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

Abstract

Background 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. Methods 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. Results 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. Conclusions 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.

1. Introduction

The prostate is a crucial organ unique to males, positioned inferior to the bladder, approximately the size of a walnut, composed of ducts with an inner layer of epithelium surrounded by stroma. Its primary functions include controlling urination and secreting prostatic fluid, which are of paramount importance to reproductive health [1]. The prostate is derived from the urogenital sinus (UGS) through a series of events including pre-budding, budding, bud elongation and branching, canalization of the solid epithelial cords, and luminal and basal epithelial cell differentiation [1]. The UGS comprises the urogenital sinus epithelium (UGE) and the urogenital sinus mesenchyme (UGM). The nascent prostate is composed of solid epithelial cords in the initial phase of branching morphogenesis. Subsequently, these epithelial cords undergo canalicular differentiation to form glandular structures, with epithelial cells differentiating into basal, luminal, and neuroendocrine lineages, ultimately leading to the formation of a complete prostatic structure [2]. Through the analysis of cellular lineage, it is evident that the entire length of the primitive urogenital sinus from which the prostate originates, is derived from the endoderm [3].

The prostate forms through epithelial budding from the UGS which is initiated by the action of androgens: during the 9–10 weeks of human embryonic development, Leydig cell-secreted dihydrotestosterone (DHT) binds to androgen receptors (AR) and then enters the nucleus to bind to androgen response elements (ARE) in chromatin, thereby activating AR downstream signals in the UGM [4]. Subsequently, mesenchymal AR activates a paracrine signal that acts on UGE to stimulate the formation of buds [5]. As a pivotal transcription factor in endodermal cells, FOXA1 functions as a pioneer factor and enhances the interaction with chromatin, thereby facilitating the binding of AR to the corresponding androgen receptor response elements and promoting transcription efficiency [6]. The high expression level