Abstract
Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank the TCP Transgenic Core and M. Gertsenstein for mouse line derivation; A. Bang for flow cytometry; the TCP Pathology Core and K. Harpal for histology analysis; M. Kownacka for providing MEFs; M. S. Shoichet for HAMC; N. Mitrousis for qPCR primers; S. Nurk for advice on Monte Carlo simulation; I. P. Michael, P. D. Tonge, B. V. Varga, C. He and R. El-Rass for experimental advice and J. S. Harding and K. C. Davidson for their proofreading of the manuscript, to J. S. Harding for the artwork in Fig. 4. This work was supported by CIHR Foundation Grant, Foundation Fighting Blindness, Canadian Research Chair and Medicine by Design (University of Toronto) funding to A.N.
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Nature thanks A. Hewitt, T. J. Kieffer and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Authors and Affiliations
Contributions
Q.L. designed and conducted most of the experiments, including targeting of mouse and human ES cells, teratoma analysis, mammary gland tumour analysis, neural in vitro differentiation, mutation rate calculation, as well as analysing the data and writing the manuscript. C.M. designed and conducted experiments, designed and constructed the CDK1–TK targeting cassette, targeted mouse and human ES cells, analysed safe-cell escapees, performed in vitro differentiation assays, analysed data, composed figures and wrote the manuscript. M.V.S. conducted the Monte Carlo simulations and analysed the data. I.G. performed the mathematical modelling. E.J.N. targeted human ES cells, differentiated RPEs and wrote the manuscript. S.H. performed the eye experiment and analysed the data. H.Y. performed in vitro differentiation into endoderm and analysed the data. C.K. performed in vitro differentiation into mesenchymal stem cells and analysed the data. P.Z. performed Southern blots. C.L. performed animal teratoma experiments. K.N. performed animal teratoma experiments, analysed the data and edited the manuscript. M.M. targeted human ES cells. H.-K.S. analysed teratoma histology. A.N. conceived and supervised the study, designed experiments, analysed the data and wrote the manuscript.
Corresponding author
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Competing interests
A.N., C.M. and Q.L. are inventors on a patent application covering the SC technology (PCT/CA2016/050256). A.N. is a co-founder and shareholder of panCELLa Inc. C.M. is a senior scientist at panCELLa Inc. The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Generation, genotyping and characterization of mouse C57BL/6N C2 Cdk1–TK/Cdk1 and Cdk1–TK/Cdk1–TK ES cells.
a, Summary of the targeting steps used to generate mouse C2 Cdk1–TK/Cdk1 and Cdk1–TK/Cdk1–TK ES cells. b, Southern blot genotyping with internal TK–mCherry probe. c, Southern blot genotyping with mouse Cdk1 genomic probe. d, In vitro GCV dose–response killing curve of mouse C2 CDK1–TK/CDK1 ES cells. Data are mean ± s.d., n = 3. e, Teratoma formation efficiency of mouse C2 Cdk1–TK-PB/Cdk1, Cdk1–TK/Cdk1 and Cdk1–TK/Cdk1–TK ES cells.
Extended Data Fig. 2 Generation, genotyping and characterization of human H1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells.
a, Generation of human H1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells. b, Southern blot genotyping of CDK1–TK/CDK1 clone Exc16, which was used in teratoma assays (Fig. 1c and Extended Data Fig. 7) and CDK1–TK/CDK1–TK clone Exc16-3C, which was used in the differentiation assays in Fig. 3. c, PCR genotyping of all the correct clones. d, Flow cytometry analysis of the CDK1–TK/CDK1 clone Exc16 and the CDK1–TK/CDK1–TK clone Exc16-3C. e, SybrGreen qPCR of human CDK1 expression in H1 wild-type cells, and cells expressing the CDK1–TK/CDK1 clone Exc16 and the CDK1–TK/CDK1–TK clone Exc16-3C. Data are mean ± s.d., n = 3. No significant difference was found between groups. f, TaqMan qPCR copy-number analysis of TKs of all clones with the correct genotype. g, The efficiency of teratoma formation in NSG mice using human H1 ES cells. h, Dose–response analysis of wild-type, CDK1–TK/CDK1 and CDK1–TK/CDK1–TK human H1 ES cells. Cells were treated with different GCV concentrations, dissociated and counted after seven days. Data are mean ± s.d., n = 3. Source data
Extended Data Fig. 3 Generation, genotyping and characterization of human CA1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells.
a, Generation of human CA1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells. b, Southern blot genotyping of human CA1 CDK1–TK/CDK1 and CDK1–TK/CDK1–TK ES cells. The plasmid concatemers are multiple copies of plasmid integration (including backbone). The ampicillin gene in the backbone contains a ScaI restriction enzyme site, which is consistent with the sizes of the band in Southern blots. c, Haematoxylin and eosin staining of a CDK1–TK/CDK1–TK CA1 ES-cell-derived teratoma. d, The efficiency of teratoma formation in NSG mice using human CA1 ES cells. e, Flow cytometry analysis shows a direct correlation between the number of CDK1–TK alleles and mCherry fluorescence levels.
