Small molecules enhance the high-efficiency generation of pancreatic ductal organoids

Abstract

Advancements in three-dimensional (3D) organoid cultures have created more physiologically relevant models for pancreatic disease research, but efficiently generating mature pancreatic ductal cells remains challenging. In this study, we develop a novel protocol to generate pancreatic ductal organoids (PDOs) with high initiation efficiency and an enrichment of pancreatic ductal cells. By utilizing a cocktail of small molecules, we optimize the culture conditions to improve organoid formation. Our findings demonstrate that this protocol facilitates the formation and expansion of PDOs derived from Sox9-positive ductal cells, including heterogeneous ductal cells and acinar cells. These organoid cultures exhibit remarkable stability, supporting long-term expansion. This system provides an efficient model with potential applications in high-throughput drug screening. Moreover, these organoids recapitulate the exocrine cell composition and may reflect the cellular plasticity between ductal and acinar cells, providing a valuable platform for investigating pancreatic diseases such as pancreatic ductal adenocarcinoma (PDAC). The model presents a promising tool for future research aimed at understanding disease mechanisms and potentially helping drug development for pancreatic disorders.

1. Introduction

The pancreas is a vital organ with dual functionality, consisting of endocrine and exocrine compartments. The endocrine compartment comprises islets containing alpha, beta, delta, epsilon and PP cells, which regulate blood glucose level by secreting hormones such as insulin and glucagon [1]. The exocrine compartment comprises acinar cells that produce digestive enzymes and ductal cells that transport these enzymes to the gastrointestinal tract for nutrient absorption [2]. Studies have shown that pancreatic ducts, which include various ductal cell types, such as large ducts, small ducts, terminal ducts, intrapancreatic ducts, and pancreaticobiliary ducts, are heterogeneous [3]. Additionally, Krt19-, Hnf1β-, and Sox9-positive cells exist in dynamic equilibrium and represent different ductal subsets [4].

Despite its critical role, the pancreas is susceptible to numerous diseases. PDAC, which primarily originates from ductal cells, is one of the most lethal malignancies due to its late diagnosis and rapid progression [5]. Cystic fibrosis affects the pancreas by altering ductal cell function [6], resulting in thick mucus that obstructs enzyme flow [7]. Pancreatitis, often triggered by enzyme blockage within ducts, causes severe inflammation and damage to both exocrine and endocrine tissues [8]. Studying the detailed mechanisms by which diseases affect pancreatic cell types is crucial for developing effective diagnostics and treatments [9,10], which require model systems that faithfully mimic pancreatic biology.

The emergence of 3D organoid cultures offers a promising new model for studying pancreatic diseases [11,12], particularly those involving ductal cell dysfunction [13]. Pancreatic organoids, engineered to mimic the cellular composition, structure, and function of the pancreas, are derived from pluripotent stem cells, embryonic pancreatic progenitors, and adult pancreatic cells [14–20]. These organoids provide a controlled, physiologically relevant model system that surpasses traditional in vitro and animal models in probing complex disease mechanisms and assessing therapeutic efficacy [21]. During organogenesis, multiple signaling pathways work in concert to regulate cell differentiation and development. Epidermal growth factor (EGF) supports normal pancreatic growth by modulating cell proliferation and development [22,23]. The precise regulation of various signaling pathways ensures proper pancreatic development [24,25]. Small molecule compounds offer targeted intervention by selectively modulating specific pathways [26,27]. Consequently, the combination of small molecule compounds with organoid culture techniques facilitates the precise reconstruction of organ-like structures in vitro, closely replicating in vivo [28,29].