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Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.

ABSTRACT: The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

SUBMITTER: Mills RJ

PROVIDER: S-EPMC5635889 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.

Mills Richard J RJ Titmarsh Drew M DM Koenig Xaver X Parker Benjamin L BL Ryall James G JG Quaife-Ryan Gregory A GA Voges Holly K HK Hodson Mark P MP Ferguson Charles C Drowley Lauren L Plowright Alleyn T AT Needham Elise J EJ Wang Qing-Dong QD Gregorevic Paul P Xin Mei M Thomas Walter G WG Parton Robert G RG Nielsen Lars K LK Launikonis Bradley S BS James David E DE Elliott David A DA Porrello Enzo R ER Hudson James E JE

Proceedings of the National Academy of Sciences of the United States of America 20170915 40

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening i ...[more]

PMID: 28916735

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Project description:Purpose: Induction of endogenous proliferation is a promising strategy for cardiac regeneration. We find that GSK3 inhibition activates a cell cycle network whereas MST1 inhibition drives the mevalonate pathway, with synergistic activation of proliferation. However, all GSK3 inhibitors tested also reduce contractile force in hCO. We screened for small molecule activators of cardiomyocyte proliferation that did not alter contractile force in hCOs. This was overcome by screening of a boutique compound library, identifying a p38 inhibitor, which activated a cell cycle network without reducing force. The screen also identified a TGFBR inhibitor that induces the mevalonate pathway and can also synergise to activate proliferation. RNA sequencing was performed to investigate underlying mechanisms of action. Methods: RNA samples were processed with Illumina TruSeq Stranded mRNA Library prep kit selecting for poly(A) taled RNA following the manufacturer’s recommendations. Libraries were quantified with Qubit HS (ThermoFisher) and Fragment Analyzer (Advances Analytical Technologies) adjusted to the appropriate concentration for sequencing. Indexed libraries were pooled and sequenced at a final concentration of 1.8 pM on an Illumina NextSeq 500 high-output run using paired-end chemistry with 75 bp read length. The sequencing data was demultiplexed using Illumina bcl2fastq2-v2.17. The quality of the reads was assessed thanks to FastQC. The reads were then processed and mapped to the human genome hg38 using the Bcbio-nextgen framework version 1.0.3. The aligner used was HISAT2 2.0.5. Raw counts were normalised and analysed with DESeq2. Results: Analysis of RNAseq data lead to the understanding that compound 3 regulated the cell cycle network, which was similar to GSK3 inhibitors, whereas compound 65 regulated the mevalonate network, which was similarly to MST1 inhibitors. Conclusions: The current study adds to a growing body of evidence that alterations in cardiomyocyte metabolism may be a cause rather than a consequence of cardiomyocyte cell cycle arrest. Controlling cellular metabolism is emerging as a key strategy in the fight against cancer but the same pathways may conversely be required for the development of cardiac regenerative therapies.

Dataset Information

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.

Publications

Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest.

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