Hepatocytes undergo punctuated expansion dynamics from a periportal stem cell niche in normal human liver

Background: Normal human liver is thought to be generally quiescent, however clonal hepatocyte expansions have been observed but neither their cellular source nor their expansion dynamics have been determined. Knowing the hepatocyte cell of origin, and their subsequent dynamics and trajectory within the human liver will provide an important basis to understand disease-associated dysregulation. Methods: Here we use in vivo lineage tracing and a combination of methylation sequence analysis to demonstrate normal human hepatocyte ancestry. We exploit next generation mitochondrial sequencing to determine hepatocyte clonal expansion dynamics across spatially-distinct areas of laser-captured, microdissected clones, in tandem with computational modelling in morphologically-normal human liver. Results: Hepatocyte clones and rare SOX9+ hepatocyte progenitors commonly associate with portal tracts and we present evidence that clones can lineage-trace with cholangiocytes, indicating the presence of a bipotential common ancestor at this niche. Within clones, we demonstrate methylation CpG sequence diversity patterns indicative of periportal, but not pericentral ancestral origins, indicating a portal to central vein expansion trajectory. Using spatial analysis of mtDNA variants by next-generation sequencing coupled with mathematical modelling and Bayesian inference across the portal-central axis, we demonstrate that patterns of mtDNA variants reveal large numbers of spatially-restricted mutations in conjunction with limited numbers of clonal mutations. Conclusions: These datasets support the existence of a periportal progenitor niche and indicate that clonal patches exhibit punctuated but slow growth, then quiesce, likely due to acute environmental stimuli. These findings crucially contribute to our understanding of hepatocyte dynamics in normal human liver.