Publicação
Generation of a reporter system for monitorization of endothelial differentiation
| Resumo: | Stem cell differentiation into endothelial cells has been an active field of research within the last years. The increased interest in this research field is a consequence of the huge clinical relevance of these cells and the associated future regenerative medicine treatments that could improve the recovery of pos-ischemic conditions and other angiogenic related states. Despite this increase in research, nowadays the differentiation strategies into the endothelial lineage are very inefficient. This inefficiency is related both to the lack of information regarding the ideal cell source to the differentiation process and to the nonstandardization of the in vitro conditions to induce the endothelial lineage commitment (growth factors, matrices, cytokines). Therefore strategies that permit a real time monitorization of the differentiation process, like viral transduction of tagged DNA, is a reliable and efficient way to evaluate these kinds of processes. In this context, the main aim of this project was to generate a lentiviral tool specific for detecting the expression of an endothelial late marker (vascular endothelial-cadherin – VEcadherin). This objective was accomplished through the ligation of the VE-cadherin promoter and a mCherry fluorescent tag. After generating the reporter system, the validation of this tool was done by infecting human umbilical vein endothelial cells (HUVECs) and induced pluripotent stem cells (iPSCs). The results show that 5 days after infection HUVECs present an increased pattern of red fluorescence and no alteration was observed within the infected iPSCs. There is already on-going work to differentiate the infected iPSCs and monitor the red fluorescence levels. Additionally, some information was generated regarding the internalization of organic nanoparticles (NPs) and adhesion and proliferation of iPSCs in possible endothelial inductive substrates. The results show that the iPSCs are able to internalize nanoparticles even when the NPs are adhered to the substrate. In conclusion, the results show the successful creation of a lentiviral construct that permits the direct monitorization of expression of mCherry associated with VE-cadherin promoter. In the future this might be a powerful tool either to be used in a stem cell context or to explore other cell sources and processes like transdifferentiation. |
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| Autores principais: | Lima, Ana Francisca Silva de |
| Assunto: | Lentivírus Endotélio |
| Ano: | 2011 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Stem cell differentiation into endothelial cells has been an active field of research within the last years. The increased interest in this research field is a consequence of the huge clinical relevance of these cells and the associated future regenerative medicine treatments that could improve the recovery of pos-ischemic conditions and other angiogenic related states. Despite this increase in research, nowadays the differentiation strategies into the endothelial lineage are very inefficient. This inefficiency is related both to the lack of information regarding the ideal cell source to the differentiation process and to the nonstandardization of the in vitro conditions to induce the endothelial lineage commitment (growth factors, matrices, cytokines). Therefore strategies that permit a real time monitorization of the differentiation process, like viral transduction of tagged DNA, is a reliable and efficient way to evaluate these kinds of processes. In this context, the main aim of this project was to generate a lentiviral tool specific for detecting the expression of an endothelial late marker (vascular endothelial-cadherin – VEcadherin). This objective was accomplished through the ligation of the VE-cadherin promoter and a mCherry fluorescent tag. After generating the reporter system, the validation of this tool was done by infecting human umbilical vein endothelial cells (HUVECs) and induced pluripotent stem cells (iPSCs). The results show that 5 days after infection HUVECs present an increased pattern of red fluorescence and no alteration was observed within the infected iPSCs. There is already on-going work to differentiate the infected iPSCs and monitor the red fluorescence levels. Additionally, some information was generated regarding the internalization of organic nanoparticles (NPs) and adhesion and proliferation of iPSCs in possible endothelial inductive substrates. The results show that the iPSCs are able to internalize nanoparticles even when the NPs are adhered to the substrate. In conclusion, the results show the successful creation of a lentiviral construct that permits the direct monitorization of expression of mCherry associated with VE-cadherin promoter. In the future this might be a powerful tool either to be used in a stem cell context or to explore other cell sources and processes like transdifferentiation. |
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