Loss of TBX3 enhances pancreatic progenitor generation from human pluripotent stem cells
Open accessAbstract
Tbx3 has been identified as a regulator of liver development in the mouse, but its function in human liver development remains unknown. TBX3 mutant human pluripotent stem cell (PSC) lines were generated using CRISPR/Cas9 genome editing. TBX3 loss led to impaired liver differentiation and an upregulation of pancreatic gene expression, including PDX1, during a hepatocyte differentiation protocol. Other pancreatic genes, including NEUROG3 and NKX2.2, displayed more open chromatin in the TBX3 mutant hepatoblasts. Using a pancreatic differentiation protocol, cells lacking TBX3 generated more pancreatic progenitors and had an enhanced pancreatic gene expression signature at the expense of hepatic gene expression. These data highlight a potential role of TBX3 in regulating hepatic and pancreatic domains during foregut patterning, with implications for enhancing the generation of pancreatic progenitors from PSCs.
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Written from the paper’s full text.
TL;DR
TBX3 loss in human pluripotent stem cells impairs hepatocyte differentiation while de-repressing pancreatic genes, and enhances generation of PDX1+/NKX6.1+ pancreatic progenitors. The authors propose TBX3 helps maintain hepatic lineage fidelity during foregut patterning.
Key findings
- CRISPR/Cas9-generated TBX3 mutant iPSC and ESC lines with a 6.9-kb deletion showed impaired hepatocyte differentiation: reduced SERPINA1 and ALBUMIN expression, fewer HNF4α+/AFP+ and AAT+/ALBUMIN+ cells, and significantly reduced CYP3A4 activity in iPSC−/− hepatocytes.
- During hepatocyte differentiation, TBX3 loss led to PDX1 expression, increased ISL1 and MNX1, and greater chromatin accessibility at the PDX1 promoter and at MNX1/ISL1 loci; MG132 treatment increased PDX1 protein in iPSC−/− cells.
- In pancreatic differentiation, iPSC−/− cells generated a higher percentage of PDX1+/NKX6.1+ PP2 cells, with higher NKX6.1 and ISL1 expression and lower hepatic gene expression than iPSC+/+ cells.
- RNA-seq/ATAC-seq analyses showed pancreatic gene signatures enriched in iPSC−/− PP1 cells and hepatic cord genes enriched in iPSC+/+ GT cells; 1,398 genes were similarly dysregulated across GT, PP1, and hepatoblast stages, and 1,787 genes had more accessible chromatin in iPSC−/− hepatoblasts versus 396 with less accessibility.
Methods
- TBX3 mutant human PSC lines were generated by CRISPR/Cas9 in CHOPi004-A iPSC and Mel1 ESC backgrounds, using two gRNAs to create a 6.9-kb deletion including the transcriptional start site and DNA binding domain.
- Cells were differentiated to hepatocytes using an Ogawa et al. protocol and to pancreatic progenitors/PP2 using a Rezania et al. protocol; analyses included qRT-PCR, western blot, flow cytometry, CYP3A4 activity, RNA-seq, and ATAC-seq.
- RNA-seq was performed on day 12 hepatoblasts and days 6 and 8 of pancreatic differentiation with three biological replicates per genotype/time point; ATAC-seq was performed on day 12 hepatoblasts with two biological replicates per genotype; GSEA used a published human embryonic dorsal pancreatic bud versus hepatic cord gene set.
Limitations
- The paper does not include a dedicated limitations section. It states that generating functionally mature terminal cell types in vitro remains difficult, and that further studies are needed on how TBX3 loss affects later pancreatic differentiation.
- Evident from the text: the study focuses on in vitro PSC differentiation and progenitor stages, without in vivo functional validation or transplantation; some pancreatic differentiation comparisons are trends or not statistically significant, such as PDX1 mRNA at PP1/PP2.
- Evident from the text: ATAC-seq used two biological replicates per genotype, and the authors note that EMT dysregulation did not appear to have clear implications for liver versus pancreas fate at the stages examined.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
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Claims the paper makes
1 of 1 cited sources checked · 1 supported · 0 flagged
- Premisecitation
Tbx3 is a regulator of liver development in the mouse.
“Tbx3 has been identified as a regulator of liver development in the mouse”
- Also stated in: § Introduction · hedged
“Tbx3 has also been implicated in liver development.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Lüdtke et al., 2009 source
- Also stated in: § Introduction · hedged
- Findingown data
Cells lacking TBX3 have an enhanced pancreatic gene expression signature at the expense of hepatic gene expression.
