Hepatic progenitor cells of biliary origin with liver repopulation capacity

May combine two claims.

Following βNF administration to Mdm2flox/flox animals lacking AhCre, no expression of p53 protein is found, confirming p53 expression is a consequence of Cre activity and Mdm2 loss.

“however following βNF administration to Mdm2flox/flox animals lacking the AhCre, we found no expression of p53 protein confirming p53 expression is a consequence of Cre activity and Mdm2 loss (Figure 1d).”

May combine two claims.

Expression of p21 protein is observed in hepatocytes following βNF administration but not in Cre− animals.

“We also observed expression of p21 protein in hepatocytes following βNF administration (Figure 1e) but not in Cre− animals (Figure 1f).”

May combine two claims.

Following βNF induction, Cre recombinase is expressed in more than 98% of hepatocytes where it renders Mdm2 inactive.

“Following induction with βNF, Cre recombinase is expressed in >98% of hepatocytes where it renders Mdm2 inactive.”

May combine two claims.

Uninduced AhCreMdm2flox/flox mice have normal hepatic architecture and do not express detectable p53 protein in hepatocytes.

“Uninduced AhCreMdm2flox/flox mice have normal hepatic architecture and do not express detectable p53 protein in hepatocytes (Figure 1b).”

May combine two claims.

Following βNF administration, ΔMdm2 mice develop jaundice after three days and require humane euthanasia by day 8.

“Following βNF administration, ΔMdm2 mice developed jaundice after three days and required humane euthanasia by day 8 (Supplementary Table 1).”

May combine two claims.

In the CDE model, there is minimal hepatocyte senescence, in contrast to advanced human diseases and rat models of HPC activation where high levels of hepatocyte senescence occur.

“We found minimal hepatocyte senescence (Supplementary figure 1b), in contrast to advanced human diseases and rat models of HPC activation where high levels of hepatocyte senescence occur29.”

• cites [29] Aravinthan, A. et al. Hepatocyte expression of the senescence marker p21 is linked to fibrosis and an adverse liver-related outcome in alcohol-related liver disease. PLoS ONE 8, e72904 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Expanded cdHPCs maintain HPC characteristics and are positive for a suite of markers (Sox9, CD44, Fn14, OPN1 and panCK) which have been used to define HPCs in vivo, and expression is maintained after 30 passages.

“Expanded cdHPCs maintained their HPC characteristics and were positive for a suite of markers (Sox9, CD44, Fn14, OPN1 and panCK) which have been used to define HPCs in vivo and the expression of these markers was maintained after 30 passages (Figure 7i, Supplementary figure 4e and f).”

May combine two claims.

19 days following induction, p53 staining in hepatocytes is more heterogeneous, with 93.5% of hepatocytes still expressing p53.

“19 days following induction, p53 staining in hepatocytes was more heterogeneous, with 93.5% of hepatocytes still expressing p53 (Figure 4a and b).”

May combine two claims.

GFP positive cells in the parenchyma are large and morphologically consistent with hepatocytes.

“GFP positive cells in the parenchyma were large and morphologically consistent with hepatocytes (Figure 8c and Supplementary figure 7a).”

May combine two claims.

Following 3-D culture of cdHPCs in Matrigel, cells form tubules with branched morphology and lumen and express the activated bile duct marker MIC1C3 and the biliary transcription factor HNF1β.

“Following 3-D culture of cdHPCs in Matrigel we observed cells forming tubules, which had a branched morphology and lumen (Supplementary figure 5c) and expressed the activated bile duct marker MIC1C3 and the biliary transcription factor HNF1β (Supplementary figure 5c).”

May combine two claims.

EpCAM+ clones are capable of generating colonies; however this is significantly impaired in EpCAM− cdHPCs, regardless of CD24 expression status.

“EpCAM+ clones were capable of generating colonies; however this was significantly impaired in EpCAM− cdHPCs, regardless of CD24 expression status (Supplementary figure 6a-c).”

May combine two claims.

The expanded cdHPC population can be maintained long-term in vitro (more than 30 passages and up to a year) and remains chromosomally stable with a normal karyotype.

“The expanded cdHPC population can be maintained long-term in vitro (more than 30 passages and up to a year) and remains chromosomally stable having a normal karyotype (Figure 7h).”

