Aurora-A overexpression is linked to development of aggressive teratomas derived from human iPS cells

Abstract

The discovery of human induced pluripotent stem cells (hiPSCs) is a promising advancement in the field of regenerative and personalized medicine. Expression of SOX2, KLF4, OCT4 and MYC transcription factors induces the nuclear reprogramming of somatic cells into hiPSCs that share striking similarities with human embryonic stem cells (hESCs). However, several studies have demonstrated that hESCs and hiPSCs could lead to teratoma formation in vivo, thus limiting their current clinical applications. Aberrant cell cycle regulation of hESCs is linked to centrosome amplification, which may account, for their enhanced chromosomal instability (CIN), and thus increase their tumorigenicity. Significantly, the tumor suppressor p53 plays a key role as a 'guardian of reprogramming', safeguarding genomic integrity during hiPSC reprogramming. Nevertheless, the molecular mechanisms leading to development of CIN during reprogramming and increased tumorigenic potential of hiPSCs remains to be fully elucidated. In the present study, we analyzed CIN in hiPSCs derived from keratinocytes and established that chromosomal and mitotic aberrations were linked to centrosome amplification, Aurora-A overexpression, abrogation of p53-mediated G1/S cell cycle checkpoint and loss of Rb tumor-suppressor function. When hiPSCs were transplanted into the kidney capsules of immunocompromised mice, they developed high-grade teratomas characterized by the presence of cells that exhibited non-uniform shapes and sizes, high nuclear pleomorphism and centrosome amplification. Significantly, ex vivo cells derived from teratomas exhibited high self-renewal capacity that was linked to Aurora-A kinase activity and gave rise to lung metastasis when injected into the tail vein of immunocompromised mice. Collectively, these findings demonstrated a high risk for malignancy of hiPSCs that exhibit Aurora-A overexpression, loss of Rb function, centrosome amplification and CIN. Based on these findings, we proposed that Aurora-A-targeted therapy could represent a promising prophylactic therapeutic strategy to decrease the likelihood of CIN and development of aggressive teratomas derived from hiPSCs.

Figure 1.

Genomic analysis of hiPSCs derived from keratinocytes and blood cells. (A) SKY technology performed in N1-HiPSCs exhibited a unique translocation between chromosomes 2 and 7. (B) SKY technology performed in DS1-hiPSCs exhibited a normal karyotype. hiPSCs, human induced pluripotent stem cells; SKY, spectral karyotyping.

Figure 2.

Analysis of G1/S cell cycle checkpoint integrity in N1-hiPSCs. (A) Immunofluorescence analysis revealed multipolar mitotic spindles in N1-hiPSCs. Mitotic spindles were labeled in green with a monoclonal antibody targeting Aurora-A. Nuclei were labeled in blue with DAPI. (B) FACS analysis revealed that treatment with 400 µM HU for 48 h induced the G1/S cell cycle arrest in N1-hiPSCs. (C) Immunofluorescence analysis revealed centrosome amplification in N1-hiPSCs treated with 400 µM HU for 48 and 24 h following HU washout. Centrioles were labeled in green with 20H5 centrin monoclonal antibody, pericentriolar material was labeled in red with a polyclonal antibody targeting pericentrin. Nuclei were labeled in blue with DAPI. (D) Graph displaying the average of the % of N1-hiPSCs untreated (control), treated with 400 µM HU for 48 and 24 h following HU washout from three independent experiments. hiPSCs, human induced pluripotent stem cells; HU, hydroxyurea.

Figure 3.

Expression of cell cycle checkpoint regulators in hiPSCs. Immunoblotting assay revealed the expression of p53, p21, p-Rb, total and phosphorylated Aurora-A before and following HU-induced genotoxic stress in N1-hiPSCs and DS1-hiPSCs. β-actin was used as a loading control. Results were derived from three independent experiments with comparable outcomes. hiPSCs, human induced pluripotent stem cells; HU, hydroxyurea.

Figure 4.

Development of teratomas from in vivo growth of N1-hiPSCs. (A) N1-hiPSCs (1×10⁶) were injected into the kidney capsule of 4-week-old SCID/beige mice to develop teratomas. (B) Animals were sacrificed after 12-week injections, and paraffin-embedded tumors were isolated for histopathological analysis. (C) Immunofluorescence analysis exhibiting centrosome phenotype in N1-derived teratomas. Centrosomes were labeled in red with a γ-tubulin monoclonal antibody. Nuclei were labeled in blue with DAPI. hiPSCs, human induced pluripotent stem cells.

Figure 5.

Tumor self-renewal and metastatic capacity of re-cultured teratoma cells. N1-hiPSC-derived teratomas were excised and re-cultured cells were termed N1-hiPSCs 1GX (first generation xenografts). (A) One thousand N1-hiPSCs 1GX were grown under non-adherent conditions and formed tumorspheres after 10 days in culture. After 48 h separate groups of tumorspheres were treated with the selective Aurora-A inhibitor, alisertib (0.5 µM) for 8 days. The formation of tumorspheres was monitored and recorded using a Zeiss light microscope. Graph displaying the average of N1-hiPSCs 1GX tumorspheres before and after treatment with alisertib. The results represented the average from three independent experiments. (B) Immunofluorescence analysis displaying Rb phosphorylation in N1-hiPSCs 1GX tumorspheres before and after treatment with alisertib. Phosphorylated Rb was labeled in green with a p-Rb monoclonal antibody. Nuclei were labeled in blue with DAPI. The results represent the average from three independent experiments. (C) MDA-MB 231 cells (1×10⁶), N1-hiPSCs and N1-hiPSCs 1GX were injected into the tail vein of 4-week-old SCID/beige mice and lung metastases were detected after 4 weeks of injections. Immunofluorescence analysis displaying lung sections where MDA-MB 231 and N1-hiPSCs 1GX are labeled in red using a human-mitochondria monoclonal antibody. Nuclei were labeled in blue with DAPI. Results were derived from three independent experiments with comparable outcomes. hiPSCs, human induced pluripotent stem cells; Rb, retinoblastoma.