“and had an enhanced pancreatic gene expression signature at the expense of hepatic gene expression.”
- Also stated in: § iPSC−/− cells are enriched for a pancreatic gene signature · plain
“At both the GT and the PP1 stages, pancreatic genes were upregulated while hepatic genes were downregulated in iPSC−/− versus iPSC+/+”
Quote in the paperSubstantial claim - Also stated in: § iPSC−/− cells are enriched for a pancreatic gene signature · plain
- Findingown data
Loss of TBX3 impairs hepatocyte differentiation from human pluripotent stem cells.
“TBX3 loss led to impaired liver differentiation and an upregulation of pancreatic gene expression, including PDX1, during a hepatocyte differentiation protocol.”
- Also stated in: § Loss of TBX3 impairs hepatocyte differentiation in human PSCs · hedged
“These results indicate that the loss of TBX3 impairs the ability of PSCs to differentiate to hepatocytes”
Quote in the paperSubstantial claim - Also stated in: § Loss of TBX3 impairs hepatocyte differentiation in human PSCs · hedged
- Findingown data
Expression of hepatocyte markers SERPINA1 and ALBUMIN is significantly reduced in iPSC−/− cells compared with iPSC+/+ cells.
“expression of hepatocyte markers SERPINA1, which encodes the α1-antitrypsin (AAT) protein, and ALBUMIN were significantly reduced in iPSC−/− cells compared with iPSC+/+ cells (Figure 1E).”
Quote in the paperSubstantial claim - Findingown data
Promoter regions of MNX1 and ISL1 are more accessible in iPSC−/− hepatoblasts than iPSC+/+ hepatoblasts.
“the promoter regions of both MNX1 and ISL1 are more accessible in iPSC−/− hepatoblasts than in iPSC+/+ hepatoblasts (Figure 2E).”
Quote in the paperSubstantial claim
- Findingown data
There is no impact on SOX2 expression during hepatocyte differentiation in iPSC−/−.
“There was no impact on SOX2 expression”
Quote in the paperNot a substantial claim - Findingown data
Expression of ISL1 and MNX1 is increased in iPSC−/− line during hepatocyte differentiation.
“expression of ISL1 (Ahlgren et al., 1997) and Motor Neuron and Pancreas Homeobox 1 (MNX1) (Li et al., 1999), genes involved in early pancreas development, was also increased in the iPSC−/− line (Figure 2C)”
Quote in the paperSubstantial claim - Findingown data
iPSC−/− hepatocytes have significantly reduced CYP3A4 activity compared with iPSC+/+ hepatocytes.
“iPSC−/− hepatocytes had significantly reduced CYP3A4 activity compared with iPSC+/+ hepatocytes”
Quote in the paperSubstantial claim - Findingown data
There is no difference in HNF4α mRNA levels between iPSC−/− and iPSC+/+ during hepatocyte differentiation.
“There was no difference in levels of hepatoblast marker HNF4α at the mRNA level (Figure 1E)”
Quote in the paperSubstantial claim - Findingown data
Loss of TBX3 upregulates pancreatic gene expression, including PDX1, during hepatocyte differentiation.
“TBX3 loss led to impaired liver differentiation and an upregulation of pancreatic gene expression, including PDX1, during a hepatocyte differentiation protocol.”
- Also stated in: § Pancreas-specific genes are upregulated in TBX3 mutant hepatoblasts · plain
“However, PDX1 was expressed in the iPSC−/− line (Figure 2A)”
Quote in the paperSubstantial claim - Also stated in: § Pancreas-specific genes are upregulated in TBX3 mutant hepatoblasts · plain
- Findingown data
AFP expression is delayed in iPSC−/− line.
“although expression at the protein level was decreased, along with delayed expression of α-fetoprotein (AFP) in the iPSC−/− line.”
Quote in the paperNot a substantial claim - Findingown data
Cells lacking TBX3 generate more pancreatic progenitors using a pancreatic differentiation protocol.
