​Temporal proteomic profiling of iPSC-derived human liver organoids reveals optimal maturation for drug metabolism and toxicology

May combine two claims.

Traditional 2D liver models fail to mimic the 3D architecture, cellular heterogeneity, and zonation of native liver, leading to discrepancies in drug metabolism and toxicity profiles and reduced predictive accuracy.

“While these model systems provide valuable insights into liver function and drug metabolism, they fail to mimic the intricate three-dimensional (3D) architecture, cellular heterogeneity, and zonation observed in the native liver. Consequently, these models often insufficiently simulate the liver microenvironment, leading to discrepancies in drug metabolism and toxicity profiles and reducing their”

• cites [7] Gerets, H. H. et al. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell. Biol. Toxicol. 28, 69–87. https://doi.org/10.1007/s10565-011-9208-4 (2012). sourcePartly supported

  The Introduction says HepG2 cells have low metabolic capacity and are less suited to testing metabolite toxicity. The Discussion reports limited sensitivity for detecting hepatotoxic compounds in the tested cell models. Neither section attributes those limitations to a lack of 3D architecture, cellular heterogeneity, or zonation; no page numbers are available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [8] Collins, S. D. et al. Hepatocellular Carcinoma (ed Tirnitz-Parker, J. E. E.) (2019).Not matched

  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).

iHLOs enable patient-specific modeling for precision medicine tailored to individual genetic and pathological conditions.

“Furthermore, iHLOs enable patient-specific modeling, providing a platform for precision medicine tailored to individual genetic and pathological conditions12,13.”

• cites [12] Mun, S. J. et al. Efficient and reproducible generation of human induced pluripotent stem cell-derived expandable liver organoids for disease modeling. Sci. Rep. 13, 22935. https://doi.org/10.1038/s41598-023-50250-w (2023). sourceSupported
• cites [13] Shinozawa, T. et al. High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids. Gastroenterology 160, 831–846. https://doi.org/10.1053/j.gastro.2020.10.002 (2021). sourcePartly supported

  The Introduction (page unavailable) says HLOs modeled genotype-specific susceptibility to bosentan-induced cholestasis and could facilitate personalized medicine. The Discussion (page unavailable) describes precision studies using donor-derived HLOs, but frames genomic stratification as a future possibility. The paper does not establish patient-specific modeling tailored to a broad range of individual pathological conditions. (LLM, conf=0.83)

  Experimental — accuracy not yet measured

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

Liver organoids express key enzymes including members of the cytochrome P450 family and hepatic transporters, often at lower levels compared to native liver tissue.

“Studies have confirmed that liver organoids express key enzymes, including members of the cytochrome P450 (CYP450) family and hepatic transporters, though often at lower levels compared to native liver tissue14,15.”

• cites [14] Howell, L. et al. Proteomic profiling of murine biliary-derived hepatic organoids and their capacity for drug disposition, bioactivation and detoxification. Arch. Toxicol. 95, 2413–2430. https://doi.org/10.1007/s00204-021-03075-3 (2021). sourceSupported
• cites [15] Harrison, S. P. et al. Scalable production of tissue-like vascularized liver organoids from human PSCs. Exp. Mol. Med. 55, 2005–2024. https://doi.org/10.1038/s12276-023-01074-1 (2023). sourcePartly supported

  The Results sections on hepatic parenchymal cells and liver-like function report CYP2A6 expression and CYP1A2/CYP3A4 activity in the organoids. Our copy does not establish expression of hepatic transporters or show that these enzymes and transporters are generally expressed at lower levels than in native liver tissue; it reports some low-expression zonation markers, but that is not a native-liver comparison. No page numbers are available in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

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

Expression of HNF1A, HNF4A, and FOXA2 typically peaks during the early progenitor stage and decreases toward the final hepatocyte-like cell stage.

“This finding is consistent with other reports that these genes’ expression typically peaks during early progenitor stage and decreases towards the final hepatocyte-like cells stage24–26.”

