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Cited by 253Liver physiology and pathologyOrgan Transplantation Techniques and OutcomesTissue Engineering and Regenerative Medicine

In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity

Open access
Zhang, Kun, Zhang, Ludi, Liu, Wenming, Ma, Xiaolong, Cen, Jin, Sun, Zhen, Wang, Chenhua, Feng, Sisi +13 more·2018·Cell Stem Cell
DOI 10.1016/j.stem.2018.10.018
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Abstract

Transplantation of human hepatocytes (HHs) holds significant potential for treating liver diseases. However, the supply of transplantable HHs is severely constrained by limited donor availability and compromised capacity for in vitro expansion. In response to chronic injury, some HHs are reprogrammed into proliferative cells that express both hepatocyte and progenitor markers, suggesting exploitable strategies for expanding HHs in vitro. Here, we report defined medium conditions that allow 10,000-fold expansion of HHs. These proliferating HHs are bi-phenotypic, partially retaining hepatic features while gaining expression of progenitor-associated genes. Importantly, these cells engraft into injured mouse liver at a level comparable to primary HHs, and they undergo maturation following transplantation in vivo or differentiation in vitro. Thus, this study provides a protocol that enables large-scale expansion of transplantable HHs, which could be further developed for modeling and treating human liver disease.

AI snapshot

Written from the paper’s full text.

TL;DR

A defined medium (HM) containing Wnt3a, combined with 5% O2 hypoxia culture, converts primary human hepatocytes into bi-phenotypic proliferating cells (ProliHHs) that expand ~10,000-fold without genetic manipulation. ProliHHs engraft Fah-deficient mouse livers at levels comparable to primary human hepatocytes (64% ± 21.8% vs 70.4% ± 21.5%) and mature in vivo.

Key findings

  • HHs cultured in HM proliferated from day 2 (77.8% ± 4.1% Ki67-positive; 21.8% ± 4.8% BrdU-positive at day 3) and were serially passaged for one month with ~300-fold expansion, then arrested after 4 passages with senescence; hypoxia (5% O2) extended proliferation by 4 additional passages for ~10,000-fold total expansion.
  • ProliHHs were bi-phenotypic: >97% ALB/AAT-positive at P3–P4, with lower ALB, HNF4A, TTR, CYP3A4 but similar-or-higher CYP1A2, CAR, C3, UGT1A1 versus freshly thawed PHHs, alongside progenitor markers (96.95% SOX9+, 99.07% CK19+, 99.24% CK7+, 98% CD133+); 1,652 of 3,482 (47.4%) LPC-upregulated genes were also upregulated in ProliHHs.
  • In FRG mice, 11 of 14 ProliHH-transplanted mice survived >4 months versus 5 of 7 PHH-transplanted mice (log-rank p = 0.72); human serum albumin reached 5.8 ± 4.5 mg/mL at 4 months versus 7.3 ± 6.1 mg/mL for PHHs (p = 0.69), and repopulation was 64% ± 21.8% versus 70.4% ± 21.5% (p = 0.66).
  • ProliHHs reverted toward mature hepatocytes in vitro (increased ALB, TTR, HNF4A, AAT; induced CYP2B6 activity and urea synthesis; reduced SOX9, CK19, CK7) and matured in vivo (repopulated cells lost CK19/CK7, showed CYP3A4/GS zonation, and in vivo transcriptome correlation with repopulated PHHs r² = 0.94 versus in vitro r² = 0.74). Late-passage (after P6) ProliHHs repopulated 29% ± 12% of FRG livers with albumin 1.9 ± 1.2 mg/mL at 20 weeks. No tumors formed at 4 months or after subcutaneous xenograft.

Methods

  • Cryopreserved human hepatocytes from 12 individuals (Celsis In Vitro Technologies) were screened across 10 media; HLIM was modified by removing Rspo1, Noggin, and forskolin to yield HM, with Wnt3a required for proliferation (CHIR99021 could not substitute). Cells were cultured in normoxia or hypoxia (5% O2, 5% CO2), and maturation used HIM in 3D for 10 days.
  • Characterization included qPCR, immunofluorescence, flow cytometry, RNA-seq (GEO: GSE112866) with PCA/hierarchical clustering and GSEA against hiPSC-derived LPCs and HLCs, plus albumin ELISA, urea synthesis, CYP2B6 and CYP2D6 (debrisoquine) metabolism assays, PAS and Oil Red O staining, and SA-β-gal staining.
  • In vivo work used Fah−/−Rag2−/−Il2rg−/− (FRG) mice with NTBC withdrawal and intrasplenic transplantation of 5 × 10^5 early- (before P4) or late-passage (after P6) ProliHHs or PHHs, with Fah immunohistochemistry used to quantify repopulation; NOD/SCID mice received subcutaneous injections for tumor assays.

