Presumptive germ cells derived from mouse pluripotent somatic cell hybrids

• DOI: 10.1016/j.diff.2009.07.003
• Publication Date: 2009-08-05T15:42:50Z
• Authors: Thais M.C. Lavagn...; Simone A.S. Fonseca; Rui C. Serafim; Virgínia S. Pereira; Enrico J.C. Santos; Soraya Abdelmassih; Alexandre Kerkis; Irina Kerkis
• Keywords: Pluripotent Stem Cells; 0301 basic medicine; 571; Reverse Transcriptase Polymerase Chain Reaction; Cell Differentiation; Hybrid Cells; Immunoenzyme Techniques; Mice; 03 medical and health sciences; Somatic cell hybrids; Germ Cells; Microscopy, Electron, Transmission; Differentiation; Germ cells; Animals; Female; RNA, Messenger; Mouse ES cells; Fusion; Cells, Cultured; In Situ Hybridization, Fluorescence; Spleen; Cell Proliferation

The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.