Do Revival Stem Cells Contribute to Liver Regeneration?

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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.