Limitations

  • Stated: the link between Wnt3a and senescence "needs to be further addressed in the future"; it remains to be determined whether mTOR inhibitors could sustain longer-term expansion; direct comparison of therapeutic effects between rat CLiPs and human ProliHHs is difficult given different recipient models and donor cells; the authors note it would be interesting to further characterize transcriptomes among PHHs, ProliHHs, and HLCs and identify key molecular mechanisms for repopulation.
  • Stated/evident: as ProliHHs expand, maturation genes decrease and progenitor genes increase, associated with decreased repopulation (late-passage cells repopulated 29% ± 12% versus 64% ± 21.8% for early passage); hypoxia-cultured ProliHHs after P6 shifted from the main cluster with an attenuated hepatic phenotype; ProliHHs did not establish the full progenitor gene profile (cluster III genes DLK1, KIT, FOXJ1 not fully upregulated).
  • Evident: expansion efficiency varied by donor age (5 young donors <11 years reached 1,000–10,000-fold at P8, whereas only 2 of 5 adult donors >37 years expanded ~1,000-fold at P5), and only 7 of 10 additional individuals' hepatocytes expanded for at least 5 passages.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.stem.2018.10.018

Cites

41 of 41 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

20 of 44 cited sources checked · 9 supported · 1 flagged · accuracy not yet measured

  • Premisecitation
    § Introduction

    Cells from these expandable organoids show poor performance in transplantation.

    “Nevertheless, cells from these expandable organoids show poor performance in transplantation (Huch et al., 2015”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Huch et al., 2015 source

      The Results section on differentiation and transplantation reports engraftment, human albumin and α-1-antitrypsin in recipient mice, and secretion levels that remained stable for over 60 days in five of six mice. Primary hepatocytes initially produced more albumin, but the levels approached those of transplanted organoids within a month. The paper therefore does not characterize the organoids’ overall transplantation performance as poor. (LLM, conf=0.83)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation
    § Introduction

    For decades, expandable HHs have been a bottleneck for their applications for research purposes and clinical therapies.

    “For decades, expandable HHs have been a bottleneck for their applications for research purposes and clinical therapies (Bhatia et al., 2014; Michalopoulos, 2014; Mitaka, 1998; Runge et al., 2000”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Bhatia et al., 2014 source
    • cites Michalopoulos, 2014 source

      The section on hepatocytes as facultative stem cells discusses cell-culture studies of hepatocyte-to-biliary-cell conversion, not a longstanding difficulty expanding human hepatocytes. The section on liver failure describes little proliferation in specific regenerative clusters, but does not link that observation to a bottleneck for research or clinical therapies. Searches for expandable human hepatocytes, in vitro expansion, and clinical applications found no passage addressing the claim; page numbers were not available in our copy. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites Mitaka, 1998 source
  • Premisecitation
    § Introduction

    Human HLCs showed limited engraftment capability in mouse livers.

    “Nevertheless, human HLCs showed limited engraftment capability in mouse livers (Gao et al., 2017; Ji et al., 2013”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Gao et al., 2017 source
    • cites Ji et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Premisecitation
    § Introduction

    In chronic liver diseases, HHs with expression of progenitor-associated markers were documented in regenerative nodules.

    “Notably, in chronic liver diseases, HHs with expression of progenitor-associated markers were also documented in regenerative nodules (Michalopoulos and Khan, 2015; Yoon et al., 2011”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites Michalopoulos and Khan, 2015 source

      The ‘Possible Origins of LPC’ section reports hepatocyte nodules in patients with micronodular cirrhosis, supporting the chronic-disease and nodule context. It does not say that hepatocytes in those nodules expressed progenitor-associated markers; its discussion of progenitor cells in regenerative clusters concerns fulminant hepatic failure instead. Page numbers were not provided in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Yoon et al., 2011 source

      The Results report EpCAM-positive hepatocytes in chronic hepatitis, with staining more extensive in cirrhotic livers; the paper identifies EpCAM as a hepatic stem/progenitor-cell marker. It does not identify those cells as HHs or specifically document their location within regenerative nodules. Its Discussion mentions regenerative nodules when reporting a separate study by Lin et al. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Findingown data
    § 1 Send email to luyingpeng@tongji.edu.cn ∙ Lijian Hui

    ProliHHs repopulate injured livers with comparable efficiency to primary hepatocytes.

