Publicação
Non-viral gene delivery to human mesenchymal stem cells using cationic liposomes
| Resumo: | Mesenchymal Stem Cells (MSC) hold a great promise for application in several therapies due to their unique biological characteristics. MSC can be easily isolated from bone marrow (BM) based on its adherence to plastic, and can be expanded in culture while maintaining the capacity to differentiate into mesoderm-lineage cell types. These cells have demonstrated immunosuppressive properties, allowing their escape from host allogeneic responses. Importantly, MSC secrete a large spectrum of bioactive molecules that provide a regenerative microenvironment for a variety of injured tissues. Despite being successfully used for the treatment of many diseases, it might be essential to enhance some of their features through gene delivery strategies to harness their full potential in cell or gene-based therapies. In this context, the main goal of this work was to develop an efficient and safe methodology to genetically-engineer mesenchymal stem cells, enhancing their therapeutic efficacy in Regenerative Medicine settings. To this end the delivery of plasmid DNA (pDNA) encoding the gene of a green fluorescent protein (pVAX-GFP) was optimized for BM MSC using Lipofectamine, a cationic liposomes-based reagent. Importantly, it was also established a Real-time Polymerase Chain Reaction (RT-PCR) method for quantification of pVAX-GFP copy numbers in lipofected MSC. The new RT-PCR method, which was considerably useful for transfection optimization, showed good reproducibility and high sensitivity, covering a wide linear range from 74 to 7.4×105 copies pDNA/cell. MSC demonstrated better transfection efficiencies when plated both at earlier passages and high cell densities (15,000-25,000 cell/cm2) with an L/D 1.25 ratio. The obtained values of transfection efficiency ranged from 1.6% to 28.3% and eGFP was expressed during seven days. As transfected MSC have shown high cell viabilities and cell recoveries while maintained their multipotency, this is an advantageous transfection strategy when it is desirable to efficiently express the therapeutic gene in a safe and transient way. |
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| Autores principais: | Mendes, Rui Daniel Martins Nunes |
| Assunto: | Biologia celular Células estaminais Técnicas biológicas Teses de mestrado |
| Ano: | 2009 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | Mesenchymal Stem Cells (MSC) hold a great promise for application in several therapies due to their unique biological characteristics. MSC can be easily isolated from bone marrow (BM) based on its adherence to plastic, and can be expanded in culture while maintaining the capacity to differentiate into mesoderm-lineage cell types. These cells have demonstrated immunosuppressive properties, allowing their escape from host allogeneic responses. Importantly, MSC secrete a large spectrum of bioactive molecules that provide a regenerative microenvironment for a variety of injured tissues. Despite being successfully used for the treatment of many diseases, it might be essential to enhance some of their features through gene delivery strategies to harness their full potential in cell or gene-based therapies. In this context, the main goal of this work was to develop an efficient and safe methodology to genetically-engineer mesenchymal stem cells, enhancing their therapeutic efficacy in Regenerative Medicine settings. To this end the delivery of plasmid DNA (pDNA) encoding the gene of a green fluorescent protein (pVAX-GFP) was optimized for BM MSC using Lipofectamine, a cationic liposomes-based reagent. Importantly, it was also established a Real-time Polymerase Chain Reaction (RT-PCR) method for quantification of pVAX-GFP copy numbers in lipofected MSC. The new RT-PCR method, which was considerably useful for transfection optimization, showed good reproducibility and high sensitivity, covering a wide linear range from 74 to 7.4×105 copies pDNA/cell. MSC demonstrated better transfection efficiencies when plated both at earlier passages and high cell densities (15,000-25,000 cell/cm2) with an L/D 1.25 ratio. The obtained values of transfection efficiency ranged from 1.6% to 28.3% and eGFP was expressed during seven days. As transfected MSC have shown high cell viabilities and cell recoveries while maintained their multipotency, this is an advantageous transfection strategy when it is desirable to efficiently express the therapeutic gene in a safe and transient way. |
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