Do Revival Stem Cells Contribute to Liver Regeneration?

肝脏是唯一具有巨大再生能力的器官，从其所需体积的30%到100%。在我的研究计划中，我对使用网络分析方法了解组织再生所涉及的途径感兴趣，肝脏是能够快速再生的组织之一。已知河马途径是动物模型肝部分切除后肝再生过程中被激活的关键途径，是组织大小的关键调节因子。合作者Wrana博士的团队最近利用单细胞图谱发现YAP通过诱导一种被称为复兴干细胞(RevSCs)的新型细胞发挥功能，这种细胞对肠道再生至关重要(Ayyaz等人，《自然》，2019)。以Clusterin(Clu)表达为标志的RevSCs在Hippo途径的控制下被瞬时诱导并功能重建Lgr5+成体干细胞室。 基本原理和假设： 对一些再生模型中单细胞动力学的研究，包括舟形虫、Axolotl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：一种罕见的瞬时干细胞对再生至关重要，我们在肠道中展示了这种干细胞是由YAP信号诱导的。我们假设河马途径通过诱导肝再生干细胞来调节正常的肝再生。 初步数据： 为了研究河马途径在正常肝再生中的作用，我的研究小组采用了肝切除后的小鼠模型。对6周龄C57BL/6小鼠施行肝部分切除(70%)，分别于术后2天(增殖高峰)和7天(再生过程结束)进行基因表达谱分析。与对照肝脏样本相比，河马途径发生了强烈的变化，突显了河马途径在正常再生中所起的关键作用。 实验方法： 为了鉴定肝脏中的RevSCs，我们首先要直接检测Clu是否也标记了肝脏的RevSC。为此，将在CluCreERT2/+小鼠中进行肝切除，在该小鼠中，三苯氧胺诱导的Cre重组酶被敲入内源Clu基因座。这些小鼠还携带rosa26-lsl-tdTomato等位基因(CluCreERT2/+；rosa26-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了RevSC后代的广泛贡献，那么接下来我们将研究如何消融RevSCs对肝脏再生的影响。 新颖性和预期意义： 本研究将明确再生干细胞在肝再生中的作用，并为探讨其在肝再生中的作用提供关键标记物。在再生肝中发现再生干细胞将有助于深入了解河马途径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.