In vitro models aiming for fertility preservation

Abstract

Fertility preservation is considered a great hope to people suffering from infertility as a consequence of developmental or genetic cause, a disease itself or its treatments sequelae. In vitro models are fundamental to enrich our knowledge about human germ cell development and its regulation, to generate and screen new therapeutic measures and might provide a tool that can be transferred to clinical use to treat infertility. There are, in principle, two strategies to start an in vitro fertility preservation protocol in male humans. First the in vitro generation of germ cells by differentiating human pluripotent stem cells and second the maturation and differentiation of human spermatogonia stem cells into more advanced stage of germ cells. However, both strategies are not suitable to be used in the clinic since the robust protocols for differentiation are not available. Additionally, reproducibility of the results between different laboratories is a big challenge faced by researchers working in the field.

Therefore, we focused in this thesis on these two strategies aiming to improve both models to reduce variability and improve reproducibility. In the first study, we addressed the variability occurring among hES cell lines when used for human germ cell differentiation. We aimed to find a suitable culture condition allowing a robust starting point for our cultures by using LN521 as a culture matrix for hES cells before using them in protocols of differentiation. Moreover, we investigated the only robust working system in rodents and aimed to translate this to the situation in humans by using normal fetal gonadal tissues as well as actual patient materials to investigate the potential use of these systems to mature human germ cells in vitro.

We found that LN521 has positive effects on hES cells growth and maintenance of their pluripotency characteristics with no influence on gonadal cells related genes expression. Furthermore, we showed that LN521 homogenized the gene expression variation among the five cell lines used in the study. In regard to in vitro germ cells maturation from spermatogonial stem cells, production of elongated spermatids was achieved when air liquid interphase organ culture method was used to cultivate mouse testicular tissues in vitro. A supplementation of 10% knockout serum replacement is shown to have positive effects on tubular maturation, germ cell proliferation and differentiation. We showed that organ culture method could be used to culture and study human first trimester gonads somatic cells in vitro as demonstrated by their ability to produce hormones in a manner similar to what is described for in vivo situation. In addition, differences in somatic cells functions in testicular tissues from different patient groups exposed to hematological and oncological treatments could be illustrated by culturing these tissues in vitro by using organ culture method.

Culturing human embryonic stem cells on LN521 could be a step forward towards future applications of human embryonic stem cells in regenerative medicine by providing more predictable and controllable system to assess the behavior of human embryonic stem cells when used in different protocols for differentiation. This would be the basis for future approaches to generate more defined in vitro protocols for differentiation of human pluripotent stem cells towards germ cells. On the other hand, organ culture method could be a useful tool to study the process of human germ cells development and to screen the effect of different substances on human gonadal cell development and function. Moreover, it can be used as quality assessment tool of cryopreserved testicular tissues, from patients exposed to hematological and oncological treatments, before considering using them for fertility preservation purposes.