Oral Virtual Presentation (Virtual only) ESA-SRB-ANZBMS 2021

A novel in vitro differentiation protocol for human embryonic gonadal organoids as a disease model for differences of sex development.   (#241)

Katie Ayers 1 2 , Svenja Pachernegg 1 , Ingrid Knarston 1 , Natalie Charitakis 1 , Gorjana Robevska 1 , Irene Ghobrial 1 , Pei Xuan Er 1 , Minoru Takasato 3 , Elizabeth Georges 1 , Anne Joergensen 4 , Alexander Combes 5 , Melissa Little 1 2 , Andrew Sinclair 1 2
  1. Murdoch Childrens Research Institute, Parkville, Vic, Australia
  2. Department of Paediatrics, The University of Melbourne, Melbourne, Australia
  3. RIKEN Center for Biosystems Dynamics Research, Kobe, Japan
  4. Department of Growth and Reproduction, Copenhagen University Hospital, Copenhagen, Denmark
  5. Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, , Monash University, Melbourne, Australia

Differences of Sex Development affect an alarming 1.7% of babies and can be caused by aberrations in the genetic pathways that control embryonic gonadal development. Currently, less than 40% of children born with a 46,XY DSD receive a genetic diagnosis, leaving 60% with either a negative genetic finding or one of uncertain significance. To improve the diagnostic rate, functional validation of novel or uncertain genetic findings is required, something that is currently extremely challenging in DSD due to the lack of a human embryonic gonadal cell line, and the near impossibility of obtaining primary human embryonic gonadal tissue. Therefore, there is an urgent need for an in vitro model of human gonadal development to study these disorders. We have addressed this by establishing a world-first protocol to differentiate human induced pluripotent stem cells (iPSCs) into early gonadal cells cultured as testis-like organoids. Our stepwise differentiation protocol uses small molecules to mimic developmental signalling, inducing iPSCs to develop into the bipotential gonad by day 7. Aggregating and culturing these cells as 3D organoids results in testis gene expression in cells that reside within tube-like structures delineated by basement membrane, reminiscent of cord-like assemblies in re-aggregated mouse testes. Transcriptomic profiling of organoids using single cell RNA sequencing shows gonadal and reproductive tissue identities, and distinct testicular cell lineages which overlap with those found in human fetal gonads. Disease modelling using an iPSC line carrying a novel genetic mutation implicated in gonadal dysgenesis results in gonad organoids with reduced growth, altered structure and accelerated cell death. Here we discuss the ongoing work to expand this methodology to include additional more mature testis cell types, to produce an organoid that fully recapitulates the human fetal testis. This innovative disease model would bring about a paradigm shift in the study of fetal gonadal health.