“Using a pancreatic differentiation protocol, cells lacking TBX3 generated more pancreatic progenitors”
- Also stated in: § Loss of TBX3 enhances pancreatic progenitor generation from PSCs · plain
“The iPSC−/− line generated a higher percentage of the PDX1+/NKX6.1+ pancreatic progenitor 2 (PP2) population compared with iPSC+/+ cells”
Quote in the paperSubstantial claim - Also stated in: § Loss of TBX3 enhances pancreatic progenitor generation from PSCs · plain
- Findingown data
HNF4α protein expression is decreased in iPSC−/− hepatoblasts.
“although expression at the protein level was decreased, along with delayed expression of α-fetoprotein (AFP) in the iPSC−/− line.”
Quote in the paperNot a substantial claim - Findingown data
PDX1 is expressed in iPSC−/− line during hepatocyte differentiation.
“However, PDX1 was expressed in the iPSC−/− line (Figure 2A)”
Quote in the paperSubstantial claim - Findingown data
Other pancreatic genes, including NEUROG3 and NKX2.2, display more open chromatin in TBX3 mutant hepatoblasts.
“Other pancreatic genes, including NEUROG3 and NKX2.2, displayed more open chromatin in the TBX3 mutant hepatoblasts.”
- Also stated in: § iPSC−/− cells are enriched for a pancreatic gene signature · plain
“some genes involved in later endocrine cell development and maturation, such as NEUROG3 and NKX2.2, are more accessible in iPSC−/− than in iPSC+/+ hepatoblasts.”
Quote in the paperSubstantial claim - Also stated in: § iPSC−/− cells are enriched for a pancreatic gene signature · plain
- Findingown data
The PDX1 promoter region is more accessible in iPSC−/− than iPSC+/+ hepatoblasts.
“the promoter region of PDX1 is more accessible in iPSC−/− than in iPSC+/+ hepatoblasts (Figure 2D).”
Quote in the paperSubstantial claim - Findingown data
Levels of hepatic genes are lower in iPSC−/− line compared with iPSC+/+ line during pancreatic differentiation.
“Levels of hepatic genes were lower in the iPSC−/− line compared with the iPSC+/+ line (Figure 3E)”
Quote in the paperSubstantial claim - Findingown data
Several pathways, including EMT, are dysregulated upon loss of TBX3, regardless of cell type.
“Several pathways, including epithelial-mesenchymal transition (EMT), were dysregulated upon loss of TBX3, regardless of cell type (Table S4D)”
Quote in the paperNot a substantial claim - Findingown data
Covariate analysis showed 1,398 genes were similarly up- or downregulated in all three developmental stages (GT, PP1, hepatoblasts).
“The covariate analysis showed 1,398 genes were similarly up- or downregulated in all three developmental stages (Figure S4E and Table S4C).”
Quote in the paperSubstantial claim - Findingown data
NKX6.1 expression is higher in iPSC−/− PP2 cells compared with iPSC+/+ PP2 cells.
“However, NKX6.1 expression was higher in iPSC−/− PP2 cells compared with iPSC+/+ PP2 cells.”
Quote in the paperSubstantial claim - Findingown data
Pathway analysis of ATAC-seq dataset showed genes involved in same pathways such as EMT identified in RNA-seq covariate analysis are differentially accessible, demonstrating good correlation between accessibility and gene expression.
“Pathway analysis of the ATAC-seq dataset showed that genes involved the same pathways such as EMT identified in the RNA-seq covariate analysis are differentially accessible, demonstrating good correlation between accessibility and gene expression (Table S4E).”
Quote in the paperNot a substantial claim - Findingown data
The top hit in pathway analysis was upregulation of Hallmark Pancreas Beta Cells.
“The top hit in the pathway analysis was upregulation of Hallmark Pancreas Beta Cells”
Quote in the paperSubstantial claim - Findingown data
Pathways relating to pancreatic development and function are upregulated in iPSC−/− PP1.
“and pathways relating to pancreatic development and function were upregulated in iPSC−/− PP1 (Table S4B).”
Quote in the paperSubstantial claim - Findingown data
NEUROG3 and NKX2.2 are more accessible in iPSC−/− than iPSC+/+ hepatoblasts.
“some genes involved in later endocrine cell development and maturation, such as NEUROG3 and NKX2.2, are more accessible in iPSC−/− than in iPSC+/+ hepatoblasts.”
Quote in the paperSubstantial claim - Findingown data
Genes involved in metabolic pathways are downregulated in iPSC−/− GT cells compared with iPSC+/+ GT cells.