• cites [24] Ng, N. H. J. et al. HNF4A and HNF1A exhibit tissue specific target gene regulation in pancreatic beta cells and hepatocytes. Nat. Commun. 15, 4288. https://doi.org/10.1038/s41467-024-48647-w (2024). sourcePartly supported

  The Results section on genome-wide targets states that HNF4A expression peaks during the early progenitor stage and decreases by the hepatocyte-like cell stage. The passages found do not establish the same expression pattern for HNF1A or FOXA2. Page numbers are not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [25] Ng, N. H. J. et al. HNF4A haploinsufficiency in MODY1 abrogates liver and pancreas differentiation from patient-derived induced pluripotent stem cells. iScience 16, 192–205. https://doi.org/10.1016/j.isci.2019.05.032 (2019). sourcePartly supported

  The Results section ('MODY1-Mediated Loss of HNF4A Transcriptional Function Affects Subsequent Hepatic and Pancreatic Development Signatures'; page unavailable) reports that HNF4A expression peaked on day 8 of differentiation into hepatocyte-like cells, before other hepatic genes peaked on days 16 or 24. Our copy does not establish the same early peak and subsequent decline for HNF1A or FOXA2, so it supports only the HNF4A part of the claim. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [26] Aghadi, M., Elgendy, R. & Abdelalim, E. M. Loss of FOXA2 induces ER stress and hepatic steatosis and alters developmental gene expression in human iPSC-derived hepatocytes. Cell. Death Dis. 13, 713. https://doi.org/10.1038/s41419-022-05158-0 (2022). sourcePartly supported

  The Results section on FOXA2 expression during differentiation reports a peak at days 6–8 followed by a decline to little or no expression at the mature hepatocyte stage. The paper does not establish the same early peak and subsequent decline for HNF1A or HNF4A; its measurements of those genes concern FOXA2-deficient cells rather than their expression over the differentiation timeline. Page numbers are not available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

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

Day 21 or around this timepoint has been identified as an important stage for liver organoid functional development in several previous studies.

“It is also noteworthy that Day 21, or around this timepoint, has been identified as an important stage for liver organoid functional development in several previous studies13,15,28,29.”

• cites [13] Shinozawa, T. et al. High-fidelity drug-induced liver injury screen using human pluripotent stem cell-derived organoids. Gastroenterology 160, 831–846. https://doi.org/10.1053/j.gastro.2020.10.002 (2021). sourcePartly supported

  Materials and methods (Large scale screening with 238 test compounds; page unavailable) says the authors seeded liver organoids into drug-screening plates at Day 21, supporting the use of functional organoids at that timepoint. It does not identify Day 21 as an important stage in their functional development or show a developmental change specifically around that day. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [15] Harrison, S. P. et al. Scalable production of tissue-like vascularized liver organoids from human PSCs. Exp. Mol. Med. 55, 2005–2024. https://doi.org/10.1038/s12276-023-01074-1 (2023). sourceSupported
• cites [28] Guan, Y. et al. Live-cell imaging of human liver fibrosis using hepatic micro-organoids. JCI Insight 10. https://doi.org/10.1172/jci.insight.187099 (2024). sourcePartly supported

  The Results section (“A high-content screening system”) calls day-21 organoids mature and reports receptor expression at that timepoint, supporting a connection between day 21 and organoid maturation. It does not identify day 21 as an important stage specifically for functional development; its day-21 evidence concerns receptor expression. (LLM, conf=0.78)

  Experimental — accuracy not yet measured
• cites [29] Thompson, W. L. & Takebe, T. Generation of multi-cellular human liver organoids from pluripotent stem cells. Methods Cell. Biol. 159, 47–68. https://doi.org/10.1016/bs.mcb.2020.03.009 (2020). sourceSupported

May combine two claims.

The mesenchymal shift by Day 45 suggests greater cellular complexity at later stages, potentially facilitating iHLO use in modeling liver pathologies such as fibrosis, cirrhosis, and liver remodeling, as indicated by increased expression of hepatic stellate cell markers DES and VIM.