    “ProliHHs repopulate injured livers with comparable efficiency to primary hepatocytes”
    • Also stated in: § 1 Send email to luyingpeng@tongji.edu.cn ∙ Lijian Hui · plain
      “these cells engraft into injured mouse liver at a level comparable to primary HHs”
    • Also stated in: § Introduction · plain
      “these cells substantially repopulated Fah-deficient mouse livers”
    Quote in the paperSubstantial claim
  • May combine two claims. 40
  • Classified as probably not a substantial claim. 21
  • The quote only partly states this claim. 3

  • May combine two claims.

    Premisecitation
    § Introduction

    The use of hepatocyte transplantation is hampered by the limited availability of donor hepatocytes and the inability to expand primary HHs in vitro.

    “However, the actual use of hepatocyte transplantation has been hampered by the limited availability of donor hepatocytes and the inability to expand primary HHs (PHHs) in vitro.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Bhatia et al., 2014 source
    • cites Michalopoulos, 2014 source

      The passages on liver failure discuss tissue obtained during liver transplantation, while the cell-culture passages discuss hepatocyte differentiation (Implications for Regenerative Mechanisms in Liver Failure; Commentary). Searches for donor-hepatocyte availability and expansion of primary human hepatocytes found no passage addressing either claimed barrier to hepatocyte transplantation. Page numbers were not available in our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites Mitaka, 1998 source

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

    Premisecitation
    § Results

    Hypoxia prevents cultured cells from senescing.

    “Because hypoxia has been reported to prevent cultured cells from senescing (Leontieva et al., 2012; Parinello, 2003”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Leontieva et al., 2012 source
    • cites Parinello, 2003 source

      The Abstract reports that mouse embryonic fibroblasts (MEFs) cultured at 3% oxygen showed little evidence of replicative senescence. But the finding does not establish that hypoxia prevents senescence in cultured cells generally; it also reports that low oxygen delayed, rather than prevented, senescence in Balb/c MEFs. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation
    § Introduction

    The repopulation efficiency of HLCs is around 2%, far lower than that of PHHs, which is around 70%.

    “The repopulation efficiency of HLCs, which is around 2%, is far lower than that of PHHs, which is around 70% (Ji et al., 2013; Rezvani et al., 2016; Sampaziotis et al., 2015”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Ji et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Rezvani et al., 2016 source

      The ‘Therapeutic efficacy of current lab-made hepatocytes’ section says lab-made hepatocytes generally repopulate injured mouse livers less effectively than primary human hepatocytes. Its iMPC-Heps subsection reports 2% repopulation for one lab-made cell type, but the iPSC-Heps subsection reports up to 20%; the copy does not establish 2% as a general figure or give the claimed 70% figure for primary hepatocytes. Page numbers were not available in the indexed passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Sampaziotis et al., 2015 source

      Section 22 says HLCs have a limited capacity to repopulate the liver in animal models, supporting the general point that their repopulation is limited. It does not give the claimed efficiencies of around 2% for HLCs or 70% for primary human hepatocytes (PHHs), or quantify the comparison between them. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation
    § Introduction

    During injury, mouse hepatocytes can be reprogrammed to proliferative biliary-like progenitor cells (HepPDs).

    “Specifically, during injury, mouse hepatocytes can be reprogrammed to proliferative biliary-like progenitor cells (HepPDs) (Tarlow et al., 2014; Yanger et al., 2013”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Tarlow et al., 2014 source
    • cites Yanger et al., 2013 source

      The Results and Discussion (page unavailable) show that, after DDC-induced injury in mice, lineage-labeled hepatocytes acquired biliary markers and morphology. The paper describes conversion toward biliary epithelial cells, but does not identify the resulting cells as HepPDs or establish that they are proliferative progenitors; it says whether proliferation is required for reprogramming remains unknown. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation
    § Introduction

    Cell plasticity plays an important role in liver regeneration.

