ESPE Abstracts

Disorders of sex development (DSD) constitute an array of rare disorders affecting the genito-urinary tract and the endocrine-reproductive system and are often identified in the newborn or adolescent. Gene mutations causing DSD are slowly being identified using high-throughput sequencing, but the interpretation of the data and ascribing causality to novel variants is challenging. This is because DSD mutations occur in multiple genes with each gene affecting a small number of individuals and suitable animal/cellular models that accurately reflect the physiopathology of DSD are lacking. To establish causality of genetic variants causing DSD and understand the mechanisms of disease development and progression we have developed multiple novel models by cellular reprogramming.

One of the novel ex-vivo cellular models we have developed involves directed differentiation of human induced pluripotent stem cells (IPSCs) into somatic cells of the XY or XX gonad using defined conditioned media. Briefly, 46,XY iPSCs are subjected to directed differentiation sequentially using a conditioned medium containing defined concentrations of bFGF, BMP4 and retinoic acid and insulin transferrin selenium. The iPSCs derived from a healthy male can differentiate to the mesoderm and intermediate mesoderm (IM), as indicated by the expression of stage appropriate markers. The IM undergoes mesenchymal-to-epithelium transition and eventually results in formation of fœtal Sertoli-like cells. These cells express Sertoli specific transcripts and have the ability to self-aggregate and form tubule-like structures similar to embryonic Sertoli cells. When these protocols are applied to iPSCs with a 46,XX chromosome complement, the resultant population shows a granulosa-like profile. We have also derived iPSCs from erythroblasts of a 46,XY individual with complete gonadal dysgenesis due to a missense mutation in NR5A1. When these cells undergo similar differentiation protocols, we observe an aberrant expression profile and an inability to form tubular structures. For optimal recapitulation of both the structure and function of the developing embryonic testis, we are developing a gonad-on-chip device that realizes the self-organization of iPSC derived Sertoli cells within a perfusible microfluidic device. Our new model is a powerful tool to investigate patient-specific atypical transcription networks as well as provide mechanistic insight into the stage specific dysregulation of signaling, associated with mutations causing DSD.