Advancements in testicular organoid research : insights for fertility preservation

Abstract

<p dir="ltr">Male childhood cancer survivors who undergo highly gonadotoxic treatments and have not yet produced mature sperm can only maintain their fertility potential by testicular tissue cryopreservation. However, no clinically proven methods currently exist to restore fertility from these cryopreserved tissues. Fertility preservation for these patients is dependent on experimental techniques, including testicular tissue or cell transplantation, as well as in vitro methods, such as testicular organoids and testicular explant tissue culture.</p><p dir="ltr">Firstly, we used a Matrigel-based 3D culture system to create prepubertal patient-derived testicular organoids and assess the impact of chemotherapy on organoid formation. Testicular cells from 11 boys were used for organoid formation and were compared with samples from 28 NORDFERTIL patients and 10 controls. Four of eleven prepubertal samples formed organoids with distinct cord-like structures and increased levels of testosterone and AMH. SOX9 expression was a key marker for successful organoid formation, and prior chemotherapy negatively affected its levels. This suggests SOX9 as a potential indicator for organoid formation and highlights the importance of Sertoli cells in maintaining testicular function and integrity.</p><p dir="ltr">Further, we collected testicular samples from a paediatric cancer patient with B- cell acute lymphoblastic leukaemia to assess the effects of leukaemia contamination on prepubertal testicular tissue. Our results showed that primary cells from the enlarged leukemic testis formed organoids with seminiferous cord- like structures similar to the testicular tissue structure observed in explant tissue culture. In addition, no signs of leukemic infiltration were observed in the cultured explant tissues or organoids.</p><p dir="ltr">Additionally, we evaluated testicular basement membrane protein composition during gonadal development and its role in germ cell maintenance. Our findings revealed that LAMA 1 is present in the testis during prenatal, prepubertal, and peripubertal stages, while LAMA 5 is exclusively found in the vasculature. Interestingly, the loss of LAMA 1 correlated with germ cell loss, underscoring its importance for testicular niche.</p><p dir="ltr">Finally, we tested a synthetic biomimetic polyisocyanopeptides (PIC) based hydrogel (Noviogel) to generate murine testicular organoids. We showed that testicular cells self-aggregated in LN111 and LN121 cultures but not in LN521.</p><p dir="ltr">Notably, only LN111-supplemented cultures produced organoids with distinct cord structures. These results demonstrate that primary mouse testicular cells can form organoids in a Matrigel-independent system and underscore the importance of specific laminin isoforms in organoid formation.</p><p dir="ltr">In summary, we demonstrated that patient-derived testicular cells can form organoids with compartmentalized cord structures. Additionally, we established a Matrigel-independent culture system using a synthetic hydrogel, paving the way for advancements in tissue engineering, drug screening, toxicology, and regenerative medicine.</p><h3>List of scientific papers</h3><p dir="ltr">I. Prior exposure to alkylating agents negatively impacts testicular organoid formation in childhood cancer patients.</p><p dir="ltr"><b>Yanhua Cui</b>, Femke Harteveld, Hajar Ali Mohammed Ba Omar, Yifan Yang, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Per Frisk, Kaisa Vepsäläinen, Cecilia Petersen, Rod T Mitchell, Jingtao Guo, João Pedro Alves-Lopes, Kirsi Jahnukainen, Jan-Bernd Stukenborg.</p><p dir="ltr">Human Reproduction open. 2024; Aug 13;2024(3): hoae049.<br><a href="https://doi.org/10.1093/hropen/hoae049" rel="noreferrer" target="_blank">https://doi.org/10.1093/hropen/hoae049</a></p><p dir="ltr"><br>II. Organoid formation and explant-tissue culture of leukaemia-infiltrated prepubertal testicular tissue.</p><p dir="ltr"><b>Yanhua Cui</b>, Jouko Lohi, Cecilia Lindskog, Kirsi Jahnukainen, Jan-Bernd Stukenborg. [Manuscript]</p><p dir="ltr">III. Spermatogonia loss correlates with LAMA 1 expression in human prepubertal testes stored for fertility preservation.</p><p dir="ltr">Magdalena Kurek, Elisabet Åkesson, Masahito Yoshihara, Elizabeth Oliver, <b>Yanhua Cui</b>, Martin Becker, João Pedro Alves-Lopes, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Cecilia Petersen, Olle Söder, Jingtao Guo, Rod T Mitchell, Kirsi Jahnukainen, Jan-Bernd Stukenborg Cells. 2021 Jan 27;10(2):241.<br><a href="https://doi.org/10.3390/cells10020241" rel="noreferrer" target="_blank">https://doi.org/10.3390/cells10020241</a></p><p dir="ltr"><br>IV. Generation of testicular organoids in synthetic defined hydrogel.</p><p dir="ltr"><b>Yanhua Cui</b>, Susana M. Chuva de Sousa Lopes, Jan-Bernd Stukenborg. [Manuscript]</p>