Publicação
Characterization of different pluripotent states, the role of PSPH and GLOD4
| Resumo: | Despite the treatments available, cancer remains a leading cause of death worldwide. The discovery of similarities between pluripotent stem cells and cancer cells has generated interest in their use as anti-cancer vaccines. Accordingly, it has already been shown that the removal of two kinase inhibitors (2i) from the culture medium of mouse embryonic stem cells (mESCs) results in the acquisition of oncogenic characteristics. Proteomic analyses have also revealed similar expression of two proteins, Phosphoserine phosphatase (PSPH) and Glyoxalase domain-containing 4 (GLOD4), between mESCs and breast cancer cells. However, their role in stem cells is currently unknown. Although embryonic stem cells (ESCs) have been described as effective immunization agents against some types of cancer, ethical concerns arise due to their embryo origin, making induced pluripotent stem cells (iPSCs) a promising alternative. Despite the low efficiency of reprogramming somatic cells into iPSCs, several studies have reported that this efficiency could be improved through adjustments in the glycolytic and oxidative pathways. Although evidence suggests that lipid metabolism also plays a crucial role in reprogramming, little is known about the influence of dietary modifications on this process. Considering these facts, we aimed to evaluate if GLOD4 and PSPH are associated with the acquisition of oncogenic characteristics through interference with stem cells´ pluripotency pathways. Furthermore, we also intended to evaluate how cells isolated from mice subjected to different diets are sensitive to reprogramming. To assess the pluripotency state of stem cells, we used the TNG-A reporter cell line in which the levels of PSPH and GLOD4 were genetically manipulated. The sensitivity of cells isolated from adult mice subjected to different diets to reprogramming was evaluated through the levels of pluripotency genes associated with different phases of the reprogramming process. The present results showed that GLOD4 and PSPH are located in the cytoplasm and nucleus of mESCs and that their localization is maintained across the different pluripotent states. After the downregulation of GLOD4 and PSPH in the cells in the primed pluripotent state, the expression of GFP and NANOG remained unaltered, suggesting that these proteins are not involved in stem cells´ pluripotency pathways. The results obtained after reprogramming, revealed a tendency towards higher reprogramming efficiency in the younger fibroblasts, with the fibroblasts subjected to low-fat and high-fat diets presenting lower reprogramming efficiency, which may indicate that these diets pose barriers to reprogramming. By evaluating the expression of pluripotency genes, we observed differences in the sensitivity of cells from mice subjected to each of the diets. However, additional studies will be needed to understand the impact of lipid content on reprogramming. The results of this work contribute to a better knowledge of the mechanisms that regulate the pluripotency of ESCs, which may be relevant for the design of new therapeutic strategies for cancer. |
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| Autores principais: | Silva, Júlia Marisa Tavares e |
| Assunto: | Pluripotent stem cells GLOD4 PSPH Reprogramming Diets Células estaminais pluripotentes GLOD4 PSPH Reprogramação Dietas |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade de Coimbra |
| Idioma: | inglês |
| Origem: | Estudo Geral - Universidade de Coimbra |
| Resumo: | Despite the treatments available, cancer remains a leading cause of death worldwide. The discovery of similarities between pluripotent stem cells and cancer cells has generated interest in their use as anti-cancer vaccines. Accordingly, it has already been shown that the removal of two kinase inhibitors (2i) from the culture medium of mouse embryonic stem cells (mESCs) results in the acquisition of oncogenic characteristics. Proteomic analyses have also revealed similar expression of two proteins, Phosphoserine phosphatase (PSPH) and Glyoxalase domain-containing 4 (GLOD4), between mESCs and breast cancer cells. However, their role in stem cells is currently unknown. Although embryonic stem cells (ESCs) have been described as effective immunization agents against some types of cancer, ethical concerns arise due to their embryo origin, making induced pluripotent stem cells (iPSCs) a promising alternative. Despite the low efficiency of reprogramming somatic cells into iPSCs, several studies have reported that this efficiency could be improved through adjustments in the glycolytic and oxidative pathways. Although evidence suggests that lipid metabolism also plays a crucial role in reprogramming, little is known about the influence of dietary modifications on this process. Considering these facts, we aimed to evaluate if GLOD4 and PSPH are associated with the acquisition of oncogenic characteristics through interference with stem cells´ pluripotency pathways. Furthermore, we also intended to evaluate how cells isolated from mice subjected to different diets are sensitive to reprogramming. To assess the pluripotency state of stem cells, we used the TNG-A reporter cell line in which the levels of PSPH and GLOD4 were genetically manipulated. The sensitivity of cells isolated from adult mice subjected to different diets to reprogramming was evaluated through the levels of pluripotency genes associated with different phases of the reprogramming process. The present results showed that GLOD4 and PSPH are located in the cytoplasm and nucleus of mESCs and that their localization is maintained across the different pluripotent states. After the downregulation of GLOD4 and PSPH in the cells in the primed pluripotent state, the expression of GFP and NANOG remained unaltered, suggesting that these proteins are not involved in stem cells´ pluripotency pathways. The results obtained after reprogramming, revealed a tendency towards higher reprogramming efficiency in the younger fibroblasts, with the fibroblasts subjected to low-fat and high-fat diets presenting lower reprogramming efficiency, which may indicate that these diets pose barriers to reprogramming. By evaluating the expression of pluripotency genes, we observed differences in the sensitivity of cells from mice subjected to each of the diets. However, additional studies will be needed to understand the impact of lipid content on reprogramming. The results of this work contribute to a better knowledge of the mechanisms that regulate the pluripotency of ESCs, which may be relevant for the design of new therapeutic strategies for cancer. |
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