In-vitro differentiation of human embryonic stem cells into primordial germ cells and male gametes

Fertility has become an important issue in the concept of quality of life. At this moment, male patients faced with infertility due to the loss of spermatogonial stem cells can only be offered cryobanking of semen as a strategy to preserve their fertility. Even though semen banking is an easy and effective technique, there remains a significant group of patients (e.g. prepubertal cancer patients, Klinefelter's patients,...) who cannot benefit from this strategy and currently, have no option to preserve their fertility. We explored alternative strategies to help these patients to be able to father their genetically-own children in the future. The research presented in this thesis can be divided into two categories: on the one hand, we investigated strategies for fertility preservation using male patients' spermatogonial stem cells (SSCs), cryobanked before the onset of stem cell loss; on the other hand strategies for fertility restoration were explored for those patients facing complete SSC loss before seeking a clinical solution.

In the fertility preservation part, we studied two strategies: the (xeno)grafting of testicular tissue pieces and the transplantation of testicular cell suspensions, using the spermatogonial stem cell transplantation (SSCT) technique.
For the xenografting, we studied the developmental potential of prepubertal and adult murine and human testicular tissue pieces, grafted to the back of immune deficient mice. Immature murine testis grafts were able to survive, proliferate and initiate spermatogenesis, while adult murine grafts showed severe sclerosis. Even though both prepubertal and adult human xenografts showed sclerosis, surviving spermatogonia could still be observed after long-term grafting.
For the SSCT, our research was mainly focused on a peculiar group of patients: prepubertal cancer patients. In this group of patients, testicular tissue might be contaminated with malignant cells, making any transplantation of tissue or cell suspensions impossible. Therefore, the ability to remove malignant cells from testicular cells is a perquisite in order to apply the SSCT technique as a clinical strategy for fertility preservation in these patients. We explored two strategies for cancer cell depletion from contaminated testicular cells. In a first protocol, we applied magnetic and fluorescence activated cell sorting (MACS and FACS), both in a mouse as in a human set-up. In a second set of experiments, a selective matrix adhesion procedure for germ cell purification from contaminated human testicular cell suspensions was investigated. In both strategies, low numbers of contaminating cells could still be detected after the completion of the cell selection protocols, proving that the efficiency of the techniques is high, but not high enough to be used as a safe strategy in a clinical setting.

As a strategy for fertility restoration, our main goal was to develop a protocol for the in vitro derivation of primordial germ cells and/or male gametes, starting from human pluripotent stem cells. We found that conditioned medium from a murine Sertoli cell line could significantly upregulate the expression of the germ cell-specific marker VASA in human embryonic stem cells (hESC) differentiating as embryoid bodies. Moreover, the easy and simple protocol that we developed proved to be as efficient as previously published protocols.
We chose hESC as a starting point because they are the current standard for pluripotent cells. However, since the derivation of hESC from the inner cell mass of human blastocysts implies that the donor embryo is destroyed, these cells are under heavy ethical scrutiny. By deriving two hESC lines from single blastomeres of 4-cell stage human embryos we made a first important step towards the derivation of hESC without the destruction of the donor embryo, neither its developmental capacity. This could make the use of hESC ethically more acceptable.

Even though we are not yet able to present a straight forward protocol to help male patients facing infertility, we hope that our work will have made a valuable contribution towards one or more possible solutions for patients in the future.