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Interrogating Canonical Wnt Signaling Pathway in Human Pluripotent Stem Cell Fate Decisions Using CRISPR-Cas9

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Abstract

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.

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Acknowledgments

This work was supported by Penn State Biomedical Engineering and Biology Departments Lab Startup funding to X.L.

Author Contributions

X.L. performed most of research and analyzed data. J.X. and X.B. provided experimental support. X.L. contributed to conception and design of experiments. X.L., J.X., X.B. and L.N.R. wrote the manuscript. All authors reviewed, edited, and approved the manuscript.

Conflict of Interest

Dr. Xiaojun Lian, Jiejia Xu, Xiaoping Bao, and Lauren N. Randolph declare that they have no conflicts of interest.

Ethical Standards

No human studies were carried out by te authors for this article. No animal studies were carried out by the authors for this article. Human embryonic stem cell work was approved by the Embryonic Stem Cell Oversight Committee.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Pennsylvania State University, University Park, PA, 16802, USA

    Xiaojun Lian & Lauren N. Randolph

  2. Department of Biology, Pennsylvania State University, University Park, PA, 16802, USA

    Xiaojun Lian

  3. The Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, PA, 16802, USA

    Xiaojun Lian & Lauren N. Randolph

  4. Department of Cell and Molecule Biology, Karolinska Institutet, 17177, Stockholm, Sweden

    Xiaojun Lian & Jiejia Xu

  5. Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI, 53706, USA

    Xiaoping Bao

Authors
  1. Xiaojun Lian
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  2. Jiejia Xu
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  3. Xiaoping Bao
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  4. Lauren N. Randolph
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Corresponding author

Correspondence to Xiaojun Lian.

Additional information

Associate Editor Michael R. King oversaw the review of this article.

Dr. Xiaojun Lance Lian currently holds a joint appointment in the Biomedical Engineering Department, Biology Department, and The Huck Institutes of the Life Sciences at the Pennsylvania State University as an assistant professor. He received a Bachelor of Science degree in Bioengineering from Zhejiang University, and a Doctor of Philosophy in Chemical Engineering from the University of Wisconsin - Madison. Most recently, Dr. Lian completed postdoctoral studies as a fellow at Harvard University and Karolinska Institutet where he specialized in Stem Cell Engineering and Regenerative Medicine. Dr. Lian provides the first demonstration of producing essentially pure populations of human heart muscle cells from pluripotent stem cells without sorting and his work was awarded the best biomedical paper of the year in PNAS and the Cozzarelli Prize of the National Academy of Sciences. His current research interests include: directed differentiation of human pluripotent stem cells, genome editing in stem cells for disease modeling, cardiovascular therapy, tissue engineering and regenerative medicine. He serves as the Principal Investigator of the Pluripotent Stem Cell Engineering Laboratory located in the Millennium Science Complex at Penn State.

This article is part of the 2016 Young Innovators Issue.

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Lian, X., Xu, J., Bao, X. et al. Interrogating Canonical Wnt Signaling Pathway in Human Pluripotent Stem Cell Fate Decisions Using CRISPR-Cas9. Cel. Mol. Bioeng. 9, 325–334 (2016). https://doi.org/10.1007/s12195-016-0453-8

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  • DOI: https://doi.org/10.1007/s12195-016-0453-8

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