May combine two claims.

Serum markers for liver injury (AST, bilirubin, alkaline phosphatase and ALT) are all elevated and serum albumin levels fall in ΔMdm2 mice, signifying injury and impaired synthetic function.

“Serum markers for liver injury (AST, bilirubin, alkaline phosphatase and ALT) were all elevated and serum albumin levels fell, signifying injury and impaired synthetic function of the livers of ΔMdm2 mice (Figure 1h-j, Supplementary figure 2e and f).”

May combine two claims.

Eight days following induction, the number of panCK positive ductular reactions significantly increases and they are found migrating as cords throughout the liver parenchyma.

“Eight days following induction of the AhCre transgene the number of panCK positive ductular reactions containing putsignificantly increases and are found migrating as cords throughout the liver parenchyma (Figure 2b and c, Supplementary figure 3a-c).”

May combine two claims.

In the CDE model, there is an expansion of a cell population that expresses the HPC markers EpCAM, CD24, and CD133; the AhCre ΔMdm2 HPCs express the same surface antigens at comparable levels.

“Here we found an expansion of a cell population that expresses the HPC markers EpCAM, CD24, and CD133 (Figure 2f) in CDE; critically our AhCre ΔMdm2 HPCs express the same surface antigens at comparable levels.”

May combine two claims.

HPCs are not affected by the Cre system in response to βNF induction, as they lack upregulation of p53.

“Furthermore, HPCs were not affected by the Cre system in response to the βNF induction, as they lacked upregulation of p53 (Figure 2j), and are proliferative following βNF administration and recombination of the Mdm2loxP locus in hepatocytes (Figure 2k).”

May combine two claims.

No significant change is observed in the expression of most HPC-related genes such as EpCAM, Ck19, Spp1, and Sox9, or Alb after long-term passaging, although Lgr5 expression increases.

“We did not observe a significant change in the expression of most HPC related genes such as EpCAM, Ck19, Spp1, and Sox9, or Alb after long term passaging although Lgr5 expression increased (Supplementary figure 4e).”

May combine two claims.

Proliferating hepatocytes positive for Ki67 are arranged in cord-like formations, with each cord of dividing hepatocytes in contact with the distal tips of the ductular reaction.

“Proliferating hepatocytes, positive for Ki67 were arranged in cord-like formations, with each cord of dividing hepatocytes being in contact with the distal tips of the ductular reaction (Figure 5a).”

May combine two claims.

AhCre+Mdm2flox/− mice have a somatic heterozygotic deficiency of the Mdm2 gene but are phenotypically normal until the second Mdm2 allele is lost.

“AhCre+Mdm2flox/− mice had a somatic heterozygotic deficiency of the Mdm2 gene, however are phenotypically normal until the second Mdm2 allele is lost.”

May combine two claims.

Following recovery of AhCre+Mdm2flox/− mice, no animals develop liver tumours or notable fibrosis.

“Following recovery of AhCre+Mdm2flox/− mice no animals developed liver tumours or notable fibrosis (Figure 4f).”

May combine two claims.

EpCAM+/CD24−/CD133− and EpCAM+/CD24+/CD133− populations form infrequent, small colonies with mesenchymal characteristics, while the EpCAM+/CD24+/CD133+ population generates frequent colonies of packed epithelial cells (cdHPCs).

“EpCAM+/CD24−/CD133− and EpCAM+/CD24+/CD133− populations formed infrequent, small colonies with mesenchymal characteristics, while the EpCAM+/CD24+/CD133+ population generated frequent colonies of packed epithelial cells, from hereon known as clonal density derived HPCs (cdHPCs) (Figure 7f and g).”

May combine two claims.

Transplanted animals have significantly improved liver histology with stronger glycogen staining, less liver scarring, and increased serum albumin levels compared to controls.

“Transplanted animals had significantly improved liver histology with stronger glycogen staining (Figure 8d top and middle panel), less liver scarring, (Figure 8d bottom panel and Figure 8e) and increased serum albumin levels compared to controls (Figure 8f).”

May combine two claims.

GFP expressing, cdHPC derived cells co-express the hepatocyte markers HNF4α and CYP2D6 and are found in patches adjacent to host GFP negative hepatocytes.