“Interestingly, the mesenchymal shift by Day 45 suggests greater cellular complexity at later stages, potentially facilitating iHLO use in modeling liver pathologies such as fibrosis, cirrhosis, and liver remodeling, as indicated by increased expression of hepatic stellate cell markers such as DES and VIM33,34.”

• cites [33] Higashi, T., Friedman, S. L. & Hoshida, Y. Hepatic stellate cells as key target in liver fibrosis. Adv. Drug Deliv Rev. 121, 27–42. https://doi.org/10.1016/j.addr.2017.05.007 (2017). sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [34] Zhang, C. Y., Yuan, W. G., He, P., Lei, J. H. & Wang, C. X. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World J. Gastroenterol. 22, 10512–10522. https://doi.org/10.3748/wjg.v22.i48.10512 (2016). sourcePartly supported

  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

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

Incorporation of patient-derived iPSCs into this system could further enhance its utility for personalized medicine, enabling the prediction of individual drug responses or disease progression.

“The incorporation of patient-derived iPSCs into this system could further enhance its utility for personalized medicine, enabling the prediction of individual drug responses or disease progression42.”

• cites [42] Bae, J., Choi, Y. S., Cho, G. & Jang, S. J. The Patient-Derived cancer organoids: promises and challenges as platforms for cancer discovery. Cancers (Basel) 14. https://doi.org/10.3390/cancers14092144 (2022). sourcePartly supported

  Section 3.3, “Precision Cancer Medicine” (page unavailable), says patient-derived cancer organoids can predict a patient’s drug response and help predict clinical outcomes. Section 3 mentions induced pluripotent stem cells as one source of organoids, but the paper does not say that incorporating patient-derived iPSCs into the proposed system would enhance it or enable prediction of disease progression. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

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

The findings align with previous proteomic studies of liver organoids demonstrating their capacity to recapitulate liver-specific phenotypes.

“The findings from current study align with previous proteomic studies of liver organoids, which have demonstrated their capacity to recapitulate liver-specific phenotypes.”

• cites [14] Howell, L. et al. Proteomic profiling of murine biliary-derived hepatic organoids and their capacity for drug disposition, bioactivation and detoxification. Arch. Toxicol. 95, 2413–2430. https://doi.org/10.1007/s00204-021-03075-3 (2021). sourceSupported
• cites [15] Harrison, S. P. et al. Scalable production of tissue-like vascularized liver organoids from human PSCs. Exp. Mol. Med. 55, 2005–2024. https://doi.org/10.1038/s12276-023-01074-1 (2023). sourceSupported

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

Kupffer cells are critical for modeling immune-mediated liver diseases.

“which are critical for modeling immune-mediated liver diseases37”

• cites [37] Ju, C. & Tacke, F. Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies. Cell. Mol. Immunol. 13, 316–327. https://doi.org/10.1038/cmi.2015.104 (2016). sourceSupported

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

Harrison et al. compared iHLO protein profiles with native liver tissue and highlighted enrichment of detoxification pathways in both systems.

“Similarly, Harrison et al. compare iHLO protein profiles with native liver tissue and highlighted enrichment of detoxification pathways in both systems, emphasizing the relevance of organoids for metabolically intensive applications15.”

• cites [15] Harrison, S. P. et al. Scalable production of tissue-like vascularized liver organoids from human PSCs. Exp. Mol. Med. 55, 2005–2024. https://doi.org/10.1038/s12276-023-01074-1 (2023). sourceSupported

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

Hepatic stellate cells, characterized by DES and VIM expressions, are central to liver fibrosis and extracellular matrix remodeling.

“Hepatic stellate cells, characterized by DES and VIM expressions, are central to liver fibrosis and extracellular matrix remodeling34,36.”

