Human pluripotent stem cells (hPSCs) offer tremendous promise in tissue engineering and cell-based therapies because of their unique combination of two properties: pluripotency and virtually unlimited proliferative potential. Currently, a lack of efficient hPSC differentiation methods impedes the use of hPSCs in biomedical translational research. To overcome this hurdle, one important prerequisite is to understand how hPSCs integrate environmental signaling cues to regulate fate choices. Wnt/β-catenin is a critical signaling pathway participating in stem cell maintenance and differentiation; however the exact role of Wnt/β-catenin signaling in hPSCs fate decisions remains controversial. Here, we engineered a single guide RNA (sgRNA) and Cas9-GFP system to overcome the low genome editing efficiency in hPSCs and achieved extremely high β-catenin knockout efficiency (up to 25%), demonstrating the robust nature of this system for hPSC genome editing. To the best of our knowledge, this represents the first reported β-catenin knockout hPSCs. Furthermore, we found that Wnt/β-catenin signaling is not required for hPSCs expansion or neuroectoderm differentiation. However, Wnt/β-catenin signaling is absolutely required to generate mesendoderm lineage which includes cardiomyocyte differentiation. The robust ability to generate gene knockout hPSC lines using the sgRNA and Cas9-GFP system should facilitate future study of gene functions in stem cell research.
All Science Journal Classification (ASJC) codes
- Modeling and Simulation
- Biochemistry, Genetics and Molecular Biology(all)