CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes
Abstract Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily...
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Nature Portfolio
2021
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oai:doaj.org-article:d5033e1c1320405bbdfe19a5a5ff8a892021-12-02T14:06:58ZCRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes10.1038/s41598-021-81860-x2045-2322https://doaj.org/article/d5033e1c1320405bbdfe19a5a5ff8a892021-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-81860-xhttps://doaj.org/toc/2045-2322Abstract Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3′ untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.Orlando ChirikianWilliam R. GoodyerElda DzilicVahid SerpooshanJan W. BuikemaWesley McKeithanHaoDi WuGuang LiSoah LeeMarkus MerkFrancisco GaldosAimee BeckAlexandre J. S. RibeiroSharon PaigeMark MercolaJoseph C. WuBeth L. PruittSean M. WuNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021) |
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DOAJ |
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DOAJ |
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EN |
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Medicine R Science Q |
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Medicine R Science Q Orlando Chirikian William R. Goodyer Elda Dzilic Vahid Serpooshan Jan W. Buikema Wesley McKeithan HaoDi Wu Guang Li Soah Lee Markus Merk Francisco Galdos Aimee Beck Alexandre J. S. Ribeiro Sharon Paige Mark Mercola Joseph C. Wu Beth L. Pruitt Sean M. Wu CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes |
| description |
Abstract Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3′ untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities. |
| format |
article |
| author |
Orlando Chirikian William R. Goodyer Elda Dzilic Vahid Serpooshan Jan W. Buikema Wesley McKeithan HaoDi Wu Guang Li Soah Lee Markus Merk Francisco Galdos Aimee Beck Alexandre J. S. Ribeiro Sharon Paige Mark Mercola Joseph C. Wu Beth L. Pruitt Sean M. Wu |
| author_facet |
Orlando Chirikian William R. Goodyer Elda Dzilic Vahid Serpooshan Jan W. Buikema Wesley McKeithan HaoDi Wu Guang Li Soah Lee Markus Merk Francisco Galdos Aimee Beck Alexandre J. S. Ribeiro Sharon Paige Mark Mercola Joseph C. Wu Beth L. Pruitt Sean M. Wu |
| author_sort |
Orlando Chirikian |
| title |
CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes |
| title_short |
CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes |
| title_full |
CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes |
| title_fullStr |
CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes |
| title_full_unstemmed |
CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes |
| title_sort |
crispr/cas9-based targeting of fluorescent reporters to human ipscs to isolate atrial and ventricular-specific cardiomyocytes |
| publisher |
Nature Portfolio |
| publishDate |
2021 |
| url |
https://doaj.org/article/d5033e1c1320405bbdfe19a5a5ff8a89 |
| work_keys_str_mv |
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