• cites [34] Zhang, C. Y., Yuan, W. G., He, P., Lei, J. H. & Wang, C. X. Liver fibrosis and hepatic stellate cells: Etiology, pathological hallmarks and therapeutic targets. World J. Gastroenterol. 22, 10512–10522. https://doi.org/10.3748/wjg.v22.i48.10512 (2016). sourcePartly supported

  The Introduction says activated hepatic stellate cells drive fibrosis and secrete proteins involved in extracellular matrix remodeling. The Etiology and Pathological Characteristics section identifies desmin (DES) as upregulated when stellate cells activate, but our copy does not identify VIM expression as a characteristic; it also confines the DES finding to activated cells. Page numbers were not available in the indexed passages. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [36] Hoffmann, C. et al. Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis. Sci. Rep. 10, 3850. https://doi.org/10.1038/s41598-020-60615-0 (2020). sourcePartly supported

  The Introduction identifies hepatic stellate cells as major contributors to fibrosis and producers of extracellular matrix proteins. The Results section on HSC-specific markers identifies desmin (DES) as an HSC marker. Our copy does not establish VIM expression as a characteristic of these cells; a search for VIM returned no matching passage. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

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

This work contributes to reducing reliance on animal testing and promoting ethical innovation in preclinical research.

“this work contributes to reducing reliance on animal testing and promoting ethical innovation in preclinical research, as emphasized by Ingelman-Sundberg and Lauschke43.”

• cites [43] Ingelman-Sundberg, M. & Lauschke, V. M. 3D human liver spheroids for translational Pharmacology and toxicology. Basic. Clin. Pharmacol. Toxicol. 130 (Suppl 1), 5–15. https://doi.org/10.1111/bcpt.13587 (2022). sourceCould not verify

  The Introduction and Conclusions describe human liver spheroids as tools for preclinical pharmacology and toxicology. Neither section says they reduce reliance on animal testing or discusses an ethical benefit; searches for animal testing, replacement, and ethics found no such passage in our copy. (LLM, conf=0.72)

  Experimental — accuracy not yet measured

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

Howell et al. quantified 4,405 proteins in murine hepatic organoids and observed differentiation-dependent upregulation of CYP450 enzymes, hepatic transporters, and other metabolic proteins.

“For instance, Howell et al. quantified 4,405 proteins in murine hepatic organoids and observed differentiation-dependent upregulation of CYP450 enzymes, hepatic transporters, and other metabolic proteins, which parallels our observations in human iHLOs14.”

• cites [14] Howell, L. et al. Proteomic profiling of murine biliary-derived hepatic organoids and their capacity for drug disposition, bioactivation and detoxification. Arch. Toxicol. 95, 2413–2430. https://doi.org/10.1007/s00204-021-03075-3 (2021). sourcePartly supported

  The Results sections on global protein expression and key hepatic proteins report 4,405 proteins identified across all samples and increased expression of multiple CYP450 enzymes, phase II enzymes, and transporters after organoid differentiation. The 4,405 count includes donor-matched liver samples, so the paper does not establish that all 4,405 were quantified in the organoids alone. Page numbers are unavailable in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Proteomic analysis focused on 19 CYP450 enzymes, 36 non-CYP Phase I enzymes, 36 Phase II enzymes, and 95 drug transporters (16 ABC transporters and 79 SLC transporters).

“Proteomic analysis focused on 19 CYP450 enzymes, 36 non-CYP Phase I enzymes, 36 Phase II enzymes, and 95 drug transporters (16 ATP-binding cassette [ABC] transporters and 79 solute carrier [SLC] transporters).”

May combine two claims.

Focal adhesion and ECM-receptor interaction pathways exhibited sustained upregulation across all phases, highlighting enhanced extracellular matrix remodeling and cell-matrix communication as organoids matured.

“Focal adhesion and ECM-receptor interaction pathways exhibited sustained upregulation across all phases, highlighting enhanced extracellular matrix remodeling and cell-matrix communication as organoids matured.”

May combine two claims.

CYP3A4 inhibition by ketoconazole yielded an IC50 of 0.83 μM in iHLOs, comparable to HepaRG cells (0.42 μM) but less potent than PHHs (0.056 μM).

“CYP3A4 inhibition by ketoconazole yielded an IC50 of 0.83 µM in iHLOs, comparable to HepaRG cells (0.42 µM) but less potent than the IC50 (0.056 µM) from PHHs, demonstrating their applicability for studying CYP-mediated drug-drug interactions (Fig. 4C).”

May combine two claims.

