Budi Nova Yuli Prasetyo, Lai Wei-Yu, Huang Yen-Hua, Ho Hong-Nerng
International Ph.D. Program in Cell Therapy and Regenerative Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Front Cell Dev Biol. 2024 Jul 8;12:1361084. doi: 10.3389/fcell.2024.1361084. eCollection 2024.
Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC-derived cholangiocytes expressed markers of mature cholangiocytes, such as , , and cystic fibrosis transmembrane conductance regulator (). Compared with the 2D culture system-generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes and and extrahepatic cholangiocytes and . Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.
特发性胆管病是影响胆管细胞的疾病，其病因不明。目前，原位肝移植是终末期肝病唯一可用的治疗方法。胆管获取受限使得胆管细胞疾病建模困难。在本研究中，通过模拟胆管细胞的胚胎发育并使用一种强大的、无饲养层和无血清方案，我们首次证明了由源自人多能干细胞（PSC）的小胆管细胞和大胆管细胞组成的独特功能性三维类器官的生成，这与传统的二维培养系统不同。在分化的第28天，人PSC衍生的胆管细胞表达成熟胆管细胞的标志物，如 、 和囊性纤维化跨膜传导调节因子（ ）。与二维培养系统产生的胆管细胞相比，三维胆管细胞类器官（CO）显示肝内胆管细胞 和 的区域特异性标志物以及肝外胆管细胞 和 的表达更高。此外，CO具有小到大的管状结构，功能分析表明它们表现出成熟胆管细胞的特征，如多药耐药蛋白1转运功能和 通道活性。除了细胞外基质支持外，表皮生长因子受体（EGFR）介导的信号调节可能参与了这种胆管细胞的成熟和分化。这些结果表明通过我们的三维类器官方案成功生成了肝内和肝外胆管细胞。结果突出了我们的三维培养系统相对于二维培养系统在促进胆管细胞功能分化和成熟方面的优势。总之，在先前工作之前，我们的研究提供了一种在经济高效的三维培养条件下人PSC小到大胆管细胞转分化的可能概念。研究结果对使用COs对胆管病患者进行有效的细胞治疗的发展具有启示意义。
