Single-Cell RNA Sequencing Pinpoints Exocrine Bipotentiality of Human Pancreatic Organoids

Objective: Despite organoid formation and characterization from different tissues have been extensively studied, pancreas remains one among the least investigated organs. Here we proposed a multifaced characterization of cell types present in human pancreatic organoids (hPO) and starting from their progenitor we described their maturation and their lineage relationships. Design: We performed unbiased transcriptome-wide scRNA-seq analysis on 3187 cells of human pancreatic organoids obtained from 3 healthy islet-depleted tissue. To support our results, we strengthened these findings with proteomic and qRT-PCR analyses performed during the long-term culture of hPO. Results: First, we described hPO focusing on their morphology and long-term culture molecular aspects and showing a well-defined epithelial organization supported by a genetic stability through all cell passages. Successively, based on single-cell RNA sequencing technology, we defined all cell types present in hPO confirming that our pancreatic organoid methodology is highly reproducible. Moreover, exploiting the latest bioinformatic tools and comparing our data with recently published ones we reshaped the conventional theory of univocal ductal nature of hPO. Finally, we defined a new strategy to sort a refined multipotent progenitor from hPO and we gave a new definition of pancreatic progenitor demonstrating its capacity to renew the hPO stem population while maintaining its heterogeneity. Conclusions: Taken together, our results give a new definition of human pancreatic organoids, which reveals the complex nature of these structures and provides a strong basis to pave the way for future study in the pancreatic field. Single cell RNA-seq profile of human pancreatic organoids obtained from 3 healthy islet-depleted tissue

研究目标：尽管不同组织的类器官（organoid）构建与表征已得到广泛研究，但胰腺仍是研究最少的器官之一。本研究针对人胰腺类器官（human pancreatic organoids，简称hPO）内的细胞类型开展多维度表征，并从其祖细胞出发，系统描述了该类器官的成熟过程与谱系关联。
实验设计：我们对从3份健康胰岛缺失组织中分离得到的人胰腺类器官的3187个细胞，开展了无偏倚全转录组层面的单细胞RNA测序（single-cell RNA sequencing，简称scRNA-seq）分析。为验证研究结果的可靠性，我们通过在hPO长期培养过程中实施的蛋白质组学与定量实时聚合酶链反应（quantitative real-time polymerase chain reaction，简称qRT-PCR）分析，进一步验证并强化了本研究的实验发现。
研究结果：首先，我们围绕hPO的形态学特征与长期培养的分子特征展开分析，证实其具备明确的上皮组织结构，且在全部细胞传代过程中均保持遗传稳定性。随后，基于scRNA-seq技术，我们明确了hPO内存在的所有细胞类型，证实本研究采用的胰腺类器官构建方法具有高度可重复性。此外，借助最新的生物信息学工具，并将本研究数据与近期已发表的同类数据进行比对，我们重塑了关于hPO单一导管属性的传统理论。最后，我们开发了一种从hPO中分选纯化多能祖细胞的新策略，并提出了胰腺祖细胞的全新定义，证实该祖细胞可在维持细胞异质性的同时，更新hPO的干细胞群体。
研究结论：综上，本研究成果为人胰腺类器官赋予了全新的定义，揭示了这类结构的复杂生物学本质，为胰腺领域的后续研究提供了坚实的理论基础。本数据集包含从3份健康胰岛缺失组织中获取的人胰腺类器官的单细胞RNA测序表达谱。