“The GFP expressing, cdHPC derived cells co-expressed the hepatocyte markers HNF4α and CYP2D6 and were found in patches adjacent to host GFP negative hepatocytes (Figure 8g and Supplementary figure 7b).”

May combine two claims.

The reduction in functionality is associated with hepatocyte necrosis by H&E staining and increased positivity of lactate dehydrogenase staining.

“This reduction in functionality is associated with hepatocyte necrosis by H&E staining which shows clear disruption of the hepatic architecture as well as increased positivity of lactate dehydrogenase staining (Figure 1k, Supplementary figure 2h).”

May combine two claims.

In AhCre+Mdm2flox/− mice, the onset of liver injury and clinical signs of acute disease occur between 14 and 21 days following AhCre activation, all delayed compared with Mdm2flox/flox.

“In AhCre+Mdm2flox/− mice the onset of liver injury and clinical signs of acute disease, demonstrated through necrosis and apoptosis as demonstrated through H&E and TUNEL staining (Figure 3a and b), together with tachypnoea, hunching, prostration, and bilirubinuria occur between and 14 and 21 days following AhCre activation all of which are delayed in the Mdm2flox/− compared with the Mdm2flox/flox”

May combine two claims.

After in vitro expansion, mean cell size reduces after 10 passages, but no significant changes in elongation assessed by cell roundness or cell width to length ratio are seen.

“After in vitro expansion, mean cell size reduced after 10 passages, however no significant changes in elongation assessed by cell roundness, or cell width to length ratio were seen (Supplementary figure 4g).”

May combine two claims.

cdHPC-derived hepatocytes are proliferating (Ki67+) in the recipient liver at 3 months following transplantation, whereas there is little proliferation in the endogenous hepatocytes of AhCre+Mdm2flox/flox recipient mice.

“cdHPCs hepatocytes were proliferating (Ki67+) in the recipient liver at 3 months following transplantation, whereas there was little proliferation in the endogenous hepatocytes of AhCre+Mdm2flox/flox recipient mice.”

May combine two claims.

Concurrent with this up-regulation, there is loss of biliary and progenitor associated genes Krt19, Epcam, Dlk1, Afp and upregulation in hepatocyte transcription factor Hnf1a.

“Concurrent with this up-regulation we found loss of biliary and progenitor associated genes Krt19, Epcam, Dlk1, Afp and upregulation in hepatocyte transcription factor Hnf1a (Supplementary figure 5e, lower histograms).”

May combine two claims.

Following Cre induction, Fn14KOΔMdm2 animals demonstrate significantly reduced survival, and all had to be euthanized within 10 days, unlike Fn14WTΔMdm2 mice in which 80% of animals survived to day 30.

“Following Cre induction, Fn14KOΔMdm2 animals demonstrated a significantly reduced survival, and all had to be euthanized within 10 days (Figure 5d), unlike Fn14WTΔMdm2 mice in which 80% of animals survived to day 30.”

May combine two claims.

cdHPCs do not express endothelial (CD31), haematopoietic (CD45) or mesenchymal (GFAP and desmin) markers.

“cdHPCs did not express endothelial (CD31), haematopoietic (CD45) or mesenchymal (GFAP and desmin) markers (Supplementary figure 5a and b).”

May combine two claims. The quote only partly states this claim.

Murine albumin increases at the transcript level and as protein in the culture medium following rWNT3A treatment.

“Murine albumin increased at the transcript level and as protein in the culture medium (Supplementary figure 5e, upper histograms).”

May combine two claims.

Only the cdHPC population can be expanded while maintaining an epithelial morphology; the other two populations fail to maintain any epithelial phenotype.

“In our assay, only the cdHPC population can be expanded whilst maintaining an epithelial morphology, the other two populations failed to maintain any epithelial phenotype.”

May combine two claims.

HPCs are proliferative following βNF administration and recombination of the Mdm2loxP locus in hepatocytes.

“Furthermore, HPCs were not affected by the Cre system in response to the βNF induction, as they lacked upregulation of p53 (Figure 2j), and are proliferative following βNF administration and recombination of the Mdm2loxP locus in hepatocytes (Figure 2k).”

May combine two claims.

