Enhancing robustness of human pluripotent stem cell differentiation to cardiomyocytes by uncovering on- and off-target differentiation trajectories via single nucleus multi-omics

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

PROJECT SUMMARY / ABSTRACT Heart disease is the leading cause of mortality globally; yet prevailing treatment modalities fail to address the fundamental issue of replacing permanently lost contractile myocardium. Human pluripotent stem cells (hPSC) have the ability to generate large numbers of human cardiomyocytes (CMs) for cell therapy, drug toxicity, and disease modeling. However, current hPSC-CM differentiation protocols suffer from significant batch-to-batch and line-to-line variability in reproducibly generating pure hPSC-CM populations. One explanation for this variability is that existing hPSC-CM differentiation protocols rely heavily on murine cardiogenesis biology, from which humans evolutionarily diverged 100 million years ago. Ethical concerns and the paucity of embryonic tissue preclude studies of in vivo human cardiogenesis. However, hPSC-CM differentiation provides the intrinsic opportunity to define the molecular events that govern efficient human cardiogenesis by temporally comparing high and low purity hPSC-CM differentiations. We have demonstrated that high and low purity hPSC-CM differentiations have distinct signal transduction, metabolic fuel consumption, and transcriptional regulation as early as the mesoderm progenitor stage using bulk transcriptomics, proteomics, and metabolomics. In this study, we propose to use single nucleus RNA and ATAC sequencing to objectively define the heterogeneous cell populations present during progenitor stages of high and low purity hPSC-CM differentiations in multiple genetically distinct cell lines. By comparing batch-to-batch and line-to-line variability in hPSC-CM differentiation purity, we expect to reveal conserved signaling pathways and gene regulatory networks that dictate human CM and non-CM cell specification. After uncovering the molecular mechanisms underlying divergent developmental cascades, we will apply this knowledge to enhance the robust generation of high purity hPSC-CMs. To accomplish this feat, we will use stage-specific signaling approaches to improve the production of high potency mesoderm and cardiac progenitors, which are poised to become high purity hPSC-CMs. After optimizing experimental perturbations at these two critical developmental stages, we will employ a combinatorial strategy to enhance the consistency of high purity hPSC-CM differentiation by merging the signaling approaches utilized to generate high potency mesoderm and cardiac progenitors. In summary, the proposed work drives progress towards improved heart disease treatment strategies by temporally investigating high and low purity hPSC-CM differentiations to unveil the molecular mechanisms of human cardiogenesis and to facilitate robust, high purity hPSC-CM biomanufacturing. The proposed work will utilize the stem cell bioengineering, cardiomyocyte biology, and single nucleus multi-omics expertise of the Sponsor, several collaborators, and outstanding research cores at the University of Wisconsin to investigate and control how human cardiogenesis unfolds in vitro. The training opportunities afforded by this proposal will facilitate extraordinary research, clinical, professional, and leadership growth, which are all essential skills to develop into a successful physician scientist.