    “Cell plasticity has lately been recognized to play an important role in liver regeneration (Kopp et al., 2016; Li et al., 2016; Michalopoulos and Khan, 2015”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Kopp et al., 2016 source
    • cites Li et al., 2016

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Michalopoulos and Khan, 2015 source

      The Conclusions describe plasticity between liver cell types, and “Possible Origins of LPC” reports that cell-type conversion can contribute to repair under severe injury or when hepatocyte replication is blocked. But that section also says that during normal liver regeneration, hepatocytes and cholangiocytes generally proliferate while retaining their identities. The unqualified claim overstates plasticity’s role across liver regeneration. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

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

    Premisecitation
    § Introduction

    High-throughput screening identified small molecules that supported HHs for one week with about 10-fold expansion.

    “Another study using high-throughput screening identified small molecules that supported HHs for one week with about 10-fold expansion (Shan et al., 2013”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Shan et al., 2013 source

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

    Premisecitation
    § Introduction

    Hepatocyte-to-HepPD conversion is likely to take place through a bi-phenotypic 'intermediate' status characterized by the co-expression of hepatocyte markers and progenitor-associated markers.

    “Hepatocyte-to-HepPD conversion is likely to take place through a bi-phenotypic “intermediate” status characterized by the co-expression of hepatocyte markers and progenitor-associated markers (Tanimizu et al., 2014; Yanger et al., 2013”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Tanimizu et al., 2014 source
    • cites Yanger et al., 2013 source

  • May combine two claims.

    Premisecitation
    § Introduction

    Oxygenated or microfabricated co-cultures allow HHs to retain metabolic function for a few weeks, but these cells lose their proliferative capacity.

    “The oxygenated or microfabricated co-cultures have been reported to allow HHs to retain metabolic function for a few weeks; however, these cells lose their proliferative capacity (Khetani and Bhatia, 2008; Kidambi et al., 2009”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Khetani and Bhatia, 2008 source
    • cites Kidambi et al., 2009 source

      The Discussion reports high metabolic activity in oxygenated co-cultures of human and rat hepatocytes. The Results document metabolic measurements through day 7 and human-hepatocyte morphology through day 9, but do not establish retained metabolic function for a few weeks. No passage found reports that these cells lose proliferative capacity. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Premisecitation
    § Introduction

    Human liver organoids generated from bile-duct-derived bipotential cells could be expanded long term in vitro.

    “Moreover, human liver organoids generated from bile-duct-derived bipotential cells could be expanded long term in vitro (Huch et al., 2015”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Huch et al., 2015 source

  • May combine two claims.

    Interpretationcitation
    § Discussion

    Wnt3a may not only activate canonical Wnt signaling but also crosstalk with other pathways, such as Hippo/YAP.

    “This may be explained by that Wnt3a not only activates the canonical Wnt signaling but also crosstalks with other pathways, such as the Hippo/YAP pathway”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Azzolin et al., 2014 source

  • May combine two claims.

    Findingown data
    § 1 Send email to luyingpeng@tongji.edu.cn ∙ Lijian Hui

    ProliHHs are bi-phenotypic, partially retaining hepatic features and expressing progenitor-associated markers.

    “Proliferating HHs (ProliHHs) display hepatic features and express progenitor markers”
    • Also stated in: § 1 Send email to luyingpeng@tongji.edu.cn ∙ Lijian Hui · plain
      “These proliferating HHs are bi-phenotypic, partially retaining hepatic features while gaining expression of progenitor-associated genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § 1 Send email to luyingpeng@tongji.edu.cn ∙ Lijian Hui

    Human hepatocytes can be serially passaged for more than one month and expanded 10,000-fold under defined medium with hypoxia.

    “Human hepatocytes can be serially passaged and expanded 10,000-fold”
    • Also stated in: § 1 Send email to luyingpeng@tongji.edu.cn ∙ Lijian Hui · plain
      “Here, we report defined medium conditions that allow 10,000-fold expansion of HHs.”
    • Also stated in: § Introduction · plain
      “In combination with hypoxia culture, HHs can proliferate continually for more than one month with 10,000-fold expansion in numbers.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § Results

    77.8% ± 4.1% of the cells were Ki67 positive on day 3.

    “We found 77.8% ± 4.1% of the cells were Ki67 positive”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    HHs cultured under hypoxic conditions showed reduced expression of cell cycle arrest genes and senescence genes and decreased number of SA-β-gal-positive cells.

    “HHs cultured under hypoxic conditions showed reduced expression of cell cycle arrest genes and senescence genes and decreased number of SA-β-gal-positive cells (Figures S3B and 1F”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Removal of Rspo1 or Noggin did not affect the proliferation of hepatocytes but did increase ALB expression.

