Studies on regeneration and fibrosis [electronic resource]

Restoration of tissue architecture and function is the pinnacle achievement of the regenerative process. The extent of the regenerative potential varies widely in metazoans; flatworms, cnidarians, and colonial ascidians are able to fully regenerate through whole body regeneration, but there exists a striking decrease in regenerative abilities in organisms with higher tissue and body complexity. Higher vertebrates, specifically mammals, retain some regenerative capacities in a number of organ systems that undergo frequent self-renewal, most notably the hematopoietic system, the intestinal crypts, and the dermis. However, when challenged with major insults, whether traumatic amputation or chronic injury, potentially leading to the exhausting of tissue resident stem cells or progenitors, mammals have mostly lost their ability to regrow organs and instead opt for the deposition of extracellular matrix proteins and collagen in place of regenerating normal parenchyma. Fibrosis has become more and more of a medical burden as numerous organ specific fibroses ultimately lead to end stage organ failure. Therefore the induction of regeneration and the inhibition of the fibrotic response have become areas of active investigation. The balance between regeneration and fibrosis is becoming an increasingly intriguing question. Whether this balance point occurred previously in evolutionary history or occurs during an organisms' lifetime is still not well understood. Here we explore new avenues in two established phenomena, mammalian liver regeneration, and peritoneal adhesion formation with the aim to better understand the underlying genetic and transcriptional changes, stem cell or progenitor identities, cellular contributions, and clonal kinetics involved in both models. We also put forward novel tools to interrogate stem cell and or progenitor fates, which provide higher resolution lineage tracing in previously unexplored tissue systems. Chronic stress or extensive traumatic injury in mammalian systems usually leads to fibrosis and or end organ failure. A notable exception is the liver, the regenerative potential of which has been well documented. Chronic injury response in the adult is thought to be characterized by initial hepatocyte proliferation and following exhaustion, the mobilization of a putative bipotent hepatocyte cholangiocyte progenitor. Acute injury, most commonly modeled by partial hepatectomy or amputation of the mammalian liver results in restoration of liver function by globalized hypertrophy and cell division across all remaining lobes, but with permanent loss of lobular morphology and architecture. Here, we identify a postnatal window in which lobular structure, architecture and function are restored following amputation. Quantifications of liver mass, enzymatic activity, histological and immunohistochemical examination of gross, cellular, and molecular morphology collectively demonstrate that damaged lobes underwent multi-lineage regeneration, reforming a lobe that is often indistinguishable from undamaged ones. Contrary to existing models; we characterize a new regenerative phenomenon primarily involving localized clonal expansions of hepatocytes. Using a multi-color fluorescent-based lineage tracing system to perform clonal analysis at single cell levels, we show that presumptive liver stem/progenitors are fate restricted, generating either hepatic or cholangiocytic clones. Further analysis on tetrachimeric mice, tracing from the blastocyst, showed that regenerating clonal distributions associatea mainly with central veins, in a pattern significantly different from that of normal development. These results illuminate an unknown endogenous program of liver regeneration that has been underappreciated in mammals and may also provide a therapeutic window in which specific transplanted cells can undergo clonal expansion and give rise to normal structure and function. The fibrotic response is thought to occur either through regenerative exhaustion, for example liver cirrhosis following chronic injury, or the over-proliferation and dysregulation of extracellular matrix and collagen producing cells. Peritoneal adhesions are fibrous tissues that tether organs to one another or to the peritoneal wall and are a significant cause of post-surgical and infectious morbidity. Extensive studies have been done and suggest that hematopoietic cells, cytokines, and fibrin deposition play a major role in promoting adhesion formation. However, the molecular pathogenesis initially promoting adhesion formation has not been well characterized. Here we identify the surface mesothelium as a primary cell type responsible for driving the adhesion formation process. Time courses of mesothelial specific stains and proliferation markers demonstrate that adhesions are formed from mesothelial cell expansion. Isolation and RNA sequencing of activated mesothelial cells in a brief time course immediately following adhesion induction suggest candidate regulators of adhesion formation. Hypoxia inducible factor 1α (HIF1α) was identified as an early regulator, and functional inhibition showed significant diminishing of adhesion formation, suggesting new therapeutic agents to prevent post-operative adhesions. Further RNA sequencing analysis of HIF1α deficient mesothelial cells following adhesion induction demonstrated upregulation of HIF1A responsive elements, including the embryonic mesothelial marker uroplakin 1B (UPK1B), and the transcription factor wilms tumor 1 (WT1), suggesting a redeployment of embryonic phenotypes upon injury. Recent expansions in lineage tracing technologies have pushed our knowledge of the contribution of stem cell fate and function towards development, homeostasis, and regeneration further. However the current technologies are either extensively resource intensive or of low resolution. Fluorescent based lineage tracing approaches such as the Rainbow, Brainbow, Confetti, or tetrachimeric mice work well to establish clones in solid organs but are limited to three to four colors, therefore requiring rigorous statistical analysis and a high number of biological replicates. Molecular approaches, such as DNA barcoding and the Sleeping Beauty Transposon solve the issue of low resolution but require high throughput sequencing technologies. Furthermore, these approaches have primary been used in studies of hematopoietic or tumor biology. The use of these methods has not been proven in solid organs, where tissue architecture and organization is essential to our understanding of their developmental, homeostatic, and regenerative dynamics. Therefore we have developed a next generation multi-color fluorescent reporter system, named "Skittles" which localizes one of five colors to the membrane and cytoplasm, thereby having the potential to recombine 625 different unique color and localization combinations. As Skittles is an expansion of the current Rainbow system, it retains much of the same architecture but includes additional incompatible LoxP variants as well as membrane and nuclear localization signals, all of which have been demonstrated to work through cell culture, confocal microscopy, and flow cytometry.

Keywords: Developmental Biology.
Advisor: Weissman Irving L. primary advisor. Thesis advisor
Beachy Philip Arden advisor. Thesis advisor
Nusse Roel
Sage Julien advisor. Thesis advisor