Inducible caspase-9 suicide gene under control of endogenous oct4 to safeguard mouse and human pluripotent stem cell therapy

Abstract

Pluripotent stem cells (PSCs) are promising in regenerative medicine. A major challenge of PSC therapy is the risk of teratoma formation because of the contamination of undifferentiated stem cells. Constitutive promoters or endogenous SOX2 promoters have been used to drive inducible caspase-9 (iCasp9) gene expression but cannot specifically eradicate undifferentiated PSCs. Here, we inserted iCasp9 gene into the endogenous OCT4 locus of human and mouse PSCs without affecting their pluripotency. A chemical inducer of dimerization (CID), AP1903, induced iCasp9 activation, which led to the apoptosis of specific undifferentiated PSCs in vitro and in vivo. Differentiated cell lineages survived because of the silence of the endogenous OCT4 gene. Human and mouse PSCs were controllable when CID was administrated within 2 weeks after PSC injection in immunodeficient mice. However, an interval longer than 2 weeks caused teratoma formation and mouse death because a mass of somatic cells already differentiated from the PSCs. In conclusion, we have developed a specific and efficient PSC suicide system that will be of value in the clinical applications of PSC-based therapy.

Keywords: OCT4 gene; apoptosis; inducible caspase-9 (iCasp9) gene; pluripotent stem cells (PSCs); teratoma formation.

Graphical abstract

Figure 1

OCT4 gene KI with iCasp9 suicide gene in human and mouse PSCs (A) Scheme for generating OCT4-iCasp9 clones using CRISPR-Cas9. T9, tdTomato-IRES-iCasp9; 9P, iCasp9-2A-Puro. (B) Sequence analysis of hPSC clones containing the iCasp9 gene in the OCT4 locus confirmed the insertion of the suicide gene. H9P, H9 cell KI with iCasp9-2A-Puro; HT9, H9 cell KI with tdTomato-IRES-iCasp9. (C) Bright-field and fluorescence images of hPSC-iCasp9 and mESC-iCasp9 clones. MT9, 129 mESC KI with tdTomato-IRES-iCasp9; M9P, 129 mESC KI with iCasp9-2A-Puro. Scale bar, 100 μm. (D) Relative mRNA levels of OCT4 and SOX2 in the indicated PSCs. N = 3. Values represent mean ± SD. (E) Immunofluorescence staining of the three germ layer markers of teratomas formed by H9P and M9P cells. Scale bar, 100 μm.

Figure 2

Induced PSC-specific apoptosis of the OCT4-iCasp9 KI system (A) Effect of AP1903 concentration on OCT4-iCasp9 PSCs by MTS assay. (B) Cell viability of OCT4-iCasp9 PSCs after 10 nM AP1903 treatment at the indicated time as determined by MTS assay. Values represent mean ± SD. (C) Scheme of OCT4-iCasp9 hESCs differentiation to human NSCs. (D) Immunofluorescence staining identification of SOX2, PAX6, and OCT4 in H9P-ESC and H9P-NSC cells with or without AP1903 treatment. Scale bar, 100 μm. (E) Bright-field images of H9P-ESC and H9P-NSC cell after 2 h of treatment with or without 10 nM AP1903 (n = 3). Scale bars, 100 μm. (F) Schematic diagram of CID inducing specific HT9-ESC apoptosis after HT9-ESC and HT9-NSC mixed culture. The culture medium is a 1:1 mixture of both cell culture media. (G) Flow cytometric analysis of HT9-ESC and HT9-NSC cultured in different proportions with or without 10 nM AP1903 (n = 3).

Figure 3

H9P cells are sensitive to CID upon transplantation in mice (A) Schematic diagram of administration conditions during the construction of teratoma by H9P cells. –2h, administration in vitro 2 h before transplantation; coin s.c., co-injection simultaneously; i.p., intraperitoneal administration; D1, the administration group 1 day after transplantation; the other groups are followed by analogy. The arrow represents the times of administration (i.e., 3 consecutive days of administration). (B) Teratoma growth curve. ∗∗∗∗p < 0.0001, a significant difference between the D21, D28, and PBS groups on D30 and other groups (n = 3). (C) Volume comparison of teratomas formed in different administration groups on D30 (n = 3). (D) Survival curves for the eight groups of recipient SCID mice. The mice were euthanized after the tumors reached a diameter greater than 2 cm (n = 5).

Figure 4

Identification of teratoma from H9P cells after CID administration (A) Immunostaining of OCT4 from teratoma section after treated with or without CID. Four time points, D7, D14, D21, and D28, were closed for detection. The CID treated groups were stained 2 days after CID administration. Arrows point to the OCT4-positive cells. Scale bar, 50 μm. (B) H&E staining of teratoma in mice at different times treated with CID. Scale bars, 100 μm. The red arrow points to the vacuole area of tissue, and white arrows point to the three germ layers of teratoma. (C) TUNEL staining in the D21 group after being treated with or without CID. Scale bars, 100 μm. (D) Cell transplantation and teratoma formation on D30. H9P and WT hESCs were injected at each side of the mice. (E) Size comparison of teratomas formed by the bilateral subcutaneous injection of WT and H9P PSCs after CID administration (n = 3). Values and error bars reflect the mean ± SD. A major challenge for PSC application is the risk of teratoma formation due to contamination of undifferentiated PSCs. Here, we have constructed the OCT4-iCasp9 safeguard to specifically eliminate undifferentiated tumorigenic cells in vitro and in vivo by CID (AP1903), which will be of value in clinical applications.