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. Overall design: 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（liver epithelial cells）——包括肝细胞（hepatocytes）与胆管上皮细胞（bile duct cells）——与内皮细胞（endothelial cells）、巨噬细胞（macrophages）和成纤维细胞（fibroblasts）构成的微环境相互交织，共同形成肝脏组织的功能单位。
稳态下，肝上皮细胞自我更新速率缓慢，但在受到损伤后具备极强的再生能力。
此前我们已证实，胆管上皮细胞可在限定性培养基中体外扩增，形成三维上皮性肝脏类器官（liver organoids），该类器官可在体外重现肝脏再生过程的诸多特征，但缺乏基质细胞（stromal cell）组分。
本研究发现，一群表达Sca1（Sca1）和Pdgfra（Pdgfra）的门脉间充质细胞亚群，对胆管上皮细胞的增殖能力具有双重调控作用。
一方面，该亚群可在无需外源性生长因子、且不依赖细胞间直接接触的条件下，促进肝脏类器官的形成与扩增；但当通过微流体包裹（microfluidics encapsulation）技术使胆管上皮细胞与SCA1+间充质细胞建立物理接触，从而让二者自发组装形成嵌合类器官（chimeric organoid）结构时，细胞增殖则会以间充质细胞剂量依赖的方式被抑制。
研究发现，在肝脏再生的不同阶段，无论是体外（in vitro）还是体内（in vivo）环境中，决定胆管上皮细胞增殖净效应的是间充质细胞与上皮细胞的相对比例，而非二者的绝对数量。
SCA1+间充质细胞调控胆管上皮细胞增殖动态的机制，至少部分通过Notch介导的细胞间接触抑制通路实现。
本研究结果凸显了上皮细胞与其间充质微环境之间的细胞接触相对比例与分布模式，在组织再生动态调控过程中的关键调控作用。
实验整体设计：从健康成年小鼠肝脏中分选得到胆管上皮细胞与间充质细胞，分别在培养0天（t=0）或体外培养15天后进行转录组谱分析。