Apoptosis levels determined through TUNEL are absent in uninduced AhCre+Mdm2flox/flox animals but clear positive cells are found throughout the parenchyma in induced mice.

“The levels of apoptosis in ΔMdm2 mice was determined through TUNEL which was absent in uninduced AhCre+Mdm2flox/flox animals however we found clear positive cells throughout the parenchyma in AhCre+Mdm2flox/flox mice which had received βNF (Figure 1l).”

May combine two claims.

In AhCre+Mdm2flox/− mice, HPC activation is more gradual than in AhCre+Mdm2flox/flox counterparts and arises from d14 post Mdm2 deletion.

“Concurrently HPC activation was more gradual than in the AhCre+Mdm2flox/flox counterparts and arose from d14 post Mdm2 deletion (Figure 3c).”

May combine two claims.

Following rWNT3A treatment, clusters of cdHPCs increase in size, become hexagonal in shape, and increase glycogen storage by PAS staining.

“Following rWNT3A treatment clusters of cdHPCs increased in size and became hexagonal in shape, and increased glycogen storage by PAS staining (Supplementary figure 5d).”

May combine two claims.

The AhCre+Mdm2flox/flox model results in massive hepatocyte injury, where more than 96% of hepatocytes upregulate p53, and activates a florid HPC response.

“The AhCre+Mdm2flox/flox model results in massive hepatocyte injury, where more than 96% of hepatocytes upregulate p53, and activates a florid HPC response.”

May combine two claims.

Within the panCK expressing ductular reactions, a subpopulation of HPCs expresses the surface markers EpCAM and CD24; EpCAM is very specific for ductular cells, while CD24 labels both HPCs and a population of hepatocytes in vivo.

“Within the panCK expressing ductular reactions, we observe a subpopulation of HPCs that express the surface markers EpCAM and CD24; whilst EpCAM is very specific for ductular cells, CD24 labels both HPCs and a population of hepatocytes in vivo (Figure 7c, Supplementary figure 3d).”

May combine two claims.

The authors succeeded in expanding HPCs in vitro under monolayer conditions while preserving their karyotypic and phenotypic stability.

“We have overcome the difficulty in expanding HPCs in vitro in monolayer conditions whilst maintaining their karyotypic and phenotypic stability.”

May combine two claims.

Transplanted HPCs produced significant structural and functional improvement of damaged liver, with reduced scarring and increased serum albumin in recipients.

“The transplanted HPCs demonstrated bi-lineage differentiation into large numbers of hepatocytes and cholangiocytes in vivo causing significant structural and functional improvement of the damaged liver, with reducced scarring and increase serum albumin in transplant recipients.”

May combine two claims.

CD45−/CD31−/Ter119−/EpCAM+/CD24+/CD133+ HPCs have high colony forming capacity and are of biliary origin.

“CD45−/CD31−/Ter119−/EpCAM+/CD24+/CD133+ HPCs have high colony forming capacity and are of biliary origin.”

May combine two claims.

Mice with Fn14 knockout do not mount an appropriate ductular reaction and have increased mortality.

“Mice with Fn14 knockout do not mount an appropriate ductular reaction, and have increased mortality.”

May combine two claims.

In AhCre+Mdm2flox/flox mice subjected to repeated hepatocyte senescence and injury plus HPC transplantation, the progeny of transplanted HPCs proliferated long-term and regenerated large areas of normal parenchyma.

“Using repeated rounds of hepatocyte senescence and injury in the AhCre+Mdm2flox/flox mice combined wih HPC transplantation we found that the progeny of the transplanted HPCs can proliferate long-term and regenerate large areas of normal parenchyma.”

• AssumptionRests on an assumption: Before this study, transplanted HPCs had limited repopulation capacity and unproven ability to improve chronically damaged liver structure.

May combine two claims.

Following CDE-recovery, there is a minor degree of tracing into hepatocytes, indicating that in this model the ductular-hepatocyte regeneration pathway is insignificant.

“Following CDE - recovery there was a minor degree of tracing into hepatocytes (Supplementary figure 1b, c), indicating that in this model the ductular–hepatocyte regeneration pathway is insignificant.”

May combine two claims.