    “Removal of Rspo1 or Noggin did not affect the proliferation of hepatocytes but did increase ALB expression (Figures S2A–S2C”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Under maturation condition, ProliHHs formed typical morphology of mature hepatocytes and showed polygonal shape.

    “Under the maturation condition, ProliHHs formed typical morphology of mature hepatocytes and showed polygonal shape”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Metabolic activity of CYP2B6 and capacity for urea synthesis were induced in ProliHHs after maturation.

    “the metabolic activity of CYP2B6 and the capacity for urea synthesis were induced in ProliHHs after maturation”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Secretion of human albumin in mouse serum increased gradually after transplantation of ProliHHs and reached 5.8 ± 4.5 mg/mL at 4 months, comparable to mice receiving PHHs (7.3 ± 6.1 mg/mL; p = 0.69).

    “The secretion of human albumin in mouse serum increased gradually after transplantation of ProliHHs (Figure 5D) and reached the levels of 5.8 ± 4.5 mg/mL at 4 months, which were comparable to mice receiving PHHs (7.3 ± 6.1 mg/mL; p = 0.69”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Wnt target genes and conserved YAP signature genes were enriched in ProliHHs.

    “Wnt target genes and conserved YAP signature genes were enriched in ProliHHs”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    When compared to PHHs, 3,482 genes were significantly upregulated in LPCs, among which 1,652 (47.4%) were also upregulated in ProliHHs.

    “When compared to PHHs, 3,482 genes were significantly upregulated in LPCs, among which 1,652 (47.4%) were also upregulated in ProliHHs”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Hepatocytes from 2 of 5 adult donors at ages >37 years could be expanded about 1,000-fold in cell number at P5 and showed cumulative growth curve similar to young donor cells.

    “hepatocytes from 2 of 5 adult donors at ages >37 years could be expanded about 1,000-fold in cell number at P5 and showed the cumulative growth curve similar to that of young donor cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Repopulated ProliHHs were Fah-positive and could be distinguished from mouse hepatocytes by pale cytoplasm staining of eosin and increased accumulation of lipid.

    “repopulated ProliHHs, which were Fah-positive, could be distinguished from mouse hepatocytes by showing the pale cytoplasm staining of eosin (Figure S6C) and increased accumulation of lipid”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    HHs lost tetraploidy and shifted to diploidy during expansion, with freshly thawed PHHs showing 67% tetraploidy and 28.6% diploidy and ProliHHs showing 8% tetraploidy and 88% diploidy.

    “HHs lost tetraploidy and shifted to diploidy during the expansion, showing 67% at tetraploidy and 28.6% at diploidy in freshly thawed PHHs and 8% at tetraploidy and 88% at diploidy in ProliHHs”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    ProliHH-transplanted mice lost body weight during the first 4 weeks after transplantation but later regained the body weight and remained stable.

    “The ProliHH-transplanted mice lost body weight during the first 4 weeks after transplantation but later regained the body weight and remained stable”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    11 of 14 FRG mice transplanted with ProliHHs at early passages (before P4) survived for more than 4 months, with survival curve similar to PHH-transplanted mice (p = 0.72).

    “11 of the 14 FRG mice that transplanted with ProliHHs at early passages (before P4) survived for more than 4 months (Figure 5A), showing similar survival curve with PHH-transplanted FRG mice (log rank test; p = 0.72).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Serum levels of ALT, AST, and TBIL were significantly reduced in both PHH-transplanted and ProliHH-transplanted FRG mice, with no significance between PHH and ProliHH.

    “Serum levels of aspartate transaminase (ALT), alanine transaminase (AST), and total bilirubin (TBIL) were significantly reduced in both PHH-transplanted and ProliHH-transplanted FRG mice (no significance between PHH and ProliHH)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Whole expression profiles showed ProliHHs cultured in normoxia still clustered close to PHHs and were separated from LPCs.

    “when whole expression profiles were analyzed, ProliHHs cultured in normoxia still clustered close to PHHs and were separated from LPCs”
    Quote in the paperSubstantial claim

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

    Findingown data
    § Results

    ProliHHs derived from new donors were bi-phenotypic, showing partial retention of hepatocyte-specific genes and functions alongside gained expression of progenitor-associated genes.

    “ProliHHs derived from the new donors were bi-phenotypic, showing partial retention of hepatocyte-specific genes and functions”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Repopulated ProliHHs remained expression of ALB and HNF4A and ceased expression of CK19 and CK7.

    “repopulated ProliHHs remained the expression of ALB and HNF4A and ceased expression of CK19 and CK7”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    During passage, some genes related to maturation were significantly reduced and genes related to progenitors were increased.

