Artificial testis to study early gonadal development and germ cell differentiation

Studying the early germ cell development is of great significance, as germ cells transfer the genetic material between generations. However, in humans, this type of studies in vivo is not applicable, for obvious ethical reasons. Interestingly, differentiating human pluripotent stem cells (hPS cells) towards germ cells in vitro was recently reported. Therefore, studying differentiation of hPS cells towards germ cells is intriguing. On the other hand, due to the advancement in cancer therapy, the survival rates of the patients receiving chemotherapy and radiotherapy are improving dramatically. However, the improved survival rates increased the demand on fertility preservation, since most of the cancer treatments are gonadotoxic. In case of pubertal and post-pubertal male patients, semen cryopreservation offers a feasible and efficient option. Meanwhile, pre-pubertal patients do not have this option. Thus, finding an option for or such patients is absolutely encouraged. Hence, we aimed in this thesis at studying the early development and differentiation of the male germ cells in vitro, in order to find a robust protocol for fertility preservation for prepubertal male patients. Specifically, we investigated whether the culture conditions and gene expression profile could be used to predict the differentiation potential of human embryonic stem (hES) cells towards male germ cells, tried to optimize the culture conditions for rat germ cell differentiation using a three-dimensional (3D) culture system, investigated the possibility of differentiating rat germ cells in vitro from immature testicular tissue, and studied the effects of long term in vitro culture on testicular tissues from pre-pubertal patients. We have found that undifferentiated hES cell lines exhibit different gene expression profiles. Furthermore, the suspension culture method resulted in downregulation of the pluripotency markers NANOG and POU5F1, compared to the culture on feeders. In addition, BMP7 stimulation resulted in an upregulation in the germ cell markers KIT and DDX4 and somatic cell markers FSHR and HS3BD1. To study the optimal culture conditions for germ cell differentiation in vitro, rat testicular cells were cultured in a 3D culture system. We found that the choice of medium has an effect on Leydig cell functionality. In addition, the germ cells were migrating outwards the cell aggregations, making the conditions unfavorable for germ cell differentiation. Exploiting a reported method for murine germ cell differentiation in vitro to other species, we have obtained round spermatids expressing Crem and Acrosin (postmeiotic markers) from rat undifferentiated spermatogonia, using the organ culture system and MEMα medium + 10% KSR. Interestingly, when we cultured human pre-pubertal testicular tissue for long term in vitro using an organ culture system, we have found that the Leydig and Sertoli cells showed viability and functionality for up to 42 days and 21 days respectively. In conclusion, we have assessed the effect of culture conditions on the differentiation potential of hES cells towards male germ cells in vitro. We have also investigated the culture conditions suitable for rat germ cell differentiation in vitro using a 3D culture system or an organ culture setup. In addition, we have also studied the effect of long term culture of human pre-pubertal testicular tissue in vitro. LIST OF SCIENTIFIC PAPERS I. Kristín Rós Kjartansdóttir, Ahmed Reda, Sarita Panula, Kelly Day, Kjell Hultenby, Olle Söder, Outi Hovatta, Jan-Bernd Stukenborg A combination of culture conditions and gene expression analysis can be used to investigate and predict hES cell differentiation potential towards male gonadal cells PLoS ONE, 2015, 10, 12 II. Ahmed Reda, Mi Hou, Luise Landreh, Kristín Rós Kjartansdóttir, Konstantin Svechnikov, Olle Söder, Jan-Bernd Stukenborg In vitro spermatogenesis – optimal culture conditions for testicular cell survival, germ cell differentiation, and steroidogenesis in rats Frontiers in Endocrinology, 2014, 5, 21 III. Ahmed Reda, Mi Hou, Timothy R Winton, Robert E Chapin, Olle Söder, JanBernd Stukenborg In vitro differentiation of rat spermatogonia into round spermatids in tissue culture Published (Epub ahead of print) in MHR, 2016, DOI: 10.1093/molehr/gaw047 IV. Jan-Bernd Stukenborg*, Ahmed Reda*, Joao Pedro Alves-Lopes*, Victoria Keros, Virpi Töhönen, Ragnar Bjarnason, Patrik Romerius, Michael Sundin, Ulrika Norén Nyström, Cecilia Langenskiöld, Rod T. Mitchell, Olle Söder, Kirsi Jahnukainen, Cecilia Petersen Long-term culture of human testicular tissue from pre-pubertal boys subjected to gonadotoxic treatment regimens Manuscript (* these authors contributed equally to the work) ADDITIONAL PUBLICATIONS NOT INCLUDED IN THE THESIS 1Kristín Rós Kjartansdóttir, Anette Gabrielsen, Ahmed Reda, Olle Söder, Rosita Bergström-Tengzelius, Claus Yding Andersen, Outi Hovatta, Jan-Bernd Stukenborg *, Jens Fedder* Differentiation of stem cells upon deprivation of exogenous FGF2: a general approach to study spontaneous differentiation of hESCs in vitro Systems Biology in Reproductive Medicine, 2012, 58: 330–338 2Jan-Bernd Stukenborg*, Kristín Rós Kjartansdóttir*, Ahmed Reda, Eugenia Colon, Jan Philipp Albersmeier, Olle Söder Male germ cell development in humans Horm Res Paediatr, 2014; 81:2–12 3Maha Al-Asmakh*, Jan-Bernd Stukenborg*, Ahmed Reda, Farhana Anuar, Mona-Lisa Strand, Lars Hedin, Sven Pettersson, Olle Söder The gut microbiota and developmental programming of the testis in mice PLoS ONE, 2014, 9, 8 (* these authors contributed equally to the work) CONTENTS