Extended Data Fig. 4 Growth graphs of mouse and human ES-cell-derived teratomas.
a, Growth of teratomas derived from mouse heterozygous safe-cell ES cells (C2 Cdk1–TK/Cdk1) b, Adult mouse with stabilized subcutaneous tissue (safe-cell ES-cell-derived dormant teratoma), 2.5 months after GCV treatment. c, Growth of teratomas derived from mouse homozygous safe-cell ES cells (C2 Cdk1–TK/Cdk1–TK). d, Growth of teratomas derived from human heterozygous safe-cell ES cells (H1 CDK1–TK/CDK1, clone Exc16); daily GCV treatment. e, Examples of teratomas from human heterozygous safe-cell ES cells showing cyst formation, images of cystic teratomas at dissection are shown next to the corresponding growth line; daily GCV treatment. The graphs with two lines represent mice that had cells injected into both flanks. The graphs with one line represent mice that had cells injected into one flank. The GCV treatment regime varies among mice because each teratoma behaves differently; we started GCV when the teratoma size started to increase. f, Growth of teratomas derived from human homozygous safe-cell ES cells (H1 CDK1–TK/CDK1–TK), GCV treatment was every other day. Images of cystic teratomas are shown next to the corresponding growth line, cysts were drained after dissection to show the difference in tumour weight due to the fluid present in the tissue. Each graph represents one mouse. a, c, d, f, All replicates of these experiments are shown. Source data
Extended Data Fig. 5 Breast cancer transplantation assay using heterozygous and homozygous safe-cell mammary tumour cells.
a, Generation of mouse lines and experimental design. b, Growth of mammary gland tumours derived from mouse Cdk1/Cdk1 and Cdk1–TK/Cdk1 mammary epithelial cells with PBS or GCV treatment. c, Growth of mammary gland tumours derived from mouse CDK1–TK/CDK1–TK mammary epithelial cells with PBS or GCV treatment. b, c, The sample sizes of each group are indicated at the bottom of the graphs.
Extended Data Fig. 6 In vitro experiments with mouse C2 Cdk1–TK/Cdk1 and Cdk1–TK/Cdk1–TK ES cells and subsequent characterization of escapees.
a, Experimental design: mCherry+ cells were selected by sorting to ensure that the starting cell population did not contain escapees. These cells were plated on six-well plates (200 cells per well, in a total of 36 wells) and allowed to grow to 14 cell doublings (this was estimated by counting cells in sample wells). The 36 cultures were then resuspended to a single-cell suspension and each was plated in a 15-cm plate (4 × 106 cells). One day after plating, selection with GCV was started and maintained until escapee colonies appeared. b, Escapee numbers obtained in 36 independent cultures growing from Cdk1–TK/Cdk1 and Cdk1–TK/Cdk1–TK ES cells. c, PCR to determine the presence of TK. d, TaqMan copy number qPCR analysis of Akap7, Sim1 and Cdk1 junction of exon 8 and 3′ UTR, Neurod, Cdk1, TK transgene and Abca on mouse chromosome 10. Data are the copy number calculated by CopyCaller Software v.2.1 and the error bars indicate the range from the minimum to the maximum number. n = 3. The same colour in the background of c and d indicates that they are from the same independent culture. n.d., not determined. e, qPCR to compare TK expression level in Cdk1–TK/Cdk1 escapee clone 2A and C2 wild-type ES cells. Data are mean ± s.e.m., n = 3. f, Summary of the copy number analysis of mouse Cdk1–TK/Cdk1 escapees. a, b, Experiments were repeated twice on a smaller scale but with similar results. d, Experiments were repeated twice with similar results.
Extended Data Fig. 7 Schematics of the possible mutation types affecting the CDL–SU allele.
a, Three types of mutations that could affect the CDL–SU allele. b, Safe-cell allele transition considered in the modelling and Monte Carlo simulations.
Extended Data Fig. 8 Quality control of batches generated from single human H1 CDK1–TK/CDK1–TK ES cells.
a, The drop of SCL due to aliquoting from a pool of cells relative to non-aliquoted batches of the same size. b, Schematics of the alleles in the CDK1–TK/CDK1–TK human ES cells used in the quality control. c, Workflow schematic of performing quality control (QC) on several ES cell batches. d, An example of the flow cytometry for the quality control of nine clonally derived batches. e, An example of PCR for the quality control of nine clonally derived batches.
