Long-term Culture of Human SSEA-4 Positive Spermatogonial Stem Cells (SSCs).

Recently we and two other groups have shown that human spermatogonial stem cells (SSCs) have the potential to become pluripotent in vitro in defined culture conditions and to differentiate into cells of the three embryonic germ layers. This discovery could open new avenues for autologous cell-based therapy in degenerative diseases, bypassing the ethical and immunological problems related to the human embryonic stem cells. In addition, human SSCs could be used to treat infertility in cancer survival children. However, in order to reprogram SSCs into pluripotency, or to preserve them for repopulation of infertile testes, the first and limiting step is to have access to a highly purified human SSC population that could be multiplied and efficiently cultured in vitro maintaining their molecular and cellular characteristics. Although various studies have attempted to identify molecular markers of human SSCs, to date there is still limited information related to the specific markers that could be used for their isolation and optimized purification that allows long-term in vitro culture of isolated human SSCs. Here using SSEA-4 as an optimal marker for isolation of a subpopulation of SSCs, we show that SSEA-4 positive cells express the highest level of SSC genes compared to other subpopulations isolated with different markers, and can be maintained in culture for over 14 passages which we were unable to obtain with other SSCs markers including GPR125 and ITGA6. In addition, we have established a new technology for cell sorting and long-term culture of human SSC-SSEA-4 positive cells that maximizes the purity and viability of the sorted cells. Our findings are crucial and could be used for the most efficient isolation, purification and long-term culture of SSCs for clinical applications in regenerative medicine, or for preparation of human SSCs for autologous treatment of infertility in cancer survival children.