Generation of In-vitro Spermatogonial Stem Cells following Genetic Manipulation of Primordial Germ-like Cells

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 differenti-ation followed by transfection. the cells were purificated based on the expres-sion 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 ef-ficiently 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-atogonia-specific 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 & 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 & 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 signifi-cantly increased (p≤0.05) at the end of differentiation stage compared to initial phase of study, this level of expression could not be tumorgenic. qpcr results of the differentiation stage showed higher expression of stra-8 in co-culture+ cocktail and co-culture groups, also, there was a significant difference (p≤0.05) in the expression of pou5f1 & 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