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Considerable effort has been made to understand the mechanisms underlying cardiomyocyte cell cycle exit and re-entry, with view to discovering therapeutics that could stimulate cardiomyocyte proliferation and heart regeneration. The advent of large compound libraries and robotic liquid handling platforms has enabled the screening of thousands of conditions in a single experiment but success of these screens depends on the appropriateness and quality of the model used. Quantification of (human) cardiomyocyte proliferation in high throughput has remained problematic because conventional antibody-based staining is costly, technically challenging and does not discriminate between cardiomyocyte division and failure in karyokinesis or cytokinesis. Live cell imaging has provided alternatives that facilitate high-throughput screening but these have other limitations. Here, we (i) review the cell cycle features of cardiomyocytes, (ii) discuss various cell cycle fluorescent reporter systems, and (iii) speculate on what could improve their predictive value in the context of cardiomyocyte proliferation. Finally, we consider how these new methods can be used in combination with state-of-the-art three-dimensional human cardiac organoid platforms to identify pro-proliferative signalling pathways that could stimulate regeneration of the human heart.<\/jats:p>","DOI":"10.1098\/rsob.240167","type":"journal-article","created":{"date-parts":[[2024,10,8]],"date-time":"2024-10-08T19:05:07Z","timestamp":1728414307000},"update-policy":"https:\/\/doi.org\/10.1098\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["Cell cycle visualization tools to study cardiomyocyte proliferation in real-time"],"prefix":"10.1098","volume":"14","author":[{"given":"Rustem","family":"Salmenov","sequence":"first","affiliation":[{"name":"Department of Anatomy and Embryology, Leiden University Medical Center, Leiden 2300RC, The Netherlands 1"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine","family":"Mummery","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Embryology, Leiden University Medical Center, Leiden 2300RC, The Netherlands 1"},{"name":"The Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), Leiden University Medical Center, Leiden 2300RC, The Netherlands 2"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0568-3475","authenticated-orcid":true,"given":"Menno","family":"ter Huurne","sequence":"additional","affiliation":[{"name":"Department of Anatomy and Embryology, Leiden University Medical Center, Leiden 2300RC, The Netherlands 1"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"175","published-online":{"date-parts":[[2024,10,9]]},"reference":[{"key":"2026022419040599400_B1","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1161\/CIR.0000000000001123","article-title":"Heart disease and stroke statistics\u20142023 update: a report from the American heart association","volume":"147","author":"Tsao","year":"2023","journal-title":"Circulation"},{"key":"2026022419040599400_B2","doi-asserted-by":"crossref","first-page":"1021","DOI":"10.1161\/CIRCRESAHA.115.306565","article-title":"Cardiac fibrosis: the fibroblast awakens","volume":"118","author":"Travers","year":"2016","journal-title":"Circ. 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