Further experiments in mice have shown that hepatocytes can change into a biliary ductular phenotype and later re-differentiate into hepatocytes.

“Further experiments in mice have shown that hepatocytes can change into a biliary ductular phenotype21, 22 then later re-differentiate into hepatocytes23.”

• cites [21] Yanger, K. et al. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation. Cell Stem Cell 15, 340–349 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [22] Yimlamai, D. et al. Hippo pathway activity influences liver cell fate. Cell 157, 1324–1338 (2014). sourceNot checked yet

  Not checked in the free run.

• cites [23] Tarlow, B. D. et al. Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes. Cell Stem Cell 15, 605–618 (2014). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In the rat, complete suppression of hepatocyte proliferation can be achieved using chemical toxins, which provokes an extensive ductular/HPC response thought to regenerate parenchyma.

“In the rat complete suppression of hepatocyte proliferation can be achieved using chemical toxins which provokes an extensive ductular/HPC response which is thought to regenerate parenchyma, although lineage tracing studies are required to formally prove this25.”

• cites [25] Golding, M. et al. Oval cell differentiation into hepatocytes in the acetylaminofluorene-treated regenerating rat liver. Hepatology 22, 1243–1253 (1995).Not checked yet

  Not checked in the free run.

May combine two claims.

Recent studies have demonstrated that HPCs arise within the liver, engraft the parenchyma, and may have some differentiation capacity but did not regenerate significant quantities of liver parenchyma.

“These studies demonstrated that HPCs arise within the liver, engraft the parenchyma14 and may have some differentiation capacity but did not regenerate significant quantities of liver parenchyma11-13.”

• cites [11] Espanol-Suner, R. et al. Liver progenitor cells yield functional hepatocytes in response to chronic liver injury in mice. Gastroenterology 143, 1564–1575 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [12] Huch, M. et al. In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–250 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [13] Jors, S. et al. Lineage fate of ductular reactions in liver injury and carcinogenesis. J. Clin. Invest. 125, 2445–2457 (2015). sourceNot checked yet

  Not checked in the free run.

• cites [14] Wang, X. et al. The origin and liver repopulating capacity of murine oval cells. Proc. Natl Acad. Sci. USA 100 (Suppl. 1), 11881–11888 (2003). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

Hepatocyte isolation is technically demanding, requires cadaveric liver donation, and hepatocytes cannot be expanded effectively in vitro.

“Hepatocytes have a high replicative potential in vivo3, 4, however hepatocyte isolation is technically demanding, requires cadaveric liver donation, and hepatocytes cannot be expanded effectively in vitro5.”

• cites [5] Hay, D. C. Cadaveric hepatocytes repopulate diseased livers: life after death. Gastroenterology 139, 729–731 (2010). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

In hepatocytes, there is a 96.8% reduction in genomic Mdm2 exon 5 following recombination, while only a 0.8% reduction in NPCs from the same cohort, indicating AhCre is highly specific in recombining ΔMdm2 in hepatocytes.

“In hepatocytes there was a 96.8% reduction in the quantity of genomic Mdm2 exon 5 following recombination, however only a 0.8% reduction in NPCs from the same cohort (Figure 1g, supplementary figure 2a-d) indicating the AhCre is highly specific in recombining ΔMdm2 in hepatocytes.”

Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

Hepatocyte cell therapy is an attractive alternative to liver transplantation.

“Hepatocyte cell therapy is an attractive alternative to liver transplantation1, 2.”

• cites [1] Horslen, S. P. & Fox, I. J. Hepatocyte transplantation. Transplantation 77, 1481–1486 (2004). sourceNot checked yet

  Not checked in the free run.

• cites [2] Horslen, S. P. et al. Isolated hepatocyte transplantation in an infant with a severe urea cycle disorder. Pediatrics 111, 1262–1267 (2003). sourceNot checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

Ductular reactions are activated in chronic liver injury and are thought to contain putative HPCs with the potential to regenerate hepatocytes and cholangiocytes.

“Ductular reactions are activated in chronic liver injury and are thought to contain putative hepatic progenitor cells (HPCs) with the potential to regenerate hepatocytes and cholangiocytes6, 7.”

