# Prostatic lineage differentiation from human embryonic stem cells through inducible expression of NKX3-1

## TL;DR

This study shows how to grow human prostate-like structures in the lab using stem cells and a key protein called NKX3-1, which could help understand prostate development and diseases.

## Contribution

The study introduces an inducible NKX3-1 cell line and a stepwise protocol for generating human prostate-like organoids from embryonic stem cells.

## Key findings

- Human embryonic stem cells can be differentiated into urogenital sinus cells expressing AR and FOXA1 but not NKX3-1.
- An inducible NKX3-1 cell line successfully generates triple-positive prostatic lineage cells.
- 3D culture combined with the protocol yields prostate-like organoids with specific structures and cell populations.

## Abstract

Understanding the lineage differentiation of human prostate not only is crucial for basic research on human developmental biology but also significantly contributes to the management of prostate-related disorders. Current knowledge mainly relies on studies on rodent models, lacking human-derived alternatives despite clinical samples may provide a snapshot at certain stage. Human embryonic stem cells can generate all the embryonic lineages including the prostate, and indeed a few studies demonstrate such possibility based on co-culture or co-transplantation with urogenital mesenchyme into mouse renal capsule.

To establish a stepwise protocol to obtain prostatic organoids in vitro from human embryonic stem cells, we apply chemicals and growth factors by mimicking the regulation network of transcription factors and signal transduction pathways, and construct cell lines carrying an inducible NKX3-1 expressing cassette, together with three-dimensional culture system. Unpaired t test was applied for statistical analyses.

We first successfully generate the definitive endoderm, hindgut, and urogenital sinus cells. The embryonic stem cell-derived urogenital sinus cells express prostatic key transcription factors AR and FOXA1, but fail to express NKX3-1. Therefore, we construct NKX3-1-inducible cell line by homologous recombination, which is eventually able to yield AR, FOXA1, and NKX3-1 triple-positive urogenital prostatic lineage cells through stepwise differentiation. Finally, combined with 3D culture we successfully derive prostate-like organoids with certain structures and prostatic cell populations.

This study reveals the crucial role of NKX3-1 in prostatic differentiation and offers the inducible NKX3-1 cell line, as well as provides a stepwise differentiation protocol to generate human prostate-like organoids, which should facilitate the studies on prostate development and disease pathogenesis.

The online version contains supplementary material available at 10.1186/s13287-024-03886-y.