Publicação
The role of DAND5 in the coordination of hiPSC-derived cardiomyocyte environmental cues: their impact on cardiomyocyte electrophysiology
| Resumo: | Cardiovascular diseases are the leading cause of death worldwide. Unfortunately, the main challenge in cardiac muscle repair lies in the human adult heart's limited regenerative capacity. Therefore, interest in regenerative stem cell therapies for heart disease and subsequent generation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged. Nowadays, the generation of hiPSC-CMs is a highly efficient procedure, however, they do not easily become fully mature compared to human adult cardiomyocytes (CMs). Recent evidences suggested, that for successful transplantation, hiPSC-CMs must mimic the structure and contractile function of CMs at an intermediated maturation stage. Cerl2 knockout mice, also known as DAND5 in humans, have been described to exhibit several heart defects, suggesting that Cerl2/DAND5 plays an essential role in the developing heart. In addition, DAND5 is known to be a multivalent antagonist that inhibits Nodal and Wnt signalling pathways during embryogenesis. According to in vitro functional analysis, a mutation in the DAND5 gene leads to a decrease in the inhibitory function of DAND5 protein, leading to extended exposure to Nodal and Wnt signalling. Since these signalling pathways influence cardiomyogenesis, we hypothesize that through DAND5 modulation, it would be possible to modulate maturity levels of hiPSC-CMs for a better implantation capability. Bearing this in mind, we used the DAND5 KO and WT corrected iPSC lines for this project and characterized their derived CMs to assess the degree of maturation. Our results show that we successfully derived and functionally characterized both hiPSC CMs lines. Overall, immunocytochemistry, RT-PCR and electrophysiology analyses show that the KO line presents a more immature phenotype when compared to the WT counterpart. In conclusion, our findings suggest that the microelectrode array method has proven to hold great promise for functional assays of hiPSC-CMs electrophysiology and could be explored to give more insight into cardiac disease research and drug safety testing. |
|---|---|
| Autores principais: | Nunes, Mafalda dos Santos Marques |
| Assunto: | Células estaminais pluripotentes humanas (hiPSC) DAND5 maturaomo dos cardiomiócitos Doenças cardíacas congénitas electrofisiologia Teses de mestrado - 2021 |
| Ano: | 2022 |
| 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: | Cardiovascular diseases are the leading cause of death worldwide. Unfortunately, the main challenge in cardiac muscle repair lies in the human adult heart's limited regenerative capacity. Therefore, interest in regenerative stem cell therapies for heart disease and subsequent generation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has emerged. Nowadays, the generation of hiPSC-CMs is a highly efficient procedure, however, they do not easily become fully mature compared to human adult cardiomyocytes (CMs). Recent evidences suggested, that for successful transplantation, hiPSC-CMs must mimic the structure and contractile function of CMs at an intermediated maturation stage. Cerl2 knockout mice, also known as DAND5 in humans, have been described to exhibit several heart defects, suggesting that Cerl2/DAND5 plays an essential role in the developing heart. In addition, DAND5 is known to be a multivalent antagonist that inhibits Nodal and Wnt signalling pathways during embryogenesis. According to in vitro functional analysis, a mutation in the DAND5 gene leads to a decrease in the inhibitory function of DAND5 protein, leading to extended exposure to Nodal and Wnt signalling. Since these signalling pathways influence cardiomyogenesis, we hypothesize that through DAND5 modulation, it would be possible to modulate maturity levels of hiPSC-CMs for a better implantation capability. Bearing this in mind, we used the DAND5 KO and WT corrected iPSC lines for this project and characterized their derived CMs to assess the degree of maturation. Our results show that we successfully derived and functionally characterized both hiPSC CMs lines. Overall, immunocytochemistry, RT-PCR and electrophysiology analyses show that the KO line presents a more immature phenotype when compared to the WT counterpart. In conclusion, our findings suggest that the microelectrode array method has proven to hold great promise for functional assays of hiPSC-CMs electrophysiology and could be explored to give more insight into cardiac disease research and drug safety testing. |
|---|