“Pathway analysis showed that genes involved in metabolic pathways were downregulated in iPSC−/− GT cells compared with iPSC+/+ GT cells (Table S4A)”
Quote in the paperNot a substantial claim - Findingown data
Loss of TBX3 increases the number of pancreatic progenitors derived from PSCs.
“We also demonstrate that loss of TBX3 increases the number of pancreatic progenitors derived from PSCs.”
Quote in the paperSubstantial claim - Findingown data
Hepatic cord-specific genes are enriched in iPSC+/+ versus iPSC−/− GT cells.
“Hepatic cord–specific genes were enriched in iPSC+/+ versus iPSC−/− GT cells”
Quote in the paperSubstantial claim - Findingown data
iPSC−/− line generates a higher percentage of PDX1+/NKX6.1+ PP2 population compared with iPSC+/+ cells.
“The iPSC−/− line generated a higher percentage of the PDX1+/NKX6.1+ pancreatic progenitor 2 (PP2) population compared with iPSC+/+ cells (Figures 3B and 3C).”
Quote in the paperSubstantial claim - Findingown data
Upregulated genes from RNA-seq covariate analysis largely overlap with genes identified by ATAC-seq as more open.
“Upregulated genes from the RNA-seq covariate analysis largely overlap with genes identified by ATAC-seq as more open (Figures S4H and S4I).”
Quote in the paperNot a substantial claim - Findingown data
In ESC−/− line, MNX1 expression is significantly increased.
“In the ESC−/− line, MNX1 expression was significantly increased (Figure S3F).”
Quote in the paperNot a substantial claim - Findingown data
Pancreatic bud-specific genes are significantly enriched in iPSC−/− PP1 population.
“and pancreatic bud–specific genes were significantly enriched in the iPSC−/− PP1 population (Figures S4B and 4D).”
Quote in the paperSubstantial claim - Findingown data
CDH2 expression is lower in iPSC−/− cells than iPSC+/+ cells at all three stages.
“CDH2 expression was lower in iPSC−/− cells than iPSC+/+ cells at all three stages (Figures 4E and 4F).”
Quote in the paperNot a substantial claim - Interpretationown data
Loss of TBX3 has implications for enhancing generation of pancreatic progenitors from PSCs.
“with implications for enhancing the generation of pancreatic progenitors from PSCs.”
- Also stated in: § iPSC−/− cells are enriched for a pancreatic gene signature · hedged
“provide a potential route to improve the generation of in vitro–derived pancreatic progenitors”
Quote in the paperSubstantial claim - Also stated in: § iPSC−/− cells are enriched for a pancreatic gene signature · hedged
- Interpretationown data
Loss of TBX3 results in loss of repression of pancreatic genes.
“suggesting a loss of repression of pancreatic genes.”
Quote in the paperSubstantial claim - Study design and conductown data
TBX3 mutant human PSC lines were generated using CRISPR/Cas9 genome editing.
“TBX3 mutant human pluripotent stem cell (PSC) lines were generated using CRISPR/Cas9 genome editing.”
- Also stated in: § Loss of TBX3 impairs hepatocyte differentiation in human PSCs · plain
“a TBX3 mutant line was generated in the CHOPi004-A iPSC background (Mukherjee et al., 2020) using CRISPR/Cas9 genome editing technology.”
Quote in the paperSubstantial claim - Also stated in: § Loss of TBX3 impairs hepatocyte differentiation in human PSCs · plain
- Interpretationown data
TBX3 may inhibit PDX1 expression via regulation of the PDX1 promoter, directly or indirectly, to prevent transcription in wild-type hepatoblasts.
“This suggests that TBX3 may inhibit PDX1 expression via regulation of the PDX1 promoter, directly or indirectly, to prevent transcription in wild-type hepatoblasts.”
Quote in the paperSubstantial claim - Interpretationown data
TBX3 is needed for hepatocyte functionality.
“indicating that the TBX3 is also needed for hepatocyte functionality (Figure 1J).”
Quote in the paperNot a substantial claim - Interpretationown data
TBX3 plays a role in regulating hepatic and pancreatic domains during foregut patterning.
“These data highlight a potential role of TBX3 in regulating hepatic and pancreatic domains during foregut patterning”
- Also stated in: § Pancreas-specific genes are upregulated in TBX3 mutant hepatoblasts · hedged
“suggesting that TBX3 may be important to maintain lineage fidelity of the hepatic domain during foregut patterning.”
