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

> **NIH NIH F30** · UNIVERSITY OF WISCONSIN-MADISON · 2024 · $40,844

## 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 inve...

## Key facts

- **NIH application ID:** 10899966
- **Project number:** 1F30HL173988-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Austin K. Feeney
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $40,844
- **Award type:** 1
- **Project period:** 2024-09-30 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10899966

## Citation

> US National Institutes of Health, RePORTER application 10899966, Enhancing robustness of human pluripotent stem cell differentiation to cardiomyocytes by uncovering on- and off-target differentiation trajectories via single nucleus multi-omics (1F30HL173988-01). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/10899966. Licensed CC0.

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