Abstract 24068: Developing a Cardiomyocyte Pipeline for Gene Edited hiPSCs
| Autor: | Kaytlyn Gerbin, Angelique Nelson, Brock Roberts, Joy Arakaki, Tanya Grancharova, Colette DeLizo, Ru Gunawardane |
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| Rok vydání: | 2017 |
| Předmět: | |
| Zdroj: | Circulation. 136 |
| ISSN: | 1524-4539 0009-7322 |
| Popis: | The Allen Institute for Cell Science (AICS) is creating an open source collection of fluorescently tagged human induced pluripotent stem cell (hiPSC) lines to model cell organization and dynamics of stem cells and differentiated hiPSC-cardiomyocytes. Understanding the sub-cellular organization and the structure/function relationships of organelles within cardiomyocytes will contribute to the development of better disease models, therapies, and regenerative medicine approaches for cardiac disease. Using the WTC human iPSC line and the CRISPR/Cas9 system, we have fluorescently tagged ~20 target genes representing key cellular organelles including a few cardiac-specific genes. Edited iPSC lines are differentiated into cardiomyocytes using established protocols with either small molecules or a combination of cytokines and small molecules, which produce high-purity monolayers of beating cardiomyocytes within 1-2 weeks. Differentiation into cardiomyocytes serves as an important quality control criterion for our gene editing efforts, but also comprises an important aspect of our predictive cell modeling efforts. We plan to study the changes in localization and organization of these tagged organelles as the stem cells differentiate into cardiomyocytes using live fluorescent cell imaging. Here, we present our cardiac differentiation methods for multiple edited hiPSC lines and the quantitative and qualitative assays used to determine the efficacy of differentiation, including myofibril contraction, cardiac protein expression, and transcriptome profiling by bulk and single cell RNAseq. Additionally, we confirm the localization of cardiac proteins such as troponin T and alpha-actinin to the myofibrils in differentiated cells using image-based assays. In experiments initiated to date we have successfully differentiated multiple gene edited iPSC lines representing major cellular structures, 2 of which are specific to cardiomyocytes (ssTNNi1 and ACTN2). Some of the gene edited hiPSC lines are fluorescently-tagged for structures including focal adhesions, actin and microtubule cytoskeleton, mitochondria, nuclear envelope, desmosomes, and endoplasmic reticulum, which are all publically available to the community. |
| Databáze: | OpenAIRE |
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