Using Gene Editing to Establish a Safeguard System for Pluripotent Stem-Cell-Based Therapies

• Published in: iScience Vol. 22; pp. 409 - 422
• Main Authors: Wu, Youjun, Chang, Tammy, Long, Yan, Huang, He, Kandeel, Fouad, Yee, Jiing-Kuan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.12.2019; Elsevier
• Subjects: Cellular Therapy; Stem Cells Research; Techniques in Genetics; Stem Cells Research; Techniques in Genetics; Cellular Therapy
• ISSN: 2589-0042; 2589-0042
• DOI: 10.1016/j.isci.2019.11.038

A major challenge in using human pluripotent stem cells (hPSCs) in therapy is the risk of teratoma formation due to contaminating undifferentiated stem cells. We used CRISPR-Cas9 for in-frame insertion of a suicide gene, iC9, into the endogenous SOX2 locus in human embryonic stem cell (ESC) line H1 for specific eradication of undifferentiated cells without affecting differentiated cells. This locus was chosen over NANOG and OCT4, two other well-characterized stem cell loci, due to significantly reduced off-target effect. We showed that undifferentiated H1-iC9 cells were induced to apoptosis by iC9 inducer AP1903, whereas differentiated cell lineages including hematopoietic cells, neurons, and islet beta-like cells were not affected. We also showed that AP1903 selectively removed undifferentiated H1-iC9 cells from a mixed cell population. This strategy therefore provides a layer of safety control before transplantation of a stem-cell-derived product in therapy. [Display omitted] •In-frame iC9 gene insertion into the SOX2 locus to target undifferentiated hESCs•The residual ESCs are selectively removed without affecting their differentiated progeny•Eradication of residual ESCs before transplantation provides an extra safety control Cellular Therapy; Techniques in Genetics; Stem Cells Research