Do Revival Stem Cells Contribute to Liver Regeneration?

肝脏是唯一具有巨大再生能力的器官，其容量可达到所需容量的30%至100%。在我的研究项目中，我对使用网络分析方法了解组织再生的途径很感兴趣，而肝脏是一个能够快速再生的组织。在动物模型中，Hippo通路是部分肝脏切除后肝脏再生过程中激活的关键通路，是组织大小的关键调节因子。合作者Wrana博士的研究小组最近利用单细胞分析发现，Yap通过诱导一种对肠道再生至关重要的新型细胞来发挥作用，他们称之为复兴干细胞（revSCs） （Ayyaz等人，Nature, 2019）。以Clusterin （Clu）表达为标志的RevSCs在Hippo通路的控制下被瞬时诱导并具有重建Lgr5+成体干细胞区室的功能。基本原理和假设：对许多再生模型的单细胞动力学研究，包括原肠、蝾螈肢体再生、小鼠肺修复和肠道，揭示了组织再生过程中的一个共同主题：一种罕见的、短暂的干细胞，对再生至关重要，我们在肠道中发现，它是由Yap信号诱导的。我们假设Hippo通路通过诱导肝脏再生干细胞介导正常肝脏再生。初步数据：为了研究Hippo通路在正常肝脏再生中的作用，我的研究小组采用了肝切除术后的小鼠模型。对6周龄的C57BL/6小鼠进行部分肝切除术（70%），并在肝切除术（PTH）后2天（增殖再生高峰）和7天（再生过程结束）记录基因表达谱。与对照肝脏样本相比，观察到Hippo通路的强烈变化，突出了Hippo通路在正常再生中的关键作用。实验方法：为了识别肝脏中的revSC，我们将首先直接检测Clu是否也标记肝脏revSC。为此，将对CluCreERT2/+小鼠进行肝切除术，其中他莫昔芬诱导的Cre重组酶被敲入内源性Clu位点。这些小鼠也携带Rosa26-lsl-tdTomato等位基因（CluCreERT2/+; Rosa26-lsl-tdTomato/+，或cludtomato），可以通过药物诱导的方式对Clu+再生干细胞及其后代进行遗传荧光标记。如果肝切除术后观察到revSC后代的广泛贡献，那么我们下一步将研究revSC消融如何影响肝脏再生。新颖性和预期意义：本研究将明确复活干细胞在肝再生中的作用，并为探索其在肝再生中的功能提供关键标志物。在再生肝脏中发现再生干细胞将有助于深入了解Hippo通路在指导正常再生中的作用。

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.