CYP3A4 activity in Day 30 iHLOs is inducible by rifampicin and inhibited by ketoconazole.

“Functional validation of CYP3A4 activity through rifampicin induction and ketoconazole inhibition demonstrated metabolic competence comparable to existing in vitro liver models, including HepaRG cells and primary human hepatocytes.”

• Also stated in: § iHLOs model for toxicology applications · plain

  “Kinetic assays showed that rifampicin significantly induced CYP3A4 activity in iHLOs over three days, while ketoconazole consistently suppressed CYP3A4 activity over the same period (Fig. 4D).”

May combine two claims.

Immunostaining confirmed ALB cellular localization on organoid surfaces in Day 45 iHLOs, suggesting continued presence of mature hepatocytes and formation of a cystic compartment.

“However, immunostaining confirmed ALB cellular localization on organoid surfaces (Fig. 1F), suggesting the continued presence of mature hepatocytes and the formation of a cystic compartment in Day 45 iHLOs.”

May combine two claims.

Day 30 iHLOs exhibited a protein expression similarity of 66.54% compared with human liver tissue, comparable to PHHs (67.26%-79.11%), but significantly higher than HepG2 (45.64%) and HepaRG cells (54.60%).

“Specifically, proteomics data showed that Day 30 iHLOs exhibited a protein expression similarity of 66.54% compared with human liver tissue, comparable to PHHs (67.26%–79.11%), but significantly higher than HepG2 (45.64%) and HepaRG cells (54.60%) (Fig. 4B).”

May combine two claims.

Primary bile acid biosynthesis was exclusively enriched at the early phase, suggesting rapid development of bile acid production function in iHLOs at this stage.

“Notably, primary bile acid biosynthesis was exclusively enriched at the early phase, suggesting a rapid development of bile acid production function in iHLOs at this stage.”

May combine two claims.

ABC transporters peaked at Day 30, with ABCB1 maintaining high expression on Day 45, emphasizing its role in drug efflux.

“ABC transporters peaked at Day 30, with ABCB1 maintaining high expression on Day 45, emphasizing its role in drug efflux (Fig. 3D).”

May combine two claims.

CYP1B1 and CYP4F12 exhibited progressive increases, reaching maximum levels at Day 45, reflecting roles in fatty acid and bile acid metabolism during later stages.

“CYP1A2 displayed stable expression across Days 7–30, while CYP1B1 and CYP4F12 exhibited progressive increases, reaching maximum levels at Day 45, reflecting roles in fatty acid and bile acid metabolism during later stages.”

May combine two claims.

PI3K-Akt signaling was significantly enriched at mid- and late phases of iHLO development, implying expansion and functional maturation of the liver organoid, along with structural organization and functional integration of different cell types.

“Moreover, PI3K-Akt signaling was significantly enriched at mid- and late phases of iHLO development, implying the expansion and functional maturation of the liver organoid, along with structural organization and functional integration of different cell types within the organoid.”

May combine two claims.

Temporal pattern-based analysis showed 660 differentially expressed proteins during the early phase (391 upregulated, 269 downregulated in Day 15), 543 during the mid phase (273 upregulated, 270 downregulated in Day 30), and 688 during the late phase (292 upregulated, 396 downregulated in Day 45).

“This Temporal pattern-based analysis showed 660 differentially expressed proteins during the early phase (391 upregulated, 269 downregulated in Day 15), 543 during the mid phase (273 upregulated, 270 downregulated in Day 30), and 688 during the late phase (292 upregulated, 396 downregulated in Day 45) (Supplementary Table 1).”

May combine two claims.

Immunostaining of Day 30 organoids confirmed the presence of CYP3A4 and ALB in hepatocytes and KRT7 in cholangiocytes, indicating co-differentiation of these lineages.

“Immunostaining of Day 30 organoids confirmed the presence of CYP3A4 and albumin (ALB) in hepatocytes and KRT7 in cholangiocytes, indicating co-differentiation of these lineages (Fig. 1E and Supplementary 2 A).”

May combine two claims.

By Day 45, reduced CYP expression correlated with the emergence of mesenchymal-like cells, although functional hepatocytes remained, as evidenced by continued ALB localization.

