The emergence of multiple testicular cell lineages in human stem cell-derived testis-like organoids
Reproductive development is a complex process orchestrated by precise gene expression and cellular interactions. Disruption to this process can result in differences of sex development (DSDs) which occur in approximately 1-2% of live births. We have previously developed a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into testis-like organoids. In this study, we performed bulk and single-cell RNA-sequencing on these organoids to investigate their transcriptional landscape. Transcriptomic analysis revealed six distinct cell clusters expressing markers associated with bipotential, early Sertoli and testicular interstitial cells. These findings provide the first comprehensive transcriptional profile of hiPSC-derived testis-like organoids. Additionally, to address the limited emergence of mature cell types, we generated an inducible NR5A1/SF1 hiPSC line, which successfully triggered the upregulation of Leydig cell markers and additional Sertoli markers upon overexpression. Our findings show that our testis-like organoids are a valuable model system for studying DSDs in vitro.
Authors
- Mirana Ramialison (ORCID: https://orcid.org/0000-0001-6315-4777)
- Fernando J. Rossello (ORCID: https://orcid.org/0000-0003-3885-8777)
- Ben Rollo.
- Denis Bienroth (ORCID: https://orcid.org/0000-0002-0574-8259)
- Natalie Charitakis (ORCID: https://orcid.org/0000-0002-9592-6257)
- Jan Terhag
- Melissa H. Little (ORCID: https://orcid.org/0000-0003-0380-2263)
- Jocelyn A. van den Bergen
- Jinchao Gu (ORCID: https://orcid.org/0009-0008-5982-465X)
- Sean B. Wilson (ORCID: https://orcid.org/0000-0002-8994-0781)
- Svenja Pachernegg (ORCID: https://orcid.org/0000-0001-6555-9399)
- Gorjana Robevska (ORCID: https://orcid.org/0000-0002-9407-8460)
- Katie L Ayers
- Eliza Martin
- Andrew H. Sinclair
- Lucas G.A. Ferreira
Institutions
- University of Copenhagen (DK)
- The University of Melbourne (AU)
- Novo Nordisk Foundation (DK)
- Australian Regenerative Medicine Institute (AU)
- Murdoch Children's Research Institute (AU)
- Monash University (AU)
Publication Details
- Journal
- Development
- Published
- 2026-02-23
- DOI
- https://doi.org/10.1242/dev.204772
- Primary Topic
- Genetic and Clinical Aspects of Sex Determination and Chromosomal Abnormalities
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Australian Government
- Novo Nordisk
- State Government of Victoria
- Novo Nordisk Fonden
- Cybec Foundation
- National Stem Cell Foundation of Australia
- Medical Research Council
- National Health and Medical Research Council