Objective        To observe the cell morphology and functional characteristics of human fibroblasts after reprogrammed by spermatogonial stem cell self-renewal gene Piwil2

and to construct an experimental cell model for studying cancer stem cells (CSC).       Methods        Lentiviral vectors carrying Piwil2-GFP and GFP were transferred into primary culture of human foreskin fibroblasts to establish the cell lines stably expressing Piwil2-GFP fusion protein and GFP after selected by puromycin. Cells morphological changes and formation of tumor spheroids were observed

chromosomal karyotypes were analyzed and tumor formation assay was operated in nude mice. The tumor characteristics

stem cell characteristics and the markers of three germ layers were identified by RT-PCR and immunohistochemistry.       Results        The morphology of Piwil2-GFP fibroblasts became round and smaller at the 5th day after transfection

and turned to be colony-like growth at the 14th day after transfection. The selected colony was resuspended in culture and appeared to form similar tumor spheroids. The cell karyotype showed hyperdiploid heterogeneity

and all the tumor spheroids-like colonies formed tumor  after transplanted into nude mice for 2 weeks. It was identified by histology and immunohistochemistry assays as a malignant tumor that expressed the markers of three germ layers. RT-PCR showed that the cells and tumors in nude mice expressed stem cell pluripotent genes such as Oct-4

Nanog

Sox2 and the markers of three germ layers.       Conclusion        According to the cell morphology

molecular phenotype of stem cells and tumorigenic cell heterogeneity after Piwil2 reprogramming

the Piwil2-GFP fibroblasts seems to have stem cell and pluripotent differentiation characteristics and significant tumor cell heterogeneity. Therefore

fibroblasts after Piwil2 reprogramming may have CSC functionality

and can be used as a model for studying the process and mechanism of CSC occurrence.