Extended Data Fig. 9 Mouse and human safe-cell homozygous CDK1–TK/CDK1–TK cells demonstrate pluripotency.
All experiments were performed using the same clone of mouse C2 or human H1 (Exc16-3C) CDK1–TK/CDK1–TK cells. a, Bright-field photograph showing mouse homozygous Cdk1–TK/Cdk1–TK ES cell morphology. b, Bright-field photograph showing human homozygous CDK1–TK/CDK1–TK ES cell morphology. c, Haematoxylin and eosin staining of a mouse Cdk1–TK/Cdk1–TK ES-cell-derived teratoma. d, Haematoxylin and eosin staining of a human CDK1–TK/CDK1–TK ES-cell-derived teratoma. e, An adult Cdk1–TK/Cdk1–TK mouse. f, OCT4 and NANOG staining of human CDK1–TK/CDK1–TK ES cells. g, qPCR characterization of CDK1–TK/CDK1–TK ES cell differentiation into RPE cells. Data are mean ± s.d., n = 3. h, Bright-field picture of human CDK1–TK/CDK1–TK ES-cell-derived RPE cells. i, ZO1 staining of human CDK1–TK/CDK1–TK ES-cell-derived RPE cells. j, Human CDK1–TK/CDK1–TK ES-cell-derived adipocytes, chondrocytes and osteocytes. k, SOX17 and FOXA2 staining of human CDK1–TK/CDK1–TK ES-cell-derived definitive endoderm. l, OCT4 (also known as POU5F1), NANOG, SOX17, FOXA2 and HOXA3 qPCR characterization of ES cell (day 0) differentiation into definitive endoderm (day 4) and pharyngeal pouch endoderm (day 8). Data are mean ± s.d., n = 3. a–d, f–i, Experiments were repeated three times with similar results. j–l, Experiments were repeated twice with similar results.
Extended Data Fig. 10 Representative images of eyes transplanted with both safe-cell RPE and safe-cell ES cells, and only with safe-cell RPE cells.
a, b, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes transplanted with safe-cell RPE and safe-cell ES cells (four-week GCV treatment). The absence of a mCherry signal indicates that ES cell growth has not occurred. b, Bottom, images of the green fluorescence channel are included to illustrate that the observed signal in the red fluorescence channel is actually autofluorescence. This experiment was repeated four times in four mice with similar results. c, Histological analysis of the eye presented in d. d, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes transplanted with safe-cell RPE and safe-cell ES cells (PBS treatment). This experiment was repeated twice in two mice with similar results. e, f, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes transplanted with safe-cell RPE and safe-cell ES cells, mCherry signal is detectable and indicates ES cell growth. GCV treatment began three weeks post-injection following an initial PBS treatment. This experiment was repeated four times in three mice with similar results. g, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes receiving only safe-cell RPE cells (four-week GCV treatment). This demonstrates that GCV treatment did not affect the RPE cells. This experiment was repeated five times in three mice with similar results. h, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes receiving only safe-cell RPE cells (four-week PBS treatment). This experiment was repeated six times in three mice with similar results. i, Fundoscopy, optical coherence tomography and fluorescence imaging of eyes receiving only HAMC (four-week GCV treatment). This experiment was repeated twice in one mouse with similar results.
Supplementary information
Supplementary Information
This file contains Supplementary Equation 1: Detailed mathematical steps used to calculate the effect of aliquoting on SCL as illustrated in Extended Data Fig. 8a
Supplementary Table
This file contains Supplementary Table 1. To generate a list of CDLCDEL candidates, we cross-referenced genes whose knock-out has an early embryonic lethal phenotype in mice (www.informatics.jax.org/phenotypes.shtml) with data from a genome-wide CRISPR/Cas9 mutagenesis screen for essential genes in human cancer cell lines41. We found 167 genes with a high-fitness score (< -1.0) that also have a known early-embryonic lethal phenotype
Supplementary Table
This file contains Supplementary Table 2: List of DNA primers that were used in this study
Supplementary Table
This file contains Supplementary Table 3: List of antibodies that were used in this study
Supplementary Table
This file contains Supplementary Table 4: Calculation of the LOH rate at the human CDK1 locus with human H1 CDK1-TK/CDK1-TK ES cells. a, Summary table. b, Detailed cell number for each individual clone
Video 1
: Bright field video of beating cardiomyocytes differentiated from CDK1-TK/CDK1-TK human H1 ES cells
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Liang, Q., Monetti, C., Shutova, M.V. et al. Linking a cell-division gene and a suicide gene to define and improve cell therapy safety. Nature 563, 701–704 (2018). https://doi.org/10.1038/s41586-018-0733-7
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DOI: https://doi.org/10.1038/s41586-018-0733-7
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