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congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD.<\/jats:p>","DOI":"10.3390\/cells12040520","type":"journal-article","created":{"date-parts":[[2023,2,6]],"date-time":"2023-02-06T04:25:28Z","timestamp":1675657528000},"page":"520","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Gene-Edited Human-Induced Pluripotent Stem Cell Lines to Elucidate DAND5 Function throughout Cardiac Differentiation"],"prefix":"10.3390","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9790-8335","authenticated-orcid":false,"given":"Jos\u00e9 M.","family":"In\u00e1cio","sequence":"first","affiliation":[{"name":"Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School\/Faculdade de Ci\u00eancias M\u00e9dicas, Universidade NOVA de Lisboa, 1150-082 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8961-3097","authenticated-orcid":false,"given":"Mafalda M.","family":"Nunes","sequence":"additional","affiliation":[{"name":"Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School\/Faculdade de Ci\u00eancias M\u00e9dicas, Universidade NOVA de Lisboa, 1150-082 Lisboa, Portugal"}]},{"given":"Micael","family":"Almeida","sequence":"additional","affiliation":[{"name":"Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School\/Faculdade de Ci\u00eancias M\u00e9dicas, Universidade NOVA de Lisboa, 1150-082 Lisboa, Portugal"}]},{"given":"Fernando","family":"Cristo","sequence":"additional","affiliation":[{"name":"Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School\/Faculdade de Ci\u00eancias M\u00e9dicas, Universidade NOVA de Lisboa, 1150-082 Lisboa, Portugal"}]},{"given":"Rui","family":"Anjos","sequence":"additional","affiliation":[{"name":"Hospital de Santa Cruz, Centro Hospitalar Lisboa Ocidental, 1449-005 Lisboa, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7384-0949","authenticated-orcid":false,"given":"Jos\u00e9 A.","family":"Belo","sequence":"additional","affiliation":[{"name":"Stem Cells and Development Laboratory, iNOVA4Health, NOVA Medical School\/Faculdade de Ci\u00eancias M\u00e9dicas, Universidade NOVA de Lisboa, 1150-082 Lisboa, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1016\/j.stemcr.2022.03.019","article-title":"Functional genomics and the future of iPSCs in disease modeling","volume":"17","author":"Brooks","year":"2022","journal-title":"Stem Cell Rep."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e139","DOI":"10.1161\/CIR.0000000000000757","article-title":"Heart disease and stroke statistics\u20142020 update: A report from the American Heart Association","volume":"141","author":"Virani","year":"2020","journal-title":"Circulation"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2561","DOI":"10.1161\/CIRCULATIONAHA.116.008731","article-title":"Endemic Cardiovascular Diseases of the Poorest Billion","volume":"133","author":"Kwan","year":"2016","journal-title":"Circulation"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2716","DOI":"10.1161\/CIRCULATIONAHA.116.023544","article-title":"Trends in Congenital Heart Disease: The Next Decade","volume":"133","author":"Triedman","year":"2016","journal-title":"Circulation"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"a008292","DOI":"10.1101\/cshperspect.a008292","article-title":"Signaling and transcriptional networks in heart development and regeneration","volume":"5","author":"Bruneau","year":"2013","journal-title":"Cold Spring Harb. 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