• cites [6] Boulter, L. et al. Macrophage-derived Wnt opposes Notch signaling to specify hepatic progenitor cell fate in chronic liver disease. Nat. Med. 18, 572–579 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [7] Boulter, L., Lu, W. Y. & Forbes, S. J. Differentiation of progenitors in the liver: a matter of local choice. J. Clin. Invest. 123, 1867–1873 (2013). sourceNot checked yet

  Not checked in the free run.

May combine two claims.

No evidence of stable nuclear p53 is found in HPCs arising in AhCre+Mdm2flox/−, indicating this long-term model faithfully recapitulates what is seen in Mdm2flox/flox mice.

“Furthermore, no evidence of stable, nuclear p53 could be found in HPCs arising in the AhCre+Mdm2flox/− indicating this long-term model faithfully recapitulates what we see in Mdm2flox/flox mice (Figure 3d and e).”

May combine two claims.

Two days after induction, there are no BrdU positive hepatocytes adjacent to BrdU positive HPCs, suggesting that proliferation of HPCs themselves does not lead to hepatocyte proliferation.

“Importantly we also found that 2 days after induction there are no BrdU positive hepatocytes adjacent to BrdU positive HPCs, suggesting that proliferation of HPCs themselves does not lead to hepatocyte proliferation. (Supplementary figure 3h).”

May combine two claims.

Proliferative BrdU positive HPCs are often found closely associated with BrdU positive hepatocytes 8 days following AhCre induction, suggesting that hepatocytes arise from BrdU positive HPCs in this model.

“Proliferative Bromodeoxiuridine (BrdU) positive HPCs were often found closely association with BrdU positive hepatocytes 8 days following AhCre induction, suggesting that hepatocytes arise from BrdU positive HPCs in this model.”

May combine two claims.

The percentage of TdTomato expressing cdHPCs remains constant before, during CDE injury, and after recovery from CDE injury, arguing against a significant non-ductular contribution to this population.

“This percentage of TdTomato expressing cdHPCs remain constant before, during CDE injury, and after recovery from CDE injury (Figure 7l and m), arguing against a significant non- ductular contribution to this population.”

May combine two claims.

Following recombination, LacZ expression is detected throughout the intestine and liver; loss of reporter is seen over 6 months, indicating that replacement of the recombined epithelium occurs from a non-recombined source.

“Following recombination LacZ expression could be detected throughout the intestine and liver. Loss of reporter was seen over 6 months indicating that replacement of the recombined epithelium occurs from a non-recombined source (Figure 4e).”

May combine two claims.

cdHPCs from Tamoxifen induced Krt19CreERTLSLTomato mice without liver injury are 70% Tomato+, demonstrating their ductular origin.

“Likewise the cdHPCs from Tamoxifen induced Krt19CreERTLSLTomato mice without liver injury were 70% Tomato+ demonstrating their ductular origin.”

May combine two claims.

Small GFP positive ductular cells express panCK and SOX9 (biliary markers), indicating bi-lineage differentiation of transplanted cdHPCs in vivo.

“Small GFP positive ductular cells were seen that expressed panCK and SOX9 (biliary markers), indicating bi-lineage differentiation of transplanted cdHPCs in vivo (Figure 8h).”

May combine two claims.

The induction of damage and repair on the background of Fn14 loss corresponds with failed ductular expansion, indicating that in the AhCre+Mdm2flox/− mouse, HPCs are critical for hepatic regeneration.

“The induction of damage and repair on the background of Fn14 loss corresponded with failed ductular expansion (Figure 5e and f), indicating that in the AhCre+Mdm2flox/− mouse, HPCs are critical for hepatic regeneration.”

May combine two claims.

This evidence suggests a role for HPCs in either supporting hepatocyte proliferation or differentiating into hepatocytes.

“This evidence, although indirect, suggested a role for HPCs in either supporting hepatocytes proliferation or differentiate into hepatocytes.”

May combine two claims.

The authors' characterisation of a defined HPC population indicates its biological significance and future therapeutic potential for treating human liver disease.

“Our characterisation of a defined population of HPCs indicates their biological significance and future therapeutic potential for the treatment of human liver disease.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figure 3a-f, 4a, c, f, 5a are representative of 40 mice over a 1-year period.

