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Induction of Human iPSC-Derived Cardiomyocyte Proliferation Revealed by Combinatorial Screening in High Density
Drew M Titmarsh1,2, Nick R Glass1, Richard J Mills2
1Australian Institute for Bioengineering &Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia.
Abstract:
Inducing cardiomyocyte proliferation in post-mitotic adult heart tissue is attracting significant attention as a therapeutic strategy to regenerate the heart after injury. Model animal screens have identified several candidate signalling pathways, however, it remains unclear as to what extent these pathways can be exploited, either individually or in combination, in the human system. The advent of human cardiac cells from directed differentiation of human pluripotent stem cells (hPSCs) now provides the ability to interrogate human cardiac biology in vitro, but it remains difficult with existing culture formats to simply and rapidly elucidate signalling pathway penetrance and interplay. To facilitate high-throughput combinatorial screening of candidate biologicals or factors driving relevant molecular pathways, we developed a high-density microbioreactor array (HDMA)--a microfluidic cell culture array containing 8100 culture chambers. We used HDMAs to combinatorially screen Wnt, Hedgehog, IGF and FGF pathway agonists. The Wnt activator CHIR99021 was identified as the most potent molecular inducer of human cardiomyocyte proliferation, inducing cell cycle activity marked by Ki67, and an increase in cardiomyocyte numbers compared to controls. The combination of human cardiomyocytes with the HDMA provides a versatile and rapid tool for stratifying combinations of factors for heart regeneration.
Insights
Researchers explored new ways to regenerate heart tissue by testing signaling pathways in human heart cells. They found that a Wnt pathway activator, CHIR99021, significantly boosted human cardiomyocyte proliferation, offering a promising therapeutic strategy for heart repair.
Area of Science:
- Cardiovascular Biology
- Regenerative Medicine
- Stem Cell Biology
Background:
- Adult cardiomyocytes are post-mitotic, limiting heart regeneration after injury.
- Identifying pathways for human cardiomyocyte proliferation is crucial for cardiac repair therapies.
- Existing in vitro models struggle to rapidly assess signaling pathway interactions in human cardiac cells.
Purpose of the Study:
- To develop a high-throughput screening method for identifying factors that induce human cardiomyocyte proliferation.
- To investigate the combinatorial effects of key signaling pathways (Wnt, Hedgehog, IGF, FGF) on human cardiomyocyte cell cycle activity.
- To evaluate the potential of specific pathway agonists for heart regeneration strategies.
Main Methods:
- Development of a high-density microbioreactor array (HDMA) with 8100 culture chambers for microfluidic cell culture.
- Combinatorial screening of Wnt, Hedgehog, IGF, and FGF pathway agonists using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs).
- Assessment of cardiomyocyte proliferation using markers like Ki67 and quantification of cardiomyocyte numbers.
Main Results:
- The Wnt pathway activator, CHIR99021, emerged as the most potent inducer of human cardiomyocyte proliferation.
- CHIR99021 treatment led to significant cell cycle activity (Ki67 positive cells) and increased cardiomyocyte numbers.
- The HDMA platform enabled rapid combinatorial screening and identification of effective pro-proliferative factors.
Conclusions:
- The HDMA platform is a versatile tool for high-throughput screening of factors influencing human cardiac biology.
- CHIR99021 demonstrates significant potential for inducing cardiomyocyte proliferation, a key step in heart regeneration.
- Combining hPSC-CMs with HDMA screening facilitates the stratification of therapeutic combinations for cardiac repair.
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