Department of Urology, Department of Biological Repositories, Frontier Science Center for Immunology and Metabolism, Medical Research Institute, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, 430071, China.
Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan, 430071, China.
Stem Cell Res Ther. 2024 Sep 2;15(1):274. doi: 10.1186/s13287-024-03886-y.
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.
了解人类前列腺的谱系分化不仅对人类发育生物学的基础研究至关重要，而且对前列腺相关疾病的治疗也有重要贡献。目前的知识主要依赖于啮齿动物模型的研究，尽管临床样本可能提供特定阶段的快照，但缺乏人类来源的替代物。人类胚胎干细胞可以产生包括前列腺在内的所有胚胎谱系，事实上，一些研究基于与泌尿生殖嵴间质的共培养或共移植到小鼠肾囊中来证明了这种可能性。
为了从人类胚胎干细胞体外获得前列腺类器官，我们通过模拟转录因子和信号转导途径的调控网络应用化学物质和生长因子，并构建携带可诱导 NKX3-1 表达盒的细胞系，以及三维培养系统。未配对 t 检验用于统计分析。
我们首先成功地生成了确定的内胚层、后肠和泌尿生殖窦细胞。胚胎干细胞衍生的泌尿生殖窦细胞表达前列腺关键转录因子 AR 和 FOXA1，但不能表达 NKX3-1。因此，我们通过同源重组构建了 NKX3-1 诱导细胞系，最终通过逐步分化能够产生 AR、FOXA1 和 NKX3-1 三重阳性的泌尿生殖前列腺谱系细胞。最后，结合 3D 培养，我们成功地获得了具有一定结构和前列腺细胞群的前列腺样类器官。
这项研究揭示了 NKX3-1 在前列腺分化中的关键作用，并提供了可诱导的 NKX3-1 细胞系，以及逐步分化方案来生成人类前列腺样类器官，这应该有助于前列腺发育和疾病发病机制的研究。