“For figure 3a-f, 4a, c, f, 5a, the images are representative for 40 mice in total over a 1-year period.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Experiments in figures 1g-j, 2c, g, h, 5c, and Supplementary figures 1a-c, 2a-h, 3e, 3f were repeated twice.

“In the cases of experiments in figures 1g-j, 2c, g, h, 5c, Supplementary figures 1a-c, 2a-h, 3e, 3f experiments were repeated twice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For representative images, whole liver lobes were examined histologically in multiple biological replicates.

“For representative images whole liver lobes were examined histologically in multiple biological replicates.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Power calculations were not routinely done, but animal numbers were selected to reflect the expected response magnitude based on variability in prior experiments.

“Power calculations were not routinely performed, however animal numbers were chosen to reflect the expected magnitude of response taking into account the variability observed in previous experiments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figures 1b-f, k-l, 2a, b, d, e, I, j, and Supplementary figures 2h, 3a-c are representative of 30 mice over a 3-year period.

“For figure 1b-f, k-l, 2a, b, d, e, I, j, Supplementary figures 2h, 3 a-c the images are representative for 30 mice in total over a 3-year period.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in Supplementary figures 5a, 6a, 6b are representative of 7 separate clonal cultures.

“For Supplementary figures 5a, 6a, 6b the images are representative for 7 separate clonal cultures.”

May combine two claims.

The authors' data indicate a facultative, functionally significant HPC population that does not contribute to homeostatic repair but becomes activated in severe liver injury with widespread hepatocyte senescence.

“Together our data suggests the existence of a facultative and functionally significant HPC population which does not contribute to homeostatic repair, but is activated in severe liver injury where hepatocyte senescence is widespread.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figure 7a-d, k, and Supplementary figure 1b, c are representative of 10 mice.

“For figure 7 a-d, k, Supplementary figure 1b, c the images are representative for 10 mice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figure 5f, g, 6c, d are representative of 7 mice.

“For figure 5f, g, 6c, d the images are representative for 7 mice.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Huch and colleagues have shown that EpCAM+ cells isolated from human liver can be expanded as organoids in vitro.

“Huch and colleagues have shown that EpCAM+ cells isolated from human liver can be expanded as organoids in vitro50.”

• cites [50] Huch, M. et al. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160, 299–312 (2015). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For figures 1g-j, 2c and Supplementary figures 2a-f, the replicates were repeated biological experiments with independent analysis, with time points varying between experiments in 1h-j, 2c and Supplementary Figures 2e,f.

“For figures 1g-j, 2c and Supplementary figures 2a-f these replicates consisted of repeated biological experiments with independent analysis, time points for analysis varied between experiments in 1h-j, 2c and Supplementary Figures 2e,f.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figure 7g, i, m, and Supplementary figures 4a, f, 5b-d are representative of 5 separate clonal cultures.

“For figure 7g, i, m, Supplementary figures 4a, f, 5b-d the images are representative for 5 separate clonal cultures.”

May combine two claims.

The proliferation of p21 positive hepatocytes is irreversibly inhibited, making it unlikely that p21 positive hepatocytes contribute to regeneration here.

“The proliferation of p21 positive hepatocytes is irreversibly inhibited49 making it unlikely that p21 positive hepatocytes contribute to regeneration here.”

• cites [49] Wu, H. et al. Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev. 10, 245–260 (1996). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Samples were randomized by a blinded third party and assessed by a separate blinded assessor, with unblinding just before final analysis.

“Samples were randomized by a 'blinded' third party before being assessed separate 'blinded' assessor. Unblinding was performed immediately prior to final data analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Species isotype staining controls were routinely performed.

“Species isotype (Santa Cruz) staining controls were routinely performed.”

Classified as probably not a substantial claim.

Cell counts were done manually on blinded slides across more than 20 consecutive non-overlapping fields at ×200 or ×400 magnification.

“Cell counts were performed manually on blinded slides and more than 20 consecutive non overlapping fields at ×200 or ×400 magnification.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For figures 2g, h and 5c and Supplementary Figures 2g, 3e, 3f, replicate experiments were repeat analyses from biological samples starting at RNA extraction.

“For figures 2g, h and 5c and Supplementary Figures 2g, 3e, 3f, replicate experiments consisted of repeat analysis from biological samples from the level of RNA extraction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in Supplementary figures 2a-c, 4a are representative of 12 mice.

