Improving cardiovascular disease modeling using human pluripotent stem cell-derived cardiac fibroblasts

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Project Summary: Annually, there are ~790,000 cases of myocardial infarction (MI) in the United States. Typically, MI progresses into heart failure where patients have a high risk of mortality within 5 years after diagnosis. While animal models provide a valuable model system of MI, interspecies differences lead to inaccurate recapitulation of human myocardium. To address this, our lab originally developed 3D human cardiac organoids through self-assembly of hPSC-CMs, human primary adult cardiac fibroblasts (adult-cFbs), endothelial cells, and stromal cells. Further, we leveraged the oxygen diffusion limitation in 3D human cardiac organoids along with chronic adrenergic stimulation to generate an organotypic model of post-MI hearts. The human cardiac infarct organoids recapitulated transcriptional, structural and functional hallmarks of post-MI myocardium. However, the use of primary, non-myocyte cell populations in our current organoids limit their potential to mimic patient-specific myocardium. To develop human isogenic cardiac organoids, we are collaborating with Dr. Sean Palecek at the University of Wisconsin-Madison to derive cardiac fibroblasts from human pluripotent stem cells (hPSC) to replace adult-cFbs in our cardiac organoid model. Dr. Palecek’s lab has developed expertise to direct hPSC differentiation into cardiac fibroblasts (hPSC-cFbs) in 2 different lineages: epicardial-derived fibroblasts (hPSC-cFb(EpiC)s) and second heart field progenitor-derived fibroblasts (hPSC- cFb(SHFP)s). While both lineages contribute to cardiac fibrosis and are functionally similar, in murine hearts, the epicardium is the predominate source of ventricular cardiac fibroblasts while a small population arise from the endocardium. In addition, the enhanced maturation may be needed for the hPSC-cFb(EpiC)s to replace human adult-cFbs, as our preliminary data that showed that prolonged culture improved cell organization of hPSC-cFb(SHFP)s in cardiac organoids when compared to that of adult-cFb organoids. The central hypothesize of this proposal is that high passage hPSC-cFb(EpiC)s will best replicate adult-cFb transcriptomics and functionality. The proposal is innovative in that, for the first time, we will identify a suitable hPSC-cFb population to replace adult-cFbs to develop an isogenic 3D organotypic model of human myocardium. Our long-term goal is to develop patient-specific cardiac organoids for in vitro disease modeling and drug testing. Accordingly, we will pursue the following two Aims: 1) Determine the effectiveness of high passage hPSC- cFb(EpiC)s to replicate the transcriptomics and functionality of adult cFbs, and 2) Determine the effectiveness of human cardiac organoids composed of high passage hPSC-cFb(EpiC)s in modeling post-MI human myocardium and responsiveness to anti-MI therapeutics. We also will perform single cell RNA-seq to examine the heterogeneity of hPSC-cFb(EpiC)s in response to our infarction protocol. Completion of this study would provide the first step towards an isogenic human myocardium model. The single cell RNA-seq studies will reveal the various roles/subpopulations of cardiac fibroblasts in post-MI human myocardium.