    “during the passage, some genes related to maturation were significantly reduced and, vice versa, genes related to progenitors were increased”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S1 (asserted), S3 (load-bearing), S4 (based).

    Findingnone given
    § Results

    Cluster III genes, such as DLK1, KIT, and FOXJ1, were not fully upregulated in ProliHHs.

    “Cluster III genes, such as the previously reported progenitor-associated markers DLK1, KIT, and FOXJ1”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Miyajima et al., 2014 source

  • May combine two claims.

    Findingown data
    § Results

    Immunofluorescent staining of ALB, CK19, and CK7 validated the qPCR data and showed no formation of mature bile duct cells.

    “Immunofluorescent staining of ALB, CK19, and CK7 validated the qPCR data and showed no formation of mature bile duct cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    PCA and hierarchical clustering revealed that ProliHHs formed an associated group and were distinct from other cells.

    “principal-component analysis (PCA) and hierarchical clustering revealed that ProliHHs formed an associated group and were distinct from other cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    In late-passage ProliHH-repopulated livers, expression of hepatocyte genes including albumin, AAT, and HNF4a was markedly increased and expression of bile duct cell gene CK19 was almost undetectable.

    “the expression of hepatocyte genes, including albumin, AAT, and HNF4a, was markedly increased (Figures S6J and S6K) and the expression of bile duct cell gene CK19 was almost undetectable”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    ProliHHs clustered closely with PHHs but remained distantly from HLCs.

    “ProliHHs clustered closely with PHHs but remained distantly from HLCs”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    ProliHHs can repopulate injured livers to around 60% and further mature in vivo.

    “By contrast, ProliHHs, which also partially maintained the mature phenotypes of hepatocytes, can repopulate injured livers around 60% and further mature in vivo, suggesting their potential for liver regenerative therapy.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Discussion

    ProliHHs generated in this study retained expression of hepatocyte genes and functions, though at levels lower than PHHs.

    “By contrast, the ProliHHs generated in our study retained the expression of hepatocyte genes and functions, though at levels lower than PHHs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Introduction

    Human-hepatocyte-derived bi-phenotypic cells are highly proliferative cells with potent capacity for repopulation.

    “These findings indicate that human-hepatocyte-derived bi-phenotypic cells are highly proliferative cells with potent capacity for repopulation.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation
    § Introduction

    This method was able to generate large numbers of functional HLCs capable of rescuing liver failure in mice after transplantation.

    “This method was able to generate large numbers of functional HLCs capable of rescuing liver failure in mice after transplantation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Ji et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

    Premisecitation
    § Introduction

    Human hepatocyte-like cells (HLCs) have been generated from human pluripotent stem cells by in vitro differentiation or from human somatic cells by direct transdifferentiation.

    “Human hepatocyte-like cells (HLCs) have been generated from human pluripotent stem cells by in vitro differentiation or from human somatic cells by direct transdifferentiation (Ji et al., 2013”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Ji et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation
    § Introduction

    Transplantation of primary hepatocytes has been evaluated as an alternative to organ transplantation both in clinics and animal models.

    “Transplantation of primary hepatocytes has been evaluated as an alternative to organ transplantation both in clinics and animal models (Dhawan et al., 2010”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dhawan et al., 2010 source

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

    Premisecitation
    § Results

    The rodent hepatocyte medium containing Y-27632, A-83-01, and CHIR99021 (YAC) was reported to induce the proliferation of rodent hepatocytes.

    “the rodent hepatocyte medium containing Y-27632, A-83-01, and CHIR99021 (YAC) medium, which was reported to induce the proliferation of rodent hepatocytes (Katsuda et al., 2017”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Katsuda et al., 2017 source

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

    Premisecitation
    § Introduction

    Efforts have been made to expand HHs by immortalization with hTERT and viral genes such as SV40, E6, and E7.

    “In other studies, effort was made to expand HHs by immortalization with hTERT and viral genes, such as SV40, E6, and E7 (Kobayashi et al., 2000; Levy et al., 2015”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kobayashi et al., 2000 source
    • cites Levy et al., 2015 source

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

    Premisecitation
    § Introduction

    Two recent studies have demonstrated the generation of expandable progenitor-like cells and duct-like cells from rodent hepatocytes in vitro.