Quote in the paperSubstantial claim - Also stated in: § Pancreas-specific genes are upregulated in TBX3 mutant hepatoblasts · hedged
- Interpretationown data
Loss of TBX3 allows for PDX1 protein expression during hepatocyte differentiation.
“This indicates that the loss of TBX3 allows for PDX1 protein expression during the hepatocyte differentiation.”
Quote in the paperSubstantial claim - Interpretationown data
Loss of TBX3 results in inappropriate expression of pancreatic genes during hepatocyte differentiation.
“The loss of TBX3 results in inappropriate expression of pancreatic genes during hepatocyte differentiation”
Quote in the paperSubstantial claim - Study design and conductown data
TBX3 is not expressed in the iPSC−/− line during differentiation at the mRNA or protein level.
“TBX3 was not expressed in the iPSC−/− line during differentiation at the mRNA or protein level (Figures 1C and 1D).”
Quote in the paperSubstantial claim - Interpretationown data
These results provide a potential route to improve generation of in vitro-derived pancreatic progenitors.
“provide a potential route to improve the generation of in vitro–derived pancreatic progenitors”
Quote in the paperSubstantial claim - Interpretationown data
Loss of TBX3 generates a purer PP2 population.
“suggesting the loss of TBX3 generates a purer PP2 population.”
Quote in the paperSubstantial claim - Interpretationown data
TBX3 has a common role in pancreas and liver development.
“suggesting a common role of TBX3 in pancreas and liver development.”
Quote in the paperSubstantial claim - Interpretationreasoning
TBX3 may be important to maintain lineage fidelity of the hepatic domain during foregut patterning.
“suggesting that TBX3 may be important to maintain lineage fidelity of the hepatic domain during foregut patterning.”
Quote in the paperSubstantial claim - Interpretationown data
De-repression of the pancreatic program is confirmed.
“confirming de-repression of the pancreatic program.”
Quote in the paperSubstantial claim - Interpretationown data
TBX3 acts as a transcriptional repressor.
“highlighting the role of TBX3 as a transcriptional repressor.”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 2
- May combine two claims. 20
- The quote only partly states this claim. 1
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataThere is a minor upregulation of CDX2 in iPSC−/− line.
“and a minor upregulation of CDX2 in the iPSC−/− line.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataIn presence of MG132, iPSC−/− cells have higher PDX1 protein levels than without MG132, while iPSC+/+ line displays no PDX1 protein expression with or without MG132.
“In the presence of MG132, iPSC−/− cells had higher PDX1 protein levels than without MG132, while the iPSC+/+ line displayed no PDX1 protein expression with or without MG132 (Figure 2B).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataTBX3 expression is induced at the definitive endoderm stage and peaks at the hepatoblast stage (day 12) in iPSC+/+ cells.
“In iPSC+/+ cells, TBX3 expression was induced at the definitive endoderm stage of differentiation and peaked at the hepatoblast stage (day 12).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataSimilar defects are seen in ESC−/− line, slightly more severe, including greater downregulation of HNF4α.
“Similar defects were seen in the ESC−/− line (Figure S2) which were slightly more severe, including a greater downregulation of HNF4α”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThere are fewer HNF4α+/AFP+ cells and AAT+/ALBUMIN+ cells in iPSC−/− line compared with iPSC+/+ line.
“At the protein level, there were fewer hepatocyte nuclear factor 4 (HNF4)α+/AFP+ cells (Figures 1F and 1G) and AAT+/ALBUMIN+ cells (Figures 1H and 1I) in the iPSC−/− line compared with the iPSC+/+ line.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataE-cadherin protein levels are higher in iPSC−/− cells than iPSC+/+ cells, while effect on N-cadherin protein is subtle.
“E-cadherin protein levels were higher in iPSC−/− cells than in iPSC+/+ cells, while the effect on N-cadherin protein was subtle (Figures S4F and S4G).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataPROX1 and CDH2 are downregulated in iPSC−/− hepatoblasts.
“PROX1, which is required for hepatoblast migration and is downregulated in Tbx3−/− mice (Lüdtke et al., 2009), and CDH2, encoding mesenchymal marker N-cadherin, were downregulated in iPSC−/− hepatoblasts”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataPDX1 mRNA levels trend higher in iPSC−/− at PP1 but do not reach statistical significance, and there is no difference at PP2.