“For Supplementary figures 2a-c, 4a the images are representative for 12 mice.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All animal experiments followed procedural guidelines, severity protocols, and had ethical approval from AWERB and the UK Home Office.

“All animal experiments were carried out under procedural guidelines, severity protocols and with ethical permission from the Animal Welfare and Ethical Review Body (AWERB) and the Home Office (UK).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Stained samples were compared with unstained and isotype controls.

“Stained samples were compared to unstained and isotype controls.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Trigger pulse width was used to exclude cell doublets from analysis and collection.

“Trigger pulse width was used to exclude cell doublets from analysis and collection.”

May combine two claims.

Although the proportion of p53 high hepatocytes was 98.9%, almost all hepatocytes (99.96%) were p21 positive, possibly reflecting either under-detection of p53 by immunohistochemistry or the occurrence of spreading senescence.

“Although the proportion of p53 high hepatocytes was 98.9%, almost all hepatocytes (99.96%) were p21 positive, possibly reflecting either under-detection of p53 by immunohistochemistry or the occurrence of spreading senescence as previosly reported in vivo and in vitro 46, 4748.”

• cites [46] Acosta, J. C. et al. A complex secretory program orchestrated by the inflammasome controls paracrine senescence. Nat. Cell Biol. 15, 978–990 (2013). sourceNot checked yet

  Not checked in the free run.

• cites [47] Nelson, G. et al. A senescent cell bystander effect: senescence-induced senescence. Aging Cell 11, 345–349 (2012). sourceNot checked yet

  Not checked in the free run.

• cites [48] Salama, R., Sadaie, M., Hoare, M. & Narita, M. Cellular senescence and its effector programs. Genes Dev. 28, 99–114 (2014). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

qPCR experiments were routinely run in technical triplicates when possible.

“Routinely qPCR experiments were performed in technical triplicates when possible.”

Classified as probably not a substantial claim. May combine two claims.

The authors used a highly stringent FACS gating strategy without a transgenic reporter to investigate multiple potential HPC populations.

“We investigated multiple potential HPC populations by using a highly stringent FACS gating strategy without the need of transgenic reporter.”

• AssumptionRests on an assumption: HPCs share markers with cholangiocytes, so a defined selection strategy is needed to reduce impurity for human translation.

May combine two claims.

Earlier FACS-based isolations of putative HPCs relied on a single selection marker or failed to fully exclude other non-parenchymal cell populations.

“Although previous studies have isolated putative HPCs using FACS isolation, these techniques are based on single selection markers37,8 or did not completely exclude other NPC populations37,52.”

• cites [37] Qiu, Q., Hernandez, J. C., Dean, A. M., Rao, P. H. & Darlington, G. J. CD24-positive cells from normal adult mouse liver are hepatocyte progenitor cells. Stem Cells Dev. 20, 2177–2188 (2011). sourceNot checked yet

  Not checked in the free run.

• cites [8] Okabe, M. et al. Potential hepatic stem cells reside in EpCAM + cells of normal and injured mouse liver. Development 136, 1951–1960 (2009). sourceNot checked yet

  Not checked in the free run.

• cites [52] Suzuki, A. et al. Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver. Hepatology 48, 1964–1978 (2008). sourceNot checked yet

  Not checked in the free run.

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figure 8 are representative of 23 mice.

“For figure 8 the images are representative for 23 mice.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

For figures 7e, f, 8a-h and Supplementary figures 4b, c, e, 5e, and 6c, flow cytometry and qPCR experiments were repeated three times, and transplantation experiments are shown as representative from three experiments.

“In the cases of experiments in figures 7e, f, 8a-h and Supplementary figures 4b, c, e, 5e, and 6c, flow cytometry and qPCR experiments were repeated for 3 times, transplantation experiments are shown as representative from 3 experiments.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The image in figure 7h is representative of 50 cells.

“For figure 7h the image is representative of 50 cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Images in figure 2k, j, 4e, and Supplementary figures 3a-d, h are representative of 6 mice.

“For figure 2k, j, 4e, Supplementary figures 3a-d, h, the images are representative for 6 mice.”