Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells

Int J Mol Sci. 2019 Feb 14;20(4):810. doi: 10.3390/ijms20040810.


Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a "thymidine block", but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.

Keywords: cytotoxic; genome editing; herpes simplex virus type 1 thymidine kinase; induced pluripotent stem cells; lentiviral vector; nucleotide metabolism.

MeSH terms

  • Apoptosis / genetics
  • CRISPR-Cas Systems / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Ganciclovir / pharmacology
  • Gene Editing
  • Gene Expression Regulation, Enzymologic / genetics
  • Gene Expression Regulation, Viral / genetics
  • Genes, Transgenic, Suicide / genetics
  • Genetic Therapy*
  • Genetic Vectors / therapeutic use
  • Humans
  • Induced Pluripotent Stem Cells / enzymology*
  • Induced Pluripotent Stem Cells / transplantation
  • Lentivirus / genetics
  • Nucleotides / biosynthesis
  • Nucleotides / genetics
  • Simplexvirus / enzymology*
  • Simplexvirus / genetics
  • Thymidine Kinase / genetics*


  • Nucleotides
  • Thymidine Kinase
  • Ganciclovir