“PDX1 mRNA levels trend higher in the iPSC−/− line at the pancreatic progenitor 1 stage (PP1, day 8) of the pancreas differentiation but do not reach statistical significance, and there was no difference at the PP2 stage (day 11).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown data1,787 genes had more accessible chromatin in iPSC−/− hepatoblasts, while 396 genes had less accessible chromatin.
“1,787 genes had more accessible chromatin in iPSC−/− hepatoblasts, while 396 genes had less accessible chromatin (Figure 4G)”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataExpression of ISL1 is markedly increased, while MNX1 trends higher in iPSC−/− cells compared with iPSC+/+ cells during pancreatic differentiation.
“expression of ISL1 was markedly increased, while MNX1 trended higher in iPSC−/− cells compared with iPSC+/+ cells (Figure 3D).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataAt both GT and PP1 stages, pancreatic genes are upregulated while hepatic genes are downregulated in iPSC−/− versus iPSC+/+.
“At both the GT and the PP1 stages, pancreatic genes were upregulated while hepatic genes were downregulated in iPSC−/− versus iPSC+/+ (Figures S4A and 4C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataCDH1 expression is higher in iPSC−/− cells than iPSC+/+ cells at GT and hepatoblast stages, with no significant difference at PP1.
“At the GT and hepatoblast stages, CDH1, encoding epithelial marker E-cadherin, expression was higher in iPSC−/− cells than iPSC+/+ cells, while there was no significant difference at the PP1 stage.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataNEUROG3 and NKX2.2 are not expressed in iPSC−/− hepatoblasts.
“Although they are more accessible, NEUROG3 and NKX2.2 are not expressed in iPSC−/− hepatoblasts (Figures 4H and 4I).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataPDX1, ISL1, and MNX1 are upregulated in iPSC−/− hepatoblasts.
“Consistent with our prior gene expression findings, PDX1, ISL1, and MNX1 were upregulated in iPSC−/− hepatoblasts (Figures S4C).”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningTBX3 is important in both mouse and human liver development.
“suggesting that TBX3 is important in both mouse and human liver development.”
Quote in the paperNot a substantial claimMay combine two claims.
Study design and conductown dataThe deletion in both alleles was verified by sequencing and the edited line has a normal karyotype.
“The deletion in both alleles was verified by sequencing and confirmed to have a normal karyotype (Figures S1A and S1B).”
Quote in the paperSubstantial claimMay combine two claims.
Study design and conductown dataThe TBX3 mutant lines carry a 6.9-kb deletion including the transcriptional start site and entire DNA binding domain, generating a nonfunctional protein.
“Two guide RNAs were designed to generate a 6.9-kb deletion that includes the transcriptional start site and the entire DNA binding domain, generating a nonfunctional protein (Figure 1A).”
Quote in the paperSubstantial claimThe quote only partly states this claim.
Interpretationown dataDownregulation of PROX1 and CDH2 suggests a migratory defect in iPSC−/− hepatoblasts.
“suggesting a migratory defect.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataLoss of TBX3 helped drive cells toward a pancreatic fate and away from a hepatic fate.
“These data further confirm that the loss of TBX3 helped drive cells toward a pancreatic fate and away from a hepatic fate.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataTBX3 is critical for proper hepatocyte development and maturation in humans, similar to the role of Tbx3 in mouse liver development.
“We established that TBX3 is critical for proper hepatocyte development and maturation in humans, similar to the role of Tbx3 in mouse liver development.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Interpretationown dataEMT may be dysregulated in liver and pancreas cells with loss of TBX3.
“Taken together, these data suggest that EMT may be dysregulated in liver and pancreas cells with the loss of TBX3.”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataTBX3 not only represses genes involved in early pancreatic development such as PDX1, but also is involved in keeping genes expressed later in pancreatic endocrine cell development in a less accessible state.
“These data suggest that TBX3 not only represses genes involved in early pancreatic development such as PDX1, but also is involved in keeping genes expressed later in pancreatic endocrine cell development in a less accessible state.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataLack of TBX3 improves differentiation efficiency and purity, as iPSC−/− generated more PP2 cells with increased expression of pancreas genes and decreased expression of hepatic genes.
“These data suggest that a lack of TBX3 improves differentiation efficiency and purity, as the iPSC−/− line generated more PP2 cells with increased expression of pancreas genes and decreased expression of hepatic genes, compared with the iPSC+/+ line.”
