Do Revival Stem Cells Contribute to Liver Regeneration?

肝脏是唯一一个具有巨大再生能力的器官，再生能力可达所需体积的30%至100%。在我的研究计划中，我有兴趣使用网络分析方法了解组织再生中涉及的途径，而肝脏是能够快速再生的组织之一。已知Hippo通路是动物模型中部分肝切除后肝再生期间激活的关键通路，并且是组织大小的关键调节因子。 合作者Wrana博士的小组最近采用单细胞分析发现，雅普通过诱导一种新型细胞发挥作用，这种细胞被称为再生干细胞（revSC），对肠道再生至关重要（Ayyaz et al.，Nature，2019）。RevSC，这是标记的异丙肾上腺素（Clu）的表达，是短暂诱导和功能，以重建Lgr 5+成人干细胞区室的控制下的Hippo途径。 原理和假设： 在一些再生模型，包括涡虫，蝾螈肢体再生，小鼠肺修复和肠的单细胞动力学的研究揭示了一个共同的主题在组织再生：一个罕见的，短暂的干细胞，是至关重要的再生，我们显示在肠是由雅普信号诱导。我们假设Hippo通路通过诱导肝再生干细胞介导正常的肝再生。 初步数据： 为了研究Hippo通路在正常肝再生中的作用，我的研究小组采用了肝切除术后的小鼠模型。对6周龄C57 BL/6小鼠进行部分肝切除术（70%），并通过肝切除术后2天（增殖再生的高峰）和7天（再生过程的结束）（PTH）分析基因表达。与对照肝脏样品相比，观察到Hippo途径的强烈变化，突出了Hippo途径在正常再生中发挥的关键作用。 实验方法： 为了鉴定肝脏中的revSC，我们将首先直接测试Clu是否也标记肝脏revSC。为此，将在CluCreERT 2/+小鼠中进行肝切除术，其中将他莫昔芬诱导型Cre重组酶敲入内源性Clu基因座。这些小鼠还携带Rosa 26-lsl-tdTomato等位基因（CluCreERT 2/+; Rosa 26-lsl-tdTomato/+或ClutdTomato），允许以药物诱导的方式对Clu+复兴干细胞及其后代进行遗传荧光标记。如果在肝切除术后观察到revSC后代的广泛贡献，那么我们接下来将研究revSC的消融如何影响肝再生。 新奇和预期意义： 本研究将明确再生干细胞在肝再生中的作用，并为探索其在肝再生中的作用提供关键标志物。再生肝脏中再生干细胞的发现将为Hippo通路在引导正常再生中的作用提供深入了解。

The liver is the only organ with a tremendous capacity to regenerate, from 30 to 100% of its required volume. In my research program, I am interested in understanding the pathways involved in tissue regeneration using a Network analysis approach, and the liver is the one tissue that is capable of rapid regeneration. The Hippo pathway is known to be the key pathway activated during liver regeneration following partial liver resection in animal models and is a critical regulator of tissue size. Collaborator Dr. Wrana's group recently employed single cell profiling to discover that Yap functions by inducing a new type of cell they called Revival stem cells (revSCs) that are critical for intestinal regeneration (Ayyaz et al., Nature, 2019). RevSCs, which are marked by Clusterin (Clu) expression, are transiently induced and function to reconstitute the Lgr5+ adult stem cell compartment under the control of the Hippo pathway. Rationale and Hypothesis: Studies of single cell dynamics in a number of regenerative models that include planaria, Axolotl limb regeneration, mouse lung repair, and intestine reveal a common theme during tissue regeneration: a rare, transient stem cell that is critical for regeneration and that we showed in intestines is induced by Yap signalling. We hypothesize that the Hippo pathway mediates normal liver regeneration via induction of liver revival stem cells. Preliminary data: To examine the role of the Hippo pathway in normal liver regeneration, my research team employed a post-hepatectomy mouse model. Partial hepatectomy (70%) was performed on 6 week-old C57BL/6 mice, and gene expression profiled by at 2 days (peak of proliferative regeneration), and 7 days (the end of the regenerative process) post-hepatectomy (PTH). Strong alterations in the Hippo pathway were observed when compared with control liver samples, highlighting the crucial role that the Hippo pathway plays in normal regeneration. Experimental Approach: In order to identify revSCs in the liver, we will first directly test if Clu also marks a liver revSC. For this, hepatectomies will be performed in CluCreERT2/+ mice in which Tamoxifen-inducible Cre recombinase was knocked in to the endogenous Clu locus. These mice, also carrying a Rosa26-lsl-tdTomato allele (CluCreERT2/+; Rosa26-lsl-tdTomato/+, or ClutdTomato) allow genetic, fluorescent labelling of Clu+ revival stem cells and their offspring in a drug-inducible manner. If extensive contribution of revSC offspring is observed post-hepatectomy, then we would next examine how ablation of revSCs affects liver regeneration. Novelty and Expected Significance: This study will definitively determine the involvement of revival stem cells in liver regeneration, and provide key markers to explore their function in liver regeneration. Discovery of revival stem cells in the regenerating liver will provide insight into the role of the Hippo pathway in guiding normal regeneration.