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

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

Chizuru Iwasawa et al. Int J Mol Sci.

Abstract

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.

Conflict of interest statement

H.O. is a compensated scientific consultant of San Bio, Co., Ltd. and K Pharma Inc. M.Nakamura is a compensated scientific consultant of K Pharma Inc. The other authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1
Transduction of human iPSCs with the lentiviral vector expressing the HSV-TK gene. (A) Schematic representation of the integrated proviral form of the lentiviral vector expressing the HSV1tk gene. HSV1tk, humanized-codons with CpG-free HSV-TK gene; EF-1α, human elongation factor 1 α subunit promoter; IRES, internal ribosomal entry site; Puror, puromycin resistance gene; ΔU3, deletion of enhancer/promoter in the U3 region of the LTR; ψ, packaging signal. (B) Puromycin-resistant 253G1 and 1210B2 iPSCs transduced with the lentiviral vector expressing the HSV1tk gene were cultured in the presence of various concentrations of GCV for 2–5 days. Cell viability was assessed by the CCK-8 assay. The percent cell viability was calculated relative to cells in the absence of GCV. There was no significant difference in the results obtained on days 2, 3, 4, and 5 of culture. Data represent the mean ± SEM (n = 4–5). *, p < 0.05; **, p < 0.01. (C) Representative images of EB formation of 253G1, 1210B2, 253G1 HSV1tk-Puro, and 1210B2 HSV1tk-Puro iPSCs on day 4 and day 14. 253G1 HSV1tk-Puro and 1210B2 HSV1tk-Puro iPSCs were cultured with 1 μg/mL puromycin (+Puro). Scale bar, 200μm.
Figure 2
Figure 2
Silencing the transgene in human iPSCs. (A) Schematic representation of the integrated proviral form of the lentiviral vector expressing the HSV-TK-1 gene. HSV-TK-1, original HSV-TK gene; hKO1, humanized-codon Kusabira-Orange fluorescent protein gene. (B) Representative images of 1210B2 iPSCs 4 days after lentiviral transduction and after the second passage. Scale bar, 200 μm. (C) hKO1-positive iPSC clones, 253G1 HSV-TK-1-hKO1 (#12, #19) and 1210B2 HSV-TK-1-hKO1 (#2H, #3), were cultured in the presence of various concentrations of GCV for 3 days. Cell viability was assessed by the CCK-8 assay. The percent cell viability was calculated relative to cells in the absence of GCV. Data represent the mean ± SEM (n = 4). *, p < 0.05. (D) Representative images of EB formation of 253G1 HSV-TK-1-hKO1 iPSCs (#12, #19) on day 14. hKO1 fluorescence signal was not detected. Scale bar, 200 μm.
Figure 3
Figure 3
Cytotoxicity of HSV-TK expression with Tet-inducible lentiviral vectors. (AC) Schematic representations of the integrated proviral forms of the Tet-inducible lentiviral vectors, which carried (A) the GFP gene, (B) the HSV-TK-1 gene, and (C) the del-HSV-TK-1 gene. del-HSV-TK-1, truncated HSV-TK gene without the cryptic promoter; TRE, Tet-responsive promoter; rtTA; reverse Tet-controlled transactivator protein gene. 253G1 iPSCs, 1210B2 iPSCs, and HeLa cells transduced with the indicated Tet-inducible lentiviral vectors were cultured in the presence of various concentrations of GCV, with or without 1 μg/mL doxycycline (Dox) for 3–4 days. Cell viability was assessed by the CCK-8 assay. The percent cell viability was calculated relative to cells in the absence of GCV without Dox. There was no significant difference in the results obtained on days 3 and 4 of culture. Data represent the mean ± SEM (n = 4–6). *, p < 0.05; **, p < 0.01.
Figure 4
Figure 4
Analysis of nucleotide metabolism in human iPSCs expressing HSV-TK and the effect of excess dT on cell viability. (A) Schematic diagram of the nucleotide synthesis pathway. Solid arrows indicate the de novo pathway. Dashed arrows indicate the salvage pathway. HSV-TK (red) phosphorylates dT, dU, dC, and dTMP. PRPP, 5-phospho-α-d-ribosyl-1-pyrophosphate; RNR, ribonucleotide reductase; TK, thymidine kinase; CD, dCMP deaminase. (B) 1210B2 iPSCs and HeLa cells transduced with the Tet-inducible lentiviral vector carrying the HSV-TK-1 gene were cultured with or without 1 μg/mL Dox. Metabolome analysis of the indicated nucleotides was performed at the indicated time points. Data are expressed as the fold change in nucleotide levels relative to corresponding cells without Dox (control). Data represent the mean ± SEM (n = 4–5). *, p < 0.05; **, p < 0.01. (C) 253G1 iPSCs, 1210B2 iPSCs, and HeLa cells were cultured in the presence of various concentrations of dT for 2–3 days. Cell viability was assessed by the CCK-8 assay. The percent cell viability was calculated relative to cells in the absence of dT. There was no significant difference in the results obtained on days 2 and 3 of culture. Data represent the mean ± SEM (n = 5–6). **, p < 0.01.

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