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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., (© 2024. The Author(s).)

Pathological or functional loss of pancreatic beta cells is the cause of diabetes. Understanding how signaling pathways regulate pancreatic lineage and searching for combinations of signal modulators to promote pancreatic differentiation will definitely facilitate the robust generation of functional beta cells for curing hyperglycemia. In this study, we first tested the effect of several potent BMP inhibitors on pancreatic differentiation using human embryonic stem cells. Next, we examined the endodermal lineage bias upon potent BMP inhibitor treatment and further checked the crosstalk between signal pathways governing endodermal lineage determination. Furthermore, we improved current pancreatic differentiation system based on the signaling pathway study. Finally, we used human-induced pluripotent stem cells to validate our finding. We found BMP inhibitors indeed not only blocked hepatic lineage but also impeded intestinal lineage from human definitive endoderm unexpectedly. Signaling pathway analysis indicated potent BMP inhibitor resulted in the decrease of WNT signal activity and inhibition of WNT could contribute to the improved pancreatic differentiation. Herein, we combined the dual inhibition of BMP and WNT signaling and greatly enhanced human pancreatic progenitor differentiation as well as beta cell generation from both embryonic stem cells and induced pluripotent stem cells. Conclusively, our present work identified the crosstalk between the BMP and WNT signal pathways during human endoderm patterning and pancreas specification, and provided an improved in vitro pancreatic directed differentiation protocol from human pluripotent stem cells. [ABSTRACT FROM AUTHOR]

Highlights from the article: Islet transplantation has been demonstrated to be able to cure diabetes caused by the loss of functional pancreatic cells; however, it is severely limited by the shortage of cadaveric islet donor [1]. In addition, the architecture of islets cannot be simply replaced by single SC- cells, thus engineered islet-like structure or islet organoid containing major cell types would be emergently required [6]. 8 W. Jiang, X. Sui, D. Zhang, CD24: A novel surface marker for pdx1-positive pancreatic progenitors derived from human embryonic stem cells, Stem Cells, 29, 2011, 609-617Jiang W, Sui X, Zhang D, et al.

Metabolic remodeling is one of the earliest events that occur during cell differentiation. Here, we define fatty acid metabolism as a key player in definitive endoderm differentiation from human embryonic stem cells. Fatty acid β-oxidation is enhanced while lipogenesis is decreased, and this is due to the phosphorylation of lipogenic enzyme acetyl-CoA carboxylase by AMPK. More importantly, inhibition of fatty acid synthesis by either its inhibitors or AMPK agonist significantly promotes human endoderm differentiation, while blockade of fatty acid oxidation impairs differentiation. Mechanistically, reduced de novo fatty acid synthesis and enhanced fatty acid β-oxidation both contribute to the accumulation of intracellular acetyl-CoA, which guarantees the acetylation of SMAD3 and further causes nuclear localization to promote endoderm differentiation. Thus, our current study identifies a fatty acid synthesis/oxidation shift during early differentiation and presents an instructive role for fatty acid metabolism in regulating human endoderm differentiation. [Display omitted] • DE cells are characterized by elevated FAO and reduced FAS • ACC is phosphorylated by AMPK, resulting in FAS reduction during DE differentiation • Elevated FAO and reduced FAS are required for endoderm differentiation • FA metabolism affects DE differentiation by interfering with the acetylation of SMAD3 Yi et al. find that fatty acid synthesis is downregulated, while fatty acid β-oxidation is enhanced during human endoderm differentiation, both of which are required for endoderm differentiation because they interfere with the acetylation of SMAD3 by accumulating acetyl-CoA and further causing the nuclear localization. [ABSTRACT FROM AUTHOR]