Periodic Reporting for period 1 - TraFICa (Transcription Factor-mediated Induction of Cardiomoycyte proliferation.)
Período documentado: 2023-05-16 hasta 2025-05-15
The adult heart has an extremely limited regenerative capacity because heart cells do not multiply (proliferate). A major ischaemic event such as a Myocardial Infarction (MI) which results in the loss of heart cells and the formation of
fibrotic scar tissue, diminishing the contractile function of the heart. This pathological process can culminate in heart failure, with the only effective treatment being heart transplantation. Therefore, regeneration of the heart is a potential therapy for treatment of ischaemic heart disease.
Firstly, TraFICa aims to develop a high throughput screening platform to study heart cell proliferation in 3D structures, grown from stem cells, that recapitulate the human heart. Next, this screening platform will be used to Identify a protein cocktail that stimulates proliferation in heart cells within these 3D structures. Finally, we will validate the protein cocktail in an ex vivo myocardial tissue culture system.
fibrotic scar tissue, diminishing the contractile function of the heart. This pathological process can culminate in heart failure, with the only effective treatment being heart transplantation. Therefore, regeneration of the heart is a potential therapy for treatment of ischaemic heart disease.
Firstly, TraFICa aims to develop a high throughput screening platform to study heart cell proliferation in 3D structures, grown from stem cells, that recapitulate the human heart. Next, this screening platform will be used to Identify a protein cocktail that stimulates proliferation in heart cells within these 3D structures. Finally, we will validate the protein cocktail in an ex vivo myocardial tissue culture system.
I have generated several cell lines (with different genetic backgrounds) that have integrated the cTnT promoter-driven FUCCI reporters at a safe harbor locus (AAVS1). These cell lines can be used for cell cycle screening purposes.
Using directed differentiation protocol were optimized to obtain cTnT+ iPSC-derived cardiomyocytes. These reporter lines allowed us to visualize and quantify the pro-proliferative effect of small molecules known for their positive effect on the cell cycle, in 2D as well as in 3D cardiac organoids, which allowed us to concurrently measure the contractile properties of these 3D cardiac tissues.
We integrated a Cas9-VPR transgene in the second AAVS1 allele. The Dox-inducible expression of Cas9-VPR was confirmed by westernblot. Functionality of the Cas9-VPR system was shown by the forced expression of the FUCCI reporters in iPSCs. Optimization lead to the activation of the cTnT-driven FUCCI transgene in roughly 25% of the iPSCs.
We next set out to exploit the Cas9-VPR system to activate the expression of transgenes in iPSC-derived FUCCI cardiomyocytes. Howevere, activation of the expression of endogenous genes mediated by the Cas9-VPR transgene expressed from the AAVS1 locus remained very inefficient, likely due to silencing of the Cas9-VPR transgene in a large fraction of the cells. Therefore, we decided to make use of lentivirus-mediated expression of Cas9-VPR. Flow cytometry confirmed in 3 independent experiments the overexpression of these endogenous genes. These results indicated that we had generated an iPSC-line that, upon cardiac differentiation, expresses both the FUCCI reporters as well as a functional Cas9-VPR system.
To further improve the system, we developed new targeting plasmidz that include a puromycyin selectable cassette and Degron-subunit, which can be used to select cells that express the Cas9-VPR protein and for the targeted degradation of the Cas9-VPR protein, respectively.
Using directed differentiation protocol were optimized to obtain cTnT+ iPSC-derived cardiomyocytes. These reporter lines allowed us to visualize and quantify the pro-proliferative effect of small molecules known for their positive effect on the cell cycle, in 2D as well as in 3D cardiac organoids, which allowed us to concurrently measure the contractile properties of these 3D cardiac tissues.
We integrated a Cas9-VPR transgene in the second AAVS1 allele. The Dox-inducible expression of Cas9-VPR was confirmed by westernblot. Functionality of the Cas9-VPR system was shown by the forced expression of the FUCCI reporters in iPSCs. Optimization lead to the activation of the cTnT-driven FUCCI transgene in roughly 25% of the iPSCs.
We next set out to exploit the Cas9-VPR system to activate the expression of transgenes in iPSC-derived FUCCI cardiomyocytes. Howevere, activation of the expression of endogenous genes mediated by the Cas9-VPR transgene expressed from the AAVS1 locus remained very inefficient, likely due to silencing of the Cas9-VPR transgene in a large fraction of the cells. Therefore, we decided to make use of lentivirus-mediated expression of Cas9-VPR. Flow cytometry confirmed in 3 independent experiments the overexpression of these endogenous genes. These results indicated that we had generated an iPSC-line that, upon cardiac differentiation, expresses both the FUCCI reporters as well as a functional Cas9-VPR system.
To further improve the system, we developed new targeting plasmidz that include a puromycyin selectable cassette and Degron-subunit, which can be used to select cells that express the Cas9-VPR protein and for the targeted degradation of the Cas9-VPR protein, respectively.
The establishment of a cardiomyocyte-specific live cell cycle reporter line that expresses the Cas9-VPR system has the potential to be widely used to study the effect of genes on the cardiomyocyte cell cycle. When used in combination with the 3D cardiac organoid platform, this system can potentially be used to not only study the pro-proliferative effect of genes, but also the effect of these genes on the contractile properties of cardiomyocytes. The generation of novel lentiviral Cas9-VPR targeting plasmids will enhance the potency of the virus-mediated expression of the Cas9-VPR constructs and will be widely applicable. This project has therefor not only contributed to our understanding on the role of genes in heart regeneration, but also generated resources that can be applied accross other fields of research.