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.