    “Two recent studies have respectively demonstrated the generation of expandable progenitor-like cells and duct-like cells from rodent hepatocytes in vitro (Katsuda et al., 2017; Wu et al., 2017”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Katsuda et al., 2017 source
    • cites Wu et al., 2017

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationcitation
    § Introduction

    Expanding HHs at the 'intermediate' status in vitro may be a suitable strategy.

    “indicating this may be a suitable strategy for expanding HHs at the “intermediate” status in vitro.”
    Quote in the paperNot a substantial claim

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

    Premisecitation
    § Introduction

    HepPDs lose hepatic features but display the characteristics of liver progenitors.

    “which lose hepatic features but display the characteristics of liver progenitors (Tarlow et al., 2014”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tarlow et al., 2014 source

  • May combine two claims.

    Interpretationown data
    § Results

    Late-passage ProliHHs largely maintain repopulation capability and could be further matured in vivo.

    “These data suggest that late-passage ProliHHs largely maintain the repopulation capability and could be further matured in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    Although ProliHHs have not established the whole gene profile of progenitor cells, they expressed a large portion of LPC genes.

    “although ProliHHs yet not established the whole gene profile of progenitor cells, they expressed a large portion of LPC genes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    ProliHHs efficiently repopulated FRG liver and had therapeutic effects in treating liver injuries.

    “These data suggested that ProliHHs efficiently repopulated FRG liver and had therapeutic effects in treating liver injuries.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning
    § Discussion

    The study provides a protocol for expansion of HHs, which may be further developed as a cellular source for the treatment of liver diseases.

    “As a proof of principle, our study provides a protocol for expansion of HHs, which may be further developed as a cellular source for the treatment of liver diseases.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Hepatocyte transplantation is a promising alternative treatment to liver transplantation, especially in treating infants and young children with inborn errors of metabolism.

  • May combine two claims.

    Interpretationown data
    § Discussion

    The protocol for ProliHHs provides insights into human hepatocyte culture and a source of cells for therapeutic purposes.

    “In conclusion, our protocol for ProliHHs provides insights into HH culture and a source of cells for therapeutic purposes.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given
    § Discussion

    No sex bias differences were detected in the mice used for transplantation.

    “A mix of 8 to 12 weeks old male and female mice were used for transplantation and no sex bias differences were detected.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisenone given
    § Discussion

    Both rat CLiPs and human ProliHHs could efficiently repopulate the diseased liver when transplanted to uPA/SCID and FRG mice, respectively.

    “both rat CLiPs and human ProliHHs could efficiently repopulate the diseased liver when transplanted to urokinase-type plasminogen activator (uPA)/severe combined immunodeficiency (SCID) and FRG mice, respectively.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given
    § Discussion

    All q-PCR data were performed with at least 2 repeats.

    “All q-PCR data were performed with at least 2 repeats.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Interpretationreasoning
    § Discussion

    The repopulation capability of ProliHHs would be of notable value for clinical indications where 10% repopulation is sufficient.

    “such repopulation capability of ProliHHs would be of notable value for these clinical indications.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning
    § Discussion

    Wnt and YAP pathways might possess important functions in induction of ProliHHs in vitro.

    “it is possible that the Wnt and YAP pathways might possess important functions in induction of ProliHHs in vitro.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing), S4 (based). May combine two claims.

    Interpretationnone given
    § Discussion

    Differences between rodent and human proliferative hepatocytes may present different stages during hepatocyte-to-progenitor conversion in vitro or may merely reflect species difference.

    “These differences between rodent and human proliferative hepatocytes may present different stages during the hepatocyte-to-progenitor conversion in vitro or it may merely reflect the species difference.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given
    § Discussion

    No sex bias differences were detected during human hepatocyte cell culture.

    “No sex bias differences were detected during cell culture.”
    Quote in the paperNot a substantial claim
58 of 58

  • The paper relies on this without support.

    The actual use of liver transplantation is limited due to organ shortages.

    “However, the actual use of liver transplantation is limited due to organ shortages.”

  • The paper relies on this without support.

    Human hepatocytes with efficient liver repopulation capability are in great demand for liver disease treatment.

    “Human hepatocytes (HHs) with efficient liver repopulation capability are in great demand for liver disease treatment.”

  • The paper relies on this without support.

    Overexpression of hTERT and viral oncogenes holds a risk of tumorigenesis after transplantation.

    “overexpression of hTERT and viral oncogenes hold a risk of tumorigenesis after transplantation.”

  • The paper relies on this without support.

    No expression profile of human HepPDs or human liver progenitor cells is available.

    “Because no expression profile of human HepPDs or human liver progenitor cells is available”

  • The paper relies on this without support.

