The ratio of periportal mesenchyme to ductal epithelium acts as a proliferative rheostat in liver regeneration

Liver epithelial cells - hepatocytes and bile duct cells - intermingle with a microenvironment of endothelial cells, macrophages and fibroblasts to form the functional unit of the tissue. In homeostasis, the liver epithelium self-renews slowly, yet it is capable of remarkable regeneration following damage. We have shown that liver ductal cells can be expanded in vitro under defined medium to form 3D epithelial 'liver organoids' that recapitulate many aspects of the regeneration process in vitro, yet lack stromal cell components. Here, we describe that a subpopulation of portal mesenchymal cells expressing Sca1 and Pdgfra, exerts a dual effect on the proliferative capacity of ductal cells. On one hand, it supports liver organoid formation and expansion in the absence of exogenous growth factors and independently of cell-to-cell contact. However, when ductal cells and SCA1+ mesenchymal cells establish physical interaction following a microfluidics encapsulation that enables the cells to self-organize into chimeric organoid structures, cell proliferation is abolished in a mesenchymal-dose dependent manner. We found that it is the ratio between mesenchyme and epithelial cells and not their absolute values that determines the net outcome of ductal proliferation both in vitro, and in vivo, during the different phases of liver regeneration. The mechanism by which Sca1+ mesenchymal cells regulate ductal cell proliferation dynamics is mediated, at least in part, by Notch cell-cell contact inhibition. Our findings underscore how the relative ratio and distribution of cell-cell contacts between the epithelium and its mesenchymal microenvironment are key regulatory processes during the dynamic regulation of tissue regeneration. RNA profile of ductal and mesenchymal cells sorted from healthy adult mouse livers and analysed at t=0 or after 15 days of in vitro culturing肝上皮细胞——包括肝细胞与胆管细胞——与由内皮细胞、巨噬细胞及成纤维细胞构成的微环境相互交织，共同组成肝脏组织的功能单位。在稳态条件下，肝上皮细胞自我更新速率缓慢，但在受到损伤后却具备极强的再生能力。本团队此前研究证实，胆管细胞可在限定性培养基中于体外扩增，形成三维上皮“肝脏类器官”，该类器官可在体外重现肝脏再生过程的诸多特征，但缺少基质细胞组分。在此项研究中，我们发现一类表达干细胞抗原1（Sca1）与血小板衍生生长因子受体α（Pdgfra）的门脉间充质细胞亚群，可对胆管细胞的增殖能力产生双重调控作用。一方面，该亚群可在无需外源性生长因子、且不依赖细胞间接触的条件下，促进肝脏类器官的形成与扩增；然而，当通过微流体包埋技术使胆管细胞与Sca1+间充质细胞建立物理接触，进而自组织形成嵌合类器官结构时，胆管细胞的增殖会以间充质细胞剂量依赖性的方式被抑制。我们发现，在肝脏再生的不同阶段，无论是体外还是体内环境中，决定胆管细胞增殖净效应的是间充质细胞与上皮细胞的比例，而非二者的绝对数量。Sca1+间充质细胞调控胆管细胞增殖动态的机制，至少部分是通过Notch细胞间接触抑制通路实现的。本研究结果证实，上皮细胞与其间充质微环境之间的细胞间接触相对比例与分布模式，是组织再生动态调控过程中的关键调控环节。本数据集包含从健康成年小鼠肝脏中分选的胆管细胞与间充质细胞的RNA表达谱，分别在培养第0天（t=0）以及体外培养15天后进行了检测分析。