Human liver stem cells originate from the canals of hering
Authors: Nimantha De Alwis, Gavin Hudson, Alastair D. Burt, Christopher P. Day, Patrick F. Chinnery
Journal: Hepatology (2009)

In collaboration with Fellous and colleagues,1 we recently described regions of healthy human liver with a deficiency of cytochrome c oxidase (COX) containing somatic mitochondrial DNA (mtDNA) mutations.2 These findings confirmed the clonal origin of the COX-deficient hepatocytes, and their distribution suggested a periportal origin. Here, we show for the first time that the hepatocytes within the COX-deficient patches originate from the periductular region (canals of Hering), where a hepatocyte stem cell population must reside. We used sequential COX–succinate dehydrogenase (SDH) histochemistry to identify regions of human liver containing cells potentially derived from a single precursor cell (Fig. 1A,B). We subsequently sequenced the entire mtDNA from different regions of the same COX-deficient region and adjacent liver. In each case, we indentified the same clonally expanded mtDNA mutation throughout the COX-deficient zone, and the mutation was not detected in adjacent normal liver (Fig. 1C). As in our previous work,1 the presence of only mutated mtDNA throughout the COX-deficient region (mtDNA homoplasmy) indicates that the mutation arose in a common precursor cell. Some of the COX-deficient regions were large, extending up to 600 μm in approximate diameter, demonstrating the extent of the region repopulated by a single precursor. All of the COX-deficient regions were contained within a single lobule, with no evidence of extension from one lobule to another. (A) Sequential cytochrome c oxidase (COX)–succinate dehydrogenase (SDH) histochemistry of human liver from a healthy control subject showing regions of COX deficiency (blue) in otherwise morphologically and histochemically normal liver (brown). (B) Higher power view showing the relationship between COX-deficient regions and the portal tract. The blue region does not encircle the portal tract, but arises from the periportal region opposite the portal vein, extending into the liver parenchyma. This is consistent with the anatomical localization of the canal of Hering. (C) Mitochondrial DNA (mtDNA) sequencing identified the somatic mutation causing the COX defect. The m.6352T→C MTCO1 gene mutation was present in the COX-deficient region, but not in the adjacent tissue with normal COX activity. The m.6352T→C mutation is predicted to change a leucine to serine in subunit 1 of COX, is highly conserved across diverse species, and has not been observed in more than 5140 humans.6 The m.6352T→C mutation thus explains the biochemical defect in this COX-deficient region. Despite a high percentage level of the mutation causing a profound biochemical defect, the hepatocytes had normal morphology. (D) Sequencing revealed multiple mtDNA mutations in some COX-deficient regions. Left panel = COX deficient region. Middle panel = regions dissected by laser capture. Right panel = mtDNA sequence variants detected. The brown column contains the sequence variants present in normal tissue (zone C in the middle panel). The blue columns contain sequence data from zones A, B, and F, which are increasingly distant from the portal tract. De novo somatic mutations are shown in red. The regions furthest from the portal tract contain additional mutations not present in the regions closest to the portal tract. This is consistent with the "streaming liver hypothesis", whereby the cells ultimately arise from the canals of Hering and migrate away from the portal tract into the liver parenchyma. Cryostat sections were 16 μm thick. Magnification: (A) ×5; (B) ×10; (C) ×40; (D) ×20. Several COX-deficient zones contained at least two somatic mtDNA mutations (Fig. 1D). Cells more distant from the portal tract had additional rare mutations not present in cells closer to the tract, and never the other way around. The most likely explanation is that, as the daughter cells migrate away from the bile duct, some acquire additional somatic mtDNA mutations. This is consistent with "the streaming liver" hypothesis, where regenerating hepatocytes arise from a stem cell population in the canal of Hering and move outward into the liver parenchyma.2 As previously observed,1 the COX-deficient zones were morphologically normal, and in some healthy aged subjects, up to 5% of the liver showed a COX defect despite having normal biochemical indices of liver function. A strong selection bias, either for or against the mtDNA mutations, therefore seems unlikely. This is supported by our observation of clonally expanded synonymous mtDNA substitutions in regions with normal COX activity, which have presumably accumulated through random genetic drift.3 The mtDNA mutations therefore appear to be a reliable marker of hepatocyte lineages. The prevailing view is that mature hepatocytes divide every year, gradually replacing adjacent liver cells. On the other hand, stem cell activation is only thought to occur after an acute insult, or after chronic liver damage when the capacity for mature hepatocyte division has been overwhelmed. Our observations indicate that the stem cell population is active in healthy liver and contributes to hepatocyte turnover. Although we found no evidence that the mtDNA mutations compromise liver cell function, there is emerging evidence that stem cell proliferation could be compromised by somatic mtDNA mutations. If correct, then this could explain why the prognosis following acute liver insult is worse in older subjects.4, 5 We are very grateful to Dr. Simon Elliot and Mr. Derek Manas for their assistance in obtaining biopsy tissue. P.F.C. is a Wellcome Trust Senior Fellow in Clinical Science who also receives funding from the Medical Research Council (UK), the UK Parkinson's Disease Society, and the UK National Institute for Health Research Biomedical Research Centre for Ageing and Age-Related Disease award to the Newcastle upon Tyne Foundation Hospitals National Health Service Trust. Nimantha De Alwis*, Gavin Hudson*, Alastair D. Burt , Christopher P. Day , Patrick F. Chinnery* , * Mitochondrial Research Group, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne, UK, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK, Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, UK.