    Hepatocyte transplantation is a promising alternative treatment to liver transplantation, especially in treating infants and young children with inborn errors of metabolism.

    “Hepatocyte transplantation is a promising alternative treatment to liver transplantation, especially in treating infants and young children with inborn errors of metabolism.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Hepatocytes isolated from a donor liver show transient viability and functionality in culture and usually decline within days due to loss of polarity, reduced expression of liver-specific genes, and cellular senescence.

    “Hepatocytes isolated from a donor liver show transient viability and functionality in culture, and usually decline within days due to loss of polarity, reduced expression of liver-specific genes, and cellular senescence [3,4].”

    From The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances (2026) · cites this paper as [4]

    The Introduction describes difficulty expanding primary human hepatocytes in vitro, and the Results report senescence and cessation of proliferation during culture. The Discussion reports reduced maturation-related gene expression during passage. The paper does not establish that donor hepatocytes usually lose viability and function within days, or that loss of polarity causes that decline; no page numbers are available in our copy. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Human primary hepatocytes show substantial donor-to-donor variability not typically observed to the same degree in inbred mouse models, and this variability can persist even within engineered expansion or maturation protocols derived from the same donor.

    “Human primary hepatocytes also show substantial donor-to-donor variability that is not typically observed to the same degree in inbred mouse models, and this variability can persist even within engineered expansion or maturation protocols derived from the same donor [4,15].”

    From The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances (2026) · cites this paper as [4]

    Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

    Experimental — accuracy not yet measured

  • Several dramatic expansion results, including near-unlimited theoretical fold-expansion and multi-month clonal proliferation, have been demonstrated almost exclusively in mouse hepatocytes, whereas human systems have generally achieved more modest expansion with measurable trade-offs in redifferentiation and repopulation capacity at higher expansion levels.

    “Several of the most dramatic expansion results reported to date—including near-unlimited theoretical fold-expansion and multi-month clonal proliferation—have been demonstrated almost exclusively in mouse hepatocytes [18,31], whereas human systems have generally achieved more modest expansion with measurable trade-offs in redifferentiation and repopulation capacity at higher expansion levels [4,17]”

    From The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances (2026) · cites this paper as [4]

Cites

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Related

Authors
Zhang, K., Zhang, L., Liu, W., Ma, X., Cen, J., Sun, Z., Wang, C., Feng, S., Zhang, Z., Yue, L., Sun, L., Zhu, Z., Chen, X., Feng, A., Wu, J., Jiang, Z., Li, P., Cheng, X., Gao, D., Peng, L., & Hui, L.
Affiliations
  • Tongji University (CN)
  • Chinese Academy of Sciences (CN)
  • Shanghai Medical College of Fudan University (CN)
Corresponding author
  • Zhang, Ludi
  • Peng, Luying
  • Hui, Lijian
ORCID
Venue
Cell Stem Cell
Publisher
Elsevier BV
Published
2018-12
Volume
23
Issue
6
Pages
806-819.e4
ISSN
  • 1875-9777 (ISSN-L)
  • 1934-5909
DOI
10.1016/j.stem.2018.10.018
OpenAlex
W2900240008
Semantic Scholar
84cdedd48412d517b6209021b162657b70af2b78
Cited by
252
References
42
Citations by year
2026: 25 · 2025: 39 · 2024: 37 · 2023: 41 · 2022: 30 · 2021: 30 · 2020: 33 · 2019: 17
FWCI
10.79
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell stem cell (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Chinese Academy of Sciences (XDA16020201)
  • Shanghai Science and Technology Committee (16JC1400202, 15JC1400200)
  • National Science Foundation of China (31630044, 31601186, 31801228, 81471948)
  • External Cooperation Program of BIC
  • CSIRO-Chinese Academy of Science (153D31KYSB20150085, 153D31KYSB20160247)
  • China Postdoctoral Science Foundation (2017M620174, 2018T110412)
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Health Sciences › Medicine › Surgery › Organ Transplantation Techniques and Outcomes
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Keywords
human hepatocytes, in vitro expansion, liver repopulation, hepatocyte transplantation, hepatic progenitor cells, defined culture medium, engraftment, hepatocyte maturation, liver disease modeling, cell therapy
MeSH terms
Animals, Cell Count, Cells, Cultured, Humans, Liver, Mice, Inbred C57BL, Mice, SCID, Mice, Inbred NOD, Mice, Knockout, Hepatocytes, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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