Abstract: PD08-12
Sources of Funding: California Institute for Regenerative Medicine (RB5-07210)_x000D_

Introduction

Spermatogonial stem cells (SSCs) are essential for the generation of sperm, an event that occurs throughout adult life. Moreover, SSCs have therapeutic potential. A prime application for SSC transplantation is cancer survivors who went through gonadotoxic therapy during their prepubertal period, and thus no mature sperm could be cryopreserved prior to treatment. Despite the importance of human SSCs, remarkably little is known about them, including the mechanisms driving their self-renewal and expansion. Here, we demonstrate effective ways to culture, expand, and manipulate gene expression in human SSCs. _x000D_

Methods

Human testicular biopsies were obtained from fertile donors and cultured either as slices (organ culture) or as dispersed cells. RHOXF2 was depleted using a RHOXF2 small hairpin (sh) RNA that we cloned into a lentiviral vector. Immunostaining, FACS, and quantitative (q) RT-PCR were used to assess gene expression. _x000D_

Results

Human testicular organ cultures exhibited a modest increase in SSC and spermatogonia markers and a dramatic decline in advanced germ cell markers (Fig. 1A). This suggests a proliferative expansion of spermatogonia, including SSCs, and loss of more differentiated germ cells. Cultured dissociated germ cells exhibited a similar pattern of marker expression except that advanced germ cell markers did not decline in level (Fig. 1B). Clusters of germ cells were observed in these cultures, possibly indicative of proliferative expansion (Fig. 1C). FACS analysis with the human SSC marker, SSEA4, revealed that both culture conditions increased the number of SSEA4+ cells, confirming expansion of SSCs/undifferentiated spermatogonia. Lentivirus infection with a RHOXF2 shRNA successfully depleted RHOXF2 expression (Fig. 1D), demonstrating the ability to manipulate gene expression in human spermatogonial cell cultures.

Conclusions

We show that human spermatogonia expressing SSC markers can be cultured and modestly expanded in vitro, either as tissue slices or dissociated cells. Our methods to culture and manipulate gene expression in human spermatogonia can be used to decipher mechanisms underlying human SSC self-renewal as a means to expand SSCs in vitro for clinical application.

Funding

California Institute for Regenerative Medicine (RB5-07210)_x000D_