Quote in the paperSubstantial claim
The paper relies on this without support.
The function of TBX3 in human liver development is unknown.
“but its function in human liver development remains unknown.”
“The role of TBX3 in human liver and pancreas development remains unclear.”
The paper relies on this without support.
The role of TBX3 in human pancreas development is unclear.
“The role of TBX3 in human liver and pancreas development remains unclear.”
The paper relies on this without support.
The function of Tbx3 in the mouse pancreas is unknown.
“Tbx3 has been detected in the mouse pancreas, both during development and in the adult, but its function remains unknown (Begum and Papaioannou, 2011).”
The paper relies on this without support.
It is difficult to generate functionally mature terminal cell types in vitro.
“However, it is still difficult to generate functionally mature terminal cell types in vitro.”
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?
TBX3 is a putative inhibitor of pancreatic lineage entry in differentiating human pluripotent stem cells.
“TBX3 is also a putative inhibitor of pancreatic lineage entry in differentiating human pluripotent stem cells [18].”
From TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration (2023) · cites this paper as [18]
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Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells
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Rezania, A., Bruin, J. E., Xu, J., Narayan, K., Fox, J., O'Neil, J., & Kieffer, T. (2013) · Stem Cells · doi:10.1002/stem.1489 · Cited by 269 · see citing works
Mechanosignalling via integrins directs fate decisions of pancreatic progenitors
Mamidi, A., Prawiro, C., Seymour, P. A., de Lichtenberg, K. H., Jackson, A., Serup, P., & Semb, H. (2018) · Nature · doi:10.1038/s41586-018-0762-2 · Cited by 226 · see citing works
Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development
Gibson‐Brown, J. J., Agulnik, S. I., Silver, L. M., Niswander, L. A., & Papaioannou, V. E. (1998) · Development · doi:10.1242/dev.125.13.2499 · Cited by 177 · see citing works
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Tbx3 Represses E-Cadherin Expression and Enhances Melanoma Invasiveness
Rodriguez, M., Aladowicz, E., Lanfrancone, L., & Goding, C. R. (2008) · Cancer Research · doi:10.1158/0008-5472.can-08-0301 · Cited by 157 · see citing works
Gouzi, M., Kim, Y. H., Katsumoto, K., Johansson, K., & Grapin‐Botton, A. (2011) · Developmental Dynamics · doi:10.1002/dvdy.22544 · Cited by 148 · see citing works
Tbx2 and Tbx3 induce atrioventricular myocardial development and endocardial cushion formation
Singh, R., Hoogaars, W. M. H., Barnett, P., Grieskamp, T., Rana, M. S., Buermans, H. P. J., Farin, H. F., Petry, M., Heallen, T. R., Martin, J. F., et al. (2011) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-011-0884-2 · Cited by 145 · see citing works
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Carlson, H. (2001) · Human Molecular Genetics · doi:10.1093/hmg/10.21.2403 · Cited by 108 · see citing works
Pcif1 modulates Pdx1 protein stability and pancreatic β cell function and survival in mice
Claiborn, K. C., Sachdeva, M. M., Cannon, C. E., Groff, D., Singer, J. D., & Stoffers, D. A. (2010) · Journal of Clinical Investigation · doi:10.1172/jci40440 · Cited by 87 · see citing works
INSGFP/w human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells
Micallef, S., Li, X.-L., Schiesser, J., Hirst, C., Yu, Q., Lim, S. M., Nostro, M., Elliott, D., Sarangi, F., Harrison, L. C., et al. (2011) · Diabetologia · doi:10.1007/s00125-011-2379-y · Cited by 86 · see citing works
TBX3 Directs Cell-Fate Decision toward Mesendoderm
Weidgang, C. E., Russell, R., Tata, P. R., Kühl, S. J., Illing, A., Müller, M., Lin, Q., Brunner, C., Boeckers, T. M., Bauer, K., et al. (2013) · Stem Cell Reports · doi:10.1016/j.stemcr.2013.08.002 · Cited by 78 · see citing works
Tiyaboonchai, A., Cardenas-Diaz, F. L., Ying, L., Maguire, J., Sim, X., Jobaliya, C. D., Gagne, A. L., Kishore, S., Stanescu, D. E., Hughes, N., et al. (2017) · Stem Cell Reports · doi:10.1016/j.stemcr.2016.12.026 · Cited by 64 · see citing works
