Generation of in-vitro spermatogonial stem cells following genetic manipulation of primordial germ-like cells

ABSTRACT

Research about potential use of stem cells for the development of germ line cells in vitro had been challenged. In the present study, we reported a novel protocol consisting of cocktail growth factor addition for germ cell differentiation followed by transfection. The cells were purificated based on the expression on the cell surface of a protein. This protein is not present in normal cells of mice and does not interfere with cellular function. This cell surface marker is efficiently recognized by monoclonal antibodies. Bone marrow mesenchymal stem cells derived primordial germ like cells were differentiated to spermatogo-nial stem like cells by inducer cocktail including Retinoic acid [RA]+Leukemia inhibitory factor [LIF]+Basic fibroblast growth factor [bFgF]. Co-culture system was used as a feeder under differentiated cells. A 400 bp fragment of sperm-atogoniaspecific Stra-8 locus was enough to direct gene expression to the germ line stem cells. Stra8-CD4HAglo construct was used for purification of pre-meiotic differentiated cells. Expression of pluripotency [Pou5F1, Nanog, c-Myc] and specific germ cell [Mvh, Piwil2, Stra-8] genes in each stage were analyzed. The purified cells expressed the known molecular markers of PGC-like cells such as Mvh, Piwil2 and Stra-8. The outcomes of qPCR showed that ratio pluripotency of genes expression in selective group significantly decreased [p?0.05] in the initial differentiation process. This results showed that ratio of Pou5F1, Nanog, c-Myc, Mvh, Piwil2 and Stra-8 expression to purified PGC-like cells were 0.41, 0.204, 1.1, 0.003, 0.184 and 2.276, respectively. Treatment of cells with RA affected up regulation of Stra-8. Although, c-Myc gene as an oncogenic gene had significantly increased [pco-culture+cocktail and co-culture groups, Also, there was a significant difference [p?0.05] in the expression of Pou5F1 and Nanog. Our results suggest that selection and purification of PGC-like cells based on Stra-8 as a pre-meiotic marker is a useful tool for getting in vitro spermatogonial stem cell. This method facilitates identification of safely differentiated germ cells in vitro