An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development

Ingrid M Knarston; Svenja Pachernegg; Gorjana Robevska; Irene Ghobrial; Pei Xuan Er; Elizabeth Georges; Minoru Takasato; Alexander N Combes; Anne Jørgensen; Melissa H Little; Andrew H Sinclair; Katie L Ayers

doi:10.1016/j.stemcr.2020.10.009

An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development

Stem Cell Reports. 2020 Dec 8;15(6):1377-1391. doi: 10.1016/j.stemcr.2020.10.009. Epub 2020 Nov 19.

Affiliations

¹ Murdoch Children's Research Institute, Melbourne, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Australia.
² Murdoch Children's Research Institute, Melbourne, Australia.
³ RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
⁴ Murdoch Children's Research Institute, Melbourne, Australia; Monash Biomedicine Discovery Institute, Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.
⁵ Department of Growth and Reproduction, Copenhagen University Hospital, Copenhagen, Denmark.
⁶ Murdoch Children's Research Institute, Melbourne, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Australia; Department of Anatomy and Neuroscience, The University of Melbourne, Melbourne, Australia.
⁷ Murdoch Children's Research Institute, Melbourne, Australia; Department of Paediatrics, The University of Melbourne, Melbourne, Australia. Electronic address: katie.ayers@mcri.edu.au.

Abstract

Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development.

Keywords: DSD; differences in sex development; disorders of sex development; embryonic development; gonad; gonadal development; human induced pluripotent stem cells; organoid culture; stem cell differentiation; testis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Antigens, Differentiation / biosynthesis*
Cell Differentiation*
Embryo, Mammalian / cytology
Embryo, Mammalian / embryology*
Humans
Male
Sertoli Cells / metabolism*
Tissue Culture Techniques

Substances

Antigens, Differentiation