The tumourigenicity of iPS cells and their differentiated derivates

Abstract

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC-derivates), iPSC-derivated cardiomyocyte (iPSC-CMs) were subcutaneously injected into the back of nude mice. Non-invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC-derivates and iPSC-CMs survived in receipts. Both iPSCs and iPSC-derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC-CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC-derivates transplanted mice, but not in iPSC-CM transplanted ones. In vitro study showed the long-term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC-like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage-specific differentiation is necessary prior to therapeutic use of iPSCs.

Fig. 1

Culture and lentiviral transduction of induced pluripotent stem cells (iPSCs). (A) It could be observed under fluorescent microscope that transduced iPSCs strongly express monomeric red fluorescent protein (mRFP) in cytoplasm. Flowcytometry analysis indicates that more than 95% iPSCs are mRFP-positive after transduction and sorting; (B) immunostaining against SSEA-1 and Sox2 show that expression of reporter gene do not adversely affect iPSC pluripotency; (C) Bioluminescent imaging shows that BLI signals were linearly correlated with iPSC number (R² = 0.95).

Fig. 2

Differentiation and enrichment of induced pluripotent stem cell (iPSC)-CMs. Cardiac differentiation from iPSCs was induced with vitamin C (vC) through embryoid bodies (EB) formation. Five days EBs were seeded onto 0.1% gelatin-coated dishes and cultured with differentiation medium supplemented with 10⁻⁴ M vC. After 14 days differentiation, iPSC-derivates were collected and enriched. (A) EB formation, cardiac differentiation and characterization of differentiated cells; (B) Marked contracting areas for micro-dissecting; (C) immune-staining of micro-dissected and dissociated cardiomyocytes; (D) RT-PCR of iPSCs, iPSC-derivates and iPSC-CMs.

Fig. 3

Tracking the engraftment and proliferation of transplanted cells with non-invasive bioluminescent imaging. (A) induced pluripotent stem cells (iPSCs) and iPSC-derivates proliferate dramatically with time after transplantation, indicating the teratoma formation; (B) Imaging of iPSC-CMs after transplantation.

Fig. 4

Teratomas formed by undifferentiated induced pluripotent stem cells (iPSCs) and iPSC-derivates. (A) visible teratomas formed by iPSCs and iPSC-derivates; (B) comparison of tumorigenic rate and teratoma size produced by different iPSC-derivates; (C) different structures in teratomas formed by iPSCs and (D) different structures in teratomas formed by iPSC-derivates. **P < 0.01 compared with iPSC-derivates.

Fig. 5

Persistent existence of pluripotent cells along induced pluripotent stem cell (iPSC) differentiation. Pluripotent cells positive for OCT4, SOX2 and NANOG remain in differentiated embryoid bodies (EBs) after 2 weeks of induced differentiation, though the ratio of pluripotent cells decrease with differentiation (bar = 100 μm).

Fig. 6

Redifferentiation of induced pluripotent stem cell (iPSC)-derivates on feeder layers. (A) Differentiated iPSC-embryoid bodies (EBs) induced by vitamin C (vC) for 14 days; (B–D) When reseeded onto feeder layers, differentiated iPSC-derivates dedifferentiated and restored to iPSC state with passaging, (B) passage1, (C) passage2, (D) passage3; (E–F) recovered iPSCs express pluripotent markers like undifferentiated iPSCs.

Fig. 7

Pluripotent gene expression in induced pluripotent stem cells (iPSCs) during induced differentiation and dedifferentiation. The expression of pluripotent genes decrease with differentiation of iPSC-embryoid bodies (EBs), but they were rapidly recovered to the level comparable to undifferentiated iPSCs (As shown Re-iPSCs). Re-iPS indicated the iPSCs recovered from 2 week-differentiated cells, which were reseeded onto feeder layers. *P < 0.01 compared with iPSCs, #P < 0.01 compared with 1 week differentiation.