Novel culture systems to model testicular function and organization

In vitro systems to model the testicular microenvironment are required to study physiology and pathology of the testis, the gonadotoxic effect of environmental and pharmaceutical chemicals, as well as to explore the mechanisms ruling spermatogonial stem cell (SSC) selfrenewal and differentiation. The knowledge produced in in vitro experiments, using animal, but most importantly utilizing human models, will contribute to the discovery of safe medical treatments and might provide translational tools to treat male infertility problems. The current models are vital to study the testicular microenvironment in vitro, but further improvements are required to more closely reconstruct the testicular organization and function found in vivo. The main focus of the research conducted in this thesis was to establish and optimize threedimensional culture conditions to further improve the current approaches to model testicular architecture and physiology in vitro. For that purpose, we explore three distinct approaches: the three-layer gradient system (3-LGS) to culture rat testicular cells suspended in extracellular matrix (ECM) components; the decellularized testicular extracellular matrix (DTM) to culture human testicular cells in their native ECM; and the organ culture system in the air-liquid interface to culture human testicular cells in their native tissue organization. During our experiments, we found that the 3-LGS promoted the reorganization of rat testicular cells into seminiferous tubule-like organoids with both a functional blood-testis barrier and the capacity to maintain proliferative undifferentiated germ cells. Moreover, the DTM allowed the generation of human testicular organoids that, despite not demonstrating characteristic testicular organization, were able to produce testosterone and inhibin B as well as to maintain spermatogonia proliferating during the entire culture period. Finally, the organ culture system served to maintain human testicular tissue in viable conditions and to demonstrate differences in terms of testicular somatic cell functionality among different patient groups exposed to chemotherapy or treatments against haematological diseases. In perspective, the rat and human organoid systems demonstrate potential to explore aspects of testicular development and toxicology, but also the possibility to, easier than before, manipulate the culture conditions to study the influence of different niche elements on SSC self-renewal and differentiation. Moreover, the organ culture system showed the aptitude to be used as a quality control tool in the assessment of bio-banked human testicular tissue and to help in determining the best-fit fertility preservation strategy for each group of patients. LIST OF SCIENTIFIC PAPERS I. João Pedro Alves-Lopes, Olle Söder & Jan-Bernd Stukenborg Use of a three-layer gradient system of cells for rat testicular organoid generation Nature Protocols, 2018, volume 13, pages 248–259 DOI:10.1038/nprot.2017. 140 II. João Pedro Alves-Lopes, Olle Söder & Jan-Bernd Stukenborg Testicular organoid generation by a novel in vitro three-layer gradient system Biomaterials, 2017, volume 130, pages 76–89 DOI: 10.1016/j.biomaterials. 2017.03. 025 III. Yoni Baert, Joery De Kock, João Pedro Alves-Lopes, Olle Söder, Jan-Bernd Stukenborg and Ellen Goossens Primary Human Testicular Cells Self-Organize into Organoids with Testicular Properties Stem Cell Reports, 2017, volume 8, pages 30–38 DOI: 10.1016/j.stemcr. 2016.11. 012 IV. João Pedro Alves-Lopes, Magdalena Kurek, Halima Albalushi, Olle Söder, Rod Mitchell, Cecilia Petersen, Kirsi Jahnukainen and Jan-Bernd Stukenborg In vitro assessment of Sertoli and Leydig cell function in boys subjected to treatment for haematological and oncological diseases Manuscript LIST OF SCIENTIFIC PAPERS (not included in the thesis) I. João Pedro Alves-Lopes and Jan-Bernd Stukenborg Testicular organoids: a new model to study the testicular microenvironment in vitro? Human Reproduction Update, 2018, volume 24, pages 176–191 DOI: 10.1093/humupd/dmx036 II. Jan-Bernd Stukenborg, João Pedro Alves-Lopes, Magdalena Kurek, Halima Albalushi, Ahmed Reda, Victoria Keros, Virpi Tohonen, Ragnar Bjarnason, Patrik Romerius, Mikael Sundin, Ulrika Noren Nystrom, Cecilia Langenskiöld, Hartmut Vogt, Lars Henningsohn, Rod Mitchell, Olle Söder, Cecilia Petersen and Kirsi Jahnukainen Spermatogonial quantity in human prepubertal testicular tissue collected for fertility preservation prior to potentially sterilizing therapy Human Reproduction, 2018 (in press) DOI:10.1093/humrep/dey240 TABLE OF CONTENTS