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Teo, A. K. K., Tsuneyoshi, N., Hoon, S. S., Tan, E. K., Stanton, L. W., Wright, C. V. E., & Dunn, N. R. (2015) · Stem Cell Reports · doi:10.1016/j.stemcr.2015.02.015 · Cited by 52 · see citing works
Cdc42/N-WASP signaling links actin dynamics to pancreatic β cell delamination and differentiation
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Krstic, M., Kolendowski, B., Cecchini, M. J., Postenka, C. O., Hassan, H. M., Andrews, J., MacMillan, C. D., Williams, K. C., Leong, H. S., Brackstone, M., et al. (2019) · The Journal of Pathology · doi:10.1002/path.5245 · Cited by 34 · see citing works
Highly Efficient CRISPR‐Cas9‐Mediated Genome Editing in Human Pluripotent Stem Cells
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Dynamic expression of Tbx2 and Tbx3 in developing mouse pancreas
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Generation of human control iPSC line CHOPi004-A from juvenile foreskin fibroblast cells
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Fetal liver development and implications for liver disease pathogenesis
Lotto, J., Stephan, T. L., & Hoodless, P. A. (2023) · Nature Reviews Gastroenterology & Hepatology · doi:10.1038/s41575-023-00775-2 · Cited by 47 · see citing works
TBX3 is dynamically expressed in pancreatic organogenesis and fine-tunes regeneration
Melzer, M. K., Schirge, S., Gout, J., Arnold, F., Srinivasan, D., Burtscher, I., Allgöwer, C., Mulaw, M., Zengerling, F., Günes, C., et al. (2023) · BMC Biology · doi:10.1186/s12915-023-01553-x · Cited by 5 · see citing works
“TBX3 is also a putative inhibitor of pancreatic lineage entry in differentiating human pluripotent stem cells [18].”
BackgroundTBX3 advances the developmental chromatin landscape toward the hepatic fate
Stephan, T. L., Cullum, R., Drissler, S., Thomas, A., Fuglerud, B. M., Clement-Ranney, M., Lotto, J., Krentz, N. A. J., Duncan, S. A., & Hoodless, P. A. (2025) · Developmental Cell · doi:10.1016/j.devcel.2025.05.014 · Cited by 1 · see citing works
Liang, Y., Peng, Y., Song, Z., Bao, X., Zhang, W., Lu, Q., Lian, H., Ye, Y., Wang, W., Chen, Y., et al. (2026) · Cellular and Molecular Gastroenterology and Hepatology · doi:10.1016/j.jcmgh.2026.101909
- Authors
- Mukherjee, S., French, D. L., & Gadue, P.
- Affiliations
- Children's Hospital of Philadelphia (US)
- Corresponding author
- Gadue, Paul
- Venue
- Stem Cell Reports
- Publisher
- Elsevier BV
- Published
- 2021-11
- Volume
- 16
- Issue
- 11
- Pages
- 2617-2627
- ISSN
- 2213-6711 (ISSN-L)
- PMC
- PMC8580886
- OpenAlex
- W3205005506
- Semantic Scholar
- 15d986e4f312a57322666ea6e796084ca5f1228f
- Cited by
- 17
- References
- 36
- Citations by year
- 2026: 3 · 2025: 3 · 2024: 4 · 2023: 7
- FWCI
- 0.94
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Stem Cell Reports (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Scholarly Commons (University of Pennsylvania) (submittedVersion)
- PubMed Central (submittedVersion, cc-by-nc-nd)
- ScholarlyCommons (University of Pennsylvania) (submittedVersion)
- Funders
- National Institutes of Health (R01 DK118155, R01 DK123162)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Developmental Biology and Gene Regulation
- Keywords
- TBX3, pancreatic progenitors, human pluripotent stem cells, liver development, hepatocyte differentiation, PDX1, Neurog3, Nkx2.2, chromatin accessibility, CRISPR/Cas9 genome editing, pancreatic differentiation
- MeSH terms
- RNA-Seq, Animals, Cell Differentiation, Cell Line, Humans, Mutation, Pancreas, Species Specificity, Blotting, Western, Reverse Transcriptase Polymerase Chain Reaction, T-Box Domain Proteins, Gene Expression Profiling, Hepatocytes, Pluripotent Stem Cells, Mice, Induced Pluripotent Stem Cells
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 2026-10-22.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-22.
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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