Spermatogonial stem cell transplantation and culture in patients

ABSTRACT Medical treatments for cancer or other conditions can cause infertility. Sperm cryopreservation is an effective option for adult men to preserve their fertility. This option, however, is not available to prepubertal boys who are not yet producing sperm. For over 20 years, centers around the world have been cryopreserving immature testicular tissues, which contain spermatogonial stem cells, for boys in anticipation that the tissues or cells can be matured in the future to produce sperm. Some of those survivors are now returning to use their tissues for reproduction. SSC transplantation and testicular tissue grafting are mature technologies that are ready for translation to the human clinic. At the University of Pittsburgh Medical Center, we are approved to deploy these technologies for patients who previously cryopreserved their testicular tissues. In the proposed research, we will test the safety and feasibility of transplanting human SSCs (hSSCs) or grafting testicular tissues back into human patients. We note, however, that technical challenges exist that limit transplant efficiency or the deployment of these technologies to all patients. First, autologous transplantation in patients with testicular or blood-borne cancers or neuroblastomas may risk re-introducing malignant cells. Second, testis biopsies from young boys are small and may contain a small number of SSCs. There is a broad consensus that we will need to propagate hSSCs ex vivo for robust regeneration of spermatogenesis upon transplantation. Long-term cultures that lead to SSC propagation are well established in mice, but its translation to hSSCs has been limited. The proposed research will develop multi-parameter sorting strategies to enrich human testis cell fractions with hSSC transplantation while removing contaminating somatic cells that can overgrow cultures and malignant cells that would be unsafe to transplant back into the patient. We will test the hypothesis that sorted human testis cell fractions with the greatest transplantation potential also have the best culture initiating potential as seen in mice. In addition, we will test the impact of donor age on hSSC culture starts as well as the impact of growth factors or other compounds, oxygen concentration, feeder cells and extracellular matrix substrates (ECM). Our multidisciplinary team has patients who are ready for first in human transplant procedures, testicular tissues from all ages of development, bioinformatic data that suggest potential ligands to test in hSSC culture and decellularized pig and human testis ECM substrates. Collectively, our efforts will test and improve the safety and feasibility of testicular cell/tissue transplant procedures in patients and establish robust, reproducible methods to maintain and expand hSSCs in culture.