Human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) applications for cell therapy and disease modeling are limited due to the cells’ lack of resemblance structurally and functionally to adult cardiomyocytes. To understand hPSC-CM maturation, we characterized two established approaches to mature cardiomyocytes—long term culture (aging of cells in a dish) and
in vivo
transplantation to an infarcted adult rat heart. RNA sequencing of hPSC-CMs from these systems demonstrated that
in vivo
transplantation is much more effective in maturing hPSC-CMs, inducing a more adult-like cardiac gene program (e.g. upregulation of
TNNI3, MYL2, SCN5A
), compared to cells kept in culture up to one year. Using this dataset, we identified candidate drivers of hPSC-CM maturation, including transcription factors and chromatin regulators, that we hypothesize are necessary to program hPSC-CMs to an adult-like state. To test the relationship between transcription factor regulation and hPSC-CM maturation, we developed a constitutive CRISPR activation (CRISPRa) pluripotent stem cell line to upregulate these transcriptional regulators upon addition of guide RNAs (gRNA). This cell line expresses nuclease-deficient Cas9 fused to the transcriptional activator VPR (dCas9-VPR), driven by the strong CAG promoter and targeted to the
AAVS1
safe harbor site. In pluripotent stem cells, target genes are upregulated up to 150-fold when gRNA is present; however, after differentiation into cardiomyocytes, dCas9-VPR transgene expression is silenced, and dCas9-VPR levels are insufficient to activate gRNA-targeted genes. To optimize CRISPRa for cardiomyocyte applications, we are generating alternative stem cell lines with dCas9-VPR targeted to the human
ROSA26
safe harbor site or driven by a cardiac-specific troponin T promoter, testing the regulation of transgene expression mediated by safe harbor site or promoter respectively. The characterization of these CRISPRa cell lines provides insights into CRISPR expression regulation and genome engineering strategies for applications in stem cells and hPSC-CMs. We will use this system to screen for maturation regulators and identify key combinations that are effective in programming hPSC-CMs towards an adult-like state.
A human pluripotent stem cell line KUMi04-A generated from a patient with chronic lymphocytic leukemia