“By Day 45, reduced CYP expression correlated with the emergence of mesenchymal-like cells, although functional hepatocytes remained, as evidenced by continued ALB localization in immunostaining analyses.”

May combine two claims.

Rifampicin significantly induced CYP3A4 activity in iHLOs over three days, while ketoconazole consistently suppressed CYP3A4 activity over the same period.

“Kinetic assays showed that rifampicin significantly induced CYP3A4 activity in iHLOs over three days, while ketoconazole consistently suppressed CYP3A4 activity over the same period (Fig. 4D).”

May combine two claims.

By Day 15, AFP notably increases and early hepatic metabolic proteins like CYP2E1 emerge, signaling the onset of hepatic differentiation.

“By Day 15, alpha-fetoprotein (AFP), a hallmark of immature hepatocytes, demonstrated a notable increase, coupled with the emergence of early hepatic metabolic proteins like CYP2E1, signaling the onset of hepatic differentiation (Fig. 1D).”

May combine two claims.

Drug-metabolizing pathways, especially cytochrome P450, varied significantly across iHLO development, with highly enriched PPAR signaling.

“In addition, drug-metabolizing pathways, especially cytochrome P450, varied significantly across iHLO development, with highly enriched PPAR signaling, a pathway critical for hepatocyte differentiation and lipid metabolism.”

May combine two claims.

Day 30 iHLOs show a decline in AFP and substantial upregulation of mature hepatocyte markers CYP3A4 and UGT1A1.

“Proteomic analysis of Day 30’s iHLOs showed a decline in AFP expression, accompanied by a substantial upregulation of markers associated with mature hepatocytes such as CYP3A4 and UDP-glucuronosyltransferase 1A1 (UGT1A1).”

May combine two claims.

The proteomic similarity of Day 30 iHLOs to PHHs, with a 33.46% expression difference from liver tissue, underscores their physiological relevance compared to HepG2 cells (54.36% difference) or HepaRG cells (45.40% difference).

“Importantly, the proteomic similarity of Day 30 iHLOs to PHHs, with a 33.46% expression difference from liver tissue, underscores their physiological relevance compared to traditional models like HepG2 cells, which exhibit a 54.36% protein difference from liver tissue, or HepaRG cells, with a 45.40% protein difference.”

May combine two claims.

iHLOs exhibited a concentration-dependent inhibition profile following ketoconazole treatment (IC50: 0.83 μM), comparable to HepaRG cells but distinct from PHHs.

“The functional assays of CYP3A4 further validate this claim: iHLOs exhibited a concentration-dependent inhibition profile following ketoconazole treatment (IC50: 0.83 µM), comparable to HepaRG cells but distinct from PHHs.”

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

Bulk proteomics provides a comprehensive view of cellular differentiation pattern.

“bulk proteomics provides a comprehensive view of cellular differentiation pattern”

May combine two claims.

iHLOs respond predictably to enzyme inducers and inhibitors, positioning them as a robust alternative to PHHs for toxicology testing.

“These results underscore the ability of iHLOs to respond predictably to enzyme inducers and inhibitors, positioning them as a robust alternative to PHHs for toxicology testing.”

May combine two claims.

Persistent endothelial cell proliferation and migration across all stages suggest uninterrupted endothelial lineage development, contributing to organoid vascularization.

“Persistent endothelial cell proliferation and migration across all stages suggest uninterrupted endothelial lineage development, further contributing to organoid vascularization (Fig. 2D and Supplementary Table 1).”

May combine two claims.

Significant epithelial cell differentiation at the late phase, coupled with morphogenesis of an epithelium, indicated that epithelial morphogenesis of the liver occurred at this stage.

“The significant epithelial cell differentiation at the late phase, coupled with the morphogenesis of an epithelium, indicated that epithelial morphogenesis of the liver occurred at this stage.”

May combine two claims.

The study delineates the dynamic progression of organoid maturation and establishes a temporal framework for selecting functional timepoints.

