(225) Modeling Early Gonad Development in Klinefelter Syndrome Patients Using Pluripotent Stem Cell-derived Organoids

Juliana I. Candelaria^1,2, Marina V. Pryzhkova^1,2, Philip W. Jordan^1,3

1. Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

2. The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA

3. Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA

 

Abstract Text:

Klinefelter syndrome (KS) (47, XXY) is the most common sex chromosomal aneuploidy disorder, affecting ~1 in 650 males and causing non-obstructive azoospermia. Although traditionally thought to initiate testicular degeneration and hypogonadism at the onset of puberty, accumulating data from fetal tissue analyses demonstrate that KS impairs fetal gonad development. Fetal KS gonads exhibit arrested germ cell development, and several studies suggest Sertoli cells are a key player in KS physiopathology. However, a better understanding of molecular mechanisms driving aberrant testis development in KS patients has been greatly hindered due to limitations in studying human fetal samples. For this reason, the advent of 3D cell-based organoid models has become an alternative approach to research fetal gonad formation. In this study, we performed stepwise differentiation of KS and normal karyotype (46,XY) male human embryonic stem cells to generate gonad-like organoids. We hypothesize that KS gonadal organoid models would reflect similar abnormal characteristics seen in fetal and adult human KS testis and be atypical compared to normal karyotype organoids. We evaluated KS and normal karyotype organoids from day 16 to day 40 of differentiation by using immunohistochemistry to determine presence or absence of male-specific gonadal cells such as Sertoli (WT1, SOX9), Leydig (CYP11A1, StAR, 3β-HSD), endothelial (VE-Cadherin, CD31), peritubular myoid (αSMA) cells, as well as tight junction proteins (ZO1, Occludin). Additionally, excess X chromosome dosage may lead to dysregulated cell signaling and function in KS testes. Therefore, we assessed the transcriptomic profiles of KS and normal karyotype organoids using RNA sequencing and examined the X chromosome inactivation (XCI) status of various cell populations. First, we found that KS gonadal organoids grow slower and are smaller compared to normal organoids. We also found that KS and normal organoids similarly show mesonephros-like tubule formation at day 16 of differentiation and express Sertoli cell markers WT1, SOX9, and tight junction marker ZO1 by day 22. Additional male-specific gonadal cell markers are currently being assessed. Ongoing RNA sequencing analyses aim to elucidate differentially expressed genes and changes in cell signaling pathways between KS and normal organoids at specific points of development. Likewise, assessing the patterning of XIST expression will provide insight into the effects of XCI on gonadal cell differentiation and organization within organoids. In this study, we present for the first time an in vitro organoid model for KS gonadogenesis. Collectively, this work extends our current knowledge of causes of infertility in KS patients and enables further study of gametogenesis failure. Overall, our work aids in the future development of novel assisted reproductive technologies such as in vitro gametogenesis and offer new gonadal organoid methods for drug, therapeutic, and toxicology screenings for human patients suffering from genetic disorders.