Abstract 2564: Characterization of dysgerminoma-like cells emerged in the process of iPSC generation from common Marmoset fibroblasts

Abstract

Abstract Recent generation of induced pluripotent stem cells (iPSCs) has made a great impact on the field of regenerative medicine. Before the clinical application of iPSCs, preclinical testing of the safety and usefulness of them must be performed using reliable animal models of various diseases. The Common Marmoset (CM, callithrix jacchus), has recently been considered one of the most useful experimental animals for medical research because of close phylogenetic similarity to humans. To generate iPSCs from CM cells, we transduced reprogramming factors including Oct3/4, Klf4, Sox2 and c-Myc into CM fibroblasts. We found that several round-shaped colonies similar to embryonic stem cells (ESCs) were formed. These cells expressed ES markers such as SSEA4 and TRA-1-60, and showed alkaline phosphatase activity. Moreover RT-PCR revealed that the cells were also positive for other ES markers such as LIN28, SALL1 and DPPA4, suggesting that these cells have been reprogrammed. Next we performed karyotype analysis, and found that these cells contained 46, X, del(4q),+mar. Although parental CM fibroblasts had abnormal chromosome called mar, majority of chromosomes were intact, indicating that chromosome 4q was lost in the process of reprogramming. We called these reprogrammed cells abnormally reprogrammed cells (ARCs). We next injected one million cells of ARCs into the testis of SCID mice. Tumors were formed approximately 6 weeks after the injection, and HE staining showed that the tumor was composed of nests and sheets of uniform round or polygonal cells with abundant, clear to faintly eosinophilic cytoplasm with well-demarcated cytoplasmic borders. In addition, immunohistochemical analysis demonstrated that the tumor cells were focally and weakly immunopositive for vimentin, and immunonegative for cytokeratin, S100, Desmin, α-smooth muscle actin and Neuron-specific enolase, and the tumor tissue appeared to be c-kit+/CD30-/CD45- by quantitative RT-PCR, all of which were characteristics of human dysgerminoma. Thus we called the tumor common marmoset dysgerminoma (CM DGs). Next we tried to culture CM DGs in vitro, and found that they could proliferate in a semifloating condition. Western blotting revealed that these tumor cells showed continuous expression of exogenous reprogramming factors. We also examined the sensitivity of CM DGs to irradiation and DNA damage agents such as mitomicin C and cisplatin, and found that they were highly sensitive to all the DNA damage treatments tested compared to the parental cells. We also found that knockdown of Sox2 or c-Myc inhibited the proliferation of CM DGs due to the increase of cell death. These results indicate that irradiation, chemotherapy using DNA damaging agents or targeting reprogramming factors such as Sox2 and c-Myc might be effective for unexpected tumors accidentally found in patients treated with the functional cells derived from iPSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2564. doi:1538-7445.AM2012-2564