool for future application in CNS regenerative medicine. |
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