“By profiling the expression of 5,736 proteins across four developmental stages, the study delineates the dynamic progression of organoid maturation and establishes a temporal framework for selecting functional timepoints.”

May combine two claims.

This study adds a temporal dimension to proteomic profiling, demonstrating how maturity-dependent protein expression impacts organoid functionality.

“This study adds to this body of work by providing a temporal dimension to proteomic profiling, demonstrating how maturity-dependent protein expression impacts organoid functionality.”

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

Day 45 organoids may offer added value for investigating liver pathologies involving mesenchymal interactions, such as fibrosis.

“Day 45 organoids may offer added value for investigating liver pathologies involving mesenchymal interactions, such as fibrosis.”

May combine two claims.

The persistence of ALB expression despite declining hepatic protein levels supports the notion of functional heterogeneity, potentially reflecting a dynamic interplay between hepatocyte populations and expanding mesenchymal cells.

“Furthermore, the persistence of ALB expression despite declining hepatic protein levels supports the notion of functional heterogeneity, potentially reflecting a dynamic interplay between hepatocyte populations and expanding mesenchymal cells.”

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

Current iHLO models are not yet ideal, and significant differences exist between various differentiation protocols.

“It should be acknowledged that current iHLO models are not yet ideal model, and significant differences exist between various differentiation protocols.”

May combine two claims.

The peak functionality of iHLOs at Day 30, evidenced by maximal expression of CYP450 enzymes like CYP3A4, CYP2B6, and CYP2C9, revealed optimal conditions for xenobiotic metabolism studies.

“The peak functionality of iHLOs at Day 30, evidenced by maximal expression of CYP450 enzymes like CYP3A4, CYP2B6, and CYP2C9, revealed optimal conditions for xenobiotic metabolism studies.”

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

Temporal proteomic profiling performed in this study defines a clear developmental roadmap for liver organoids.

“the temporal proteomic profiling performed in this study defines a clear developmental roadmap for liver organoids”

Classified as probably not a substantial claim.

Temporal proteomic profiling offers actionable insights for optimizing their use in drug metabolism, toxicological testing, and disease modeling.

“offering actionable insights for optimizing their use in drug metabolism, toxicological testing, and disease modeling”

May combine two claims.

Temporal profiling revealed critical insights into the developmental trajectory of hepatocyte and cholangiocyte differentiation, in addition to identifying increased cellular heterogeneity by Day 45.

“The temporal profiling revealed critical insights into the developmental trajectory of hepatocyte and cholangiocyte differentiation, in addition to identifying increased cellular heterogeneity by Day 45.”

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

CYP3A4 was extensively validated in functional assays.

“CYP3A4 was extensively validated in functional assays”

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

The mass spectrometry proteomics data from this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD063438.

“The mass spectrometry proteomics data from this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD063438.”

May combine two claims.

PHHs remain the gold standard for in vitro liver models, but their availability, variability, and rapid dedifferentiation underscore the value of iHLOs as a scalable, reproducible alternative.

“Although PHHs remain the gold standard for in vitro liver models, their availability, variability, and rapid dedifferentiation underscore the values of iHLOs as a scalable, reproducible alternative.”

• AssumptionRests on an assumption: PHHs have limited availability, variability, and rapid dedifferentiation.

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

All data were presented as mean ± standard deviation (SD) from at least three independent experiments unless otherwise stated.

“All data were presented as mean ± standard deviation (SD) from at least three independent experiments unless otherwise stated”

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

This study supports iHLO adoption for drug discovery and preclinical testing.

“this study supports iHLO adoption for drug discovery and preclinical testing”

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

The shift toward mesenchymal phenotypes at Day 45 highlights an unexplored research avenue.

“Interestingly, the shift toward mesenchymal phenotypes at Day 45 highlights an unexplored research avenue.”

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

Labeling efficiency was determined, with a minimum 95% efficiency required for further processing.

“Labeling efficiency was determined by analyzing a small aliquot (1%) of the sample, with a minimum 95% efficiency required for further processing.”

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

The false positive discovery rates (FDR) were set at 1%.

“The false positive discovery rates (FDR) were set at 1%.”