Stage-specific regulation of the WNT/β-catenin pathway enhances differentiation of hESCs into hepatocytes
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PROVIDER: PRJNA526457 | ENA |
REPOSITORIES: ENA
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Project description:Background & Aims Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported, but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development. Methods We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes, using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally, induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes. Results Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1), we showed that inhibition of the WNT/β-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut, and then hepatic gut cells. In contrast, during the 4th stage of differentiation, we found that activation of the WNT/β-catenin pathway allowed generation of proliferative bipotent hepatoblasts, which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-β and NOTCH signaling. Conclusion Here, we show that stage-specific regulation of the WNT/β-catenin pathway results in improved differentiation of hESCs to functional hepatocytes.
2019-03-12 | GSE128102 | GEO
Project description:Hepatocytes differentiated from human embryonic stem cells (hESCs) have the potential to overcome the shortage of primary hepatocytes for clinical use and drug development. Many strategies for this process have been reported, but the functionality of the resulting cells is incomplete. We hypothesize that the functionality of hPSC-derived hepatocytes might be improved by making the differentiation method more similar to normal in vivo hepatic development.We tested combinations of growth factors and small molecules targeting candidate signaling pathways culled from the literature to identify optimal conditions for differentiation of hESCs to hepatocytes, using qRT-PCR for stage-specific markers to identify the best conditions. Immunocytochemistry was then used to validate the selected conditions. Finally, induction of expression of metabolic enzymes in terminally differentiated cells was used to assess the functionality of the hESC-derived hepatocytes.Optimal differentiation of hESCs was attained using a 5-stage protocol. After initial induction of definitive endoderm (stage 1), we showed that inhibition of the WNT/?-catenin pathway during the 2nd and 3rd stages of differentiation was required to specify first posterior foregut, and then hepatic gut cells. In contrast, during the 4th stage of differentiation, we found that activation of the WNT/?-catenin pathway allowed generation of proliferative bipotent hepatoblasts, which then were efficiently differentiated into hepatocytes in the 5th stage by dual inhibition of TGF-? and NOTCH signaling.Here, we show that stage-specific regulation of the WNT/?-catenin pathway results in improved differentiation of hESCs to functional hepatocytes.
| S-EPMC5010388 | biostudies-literature
Project description:The liver has a remarkable capacity to regenerate, which is sustained by the ability of hepatocytes to act as facultative stem cells that, while normally quiescent, re-enter the cell cycle upon injury. In rodents, growth factor signaling is indispensable, whereas Wnt/β-catenin is not required for effective tissue repair. However, molecular networks controlling human liver regeneration remain unclear.
2024-05-22 | PXD036100 | Pride
Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin-driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. Here, we combined RNA sequencing with CUT&RUN-LoV-U against β-catenin to assess the correlation between β-catenin recruitment to target loci and its effect on target gene expression. To this end, we performed a bulk RNA sequencing analysis on human embryonic stem cells (hESCs) treated with the the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days, and compared them to untreated hESCs. We then correlated the observed gene expression changes with β-catenin binding events identified from a separate experiment (see “Related Accession Number”). We observed that β-catenin binding is associated with both activation and repression of cell-specific gene expression programs, underscoring how Wnt/b-catenin drives complex cell behaviors.
2022-09-30 | E-MTAB-12075 | biostudies-arrayexpress
Project description:The Wnt/β-catenin signaling pathway is a critical regulator of development and stem cell maintenance. Mounting evidence suggests that the context-specific outcome of Wnt signaling is determined by the collaborative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. The contribution of FOX transcription factors to Wnt signaling has not been investigated in a systemic manner. Here, by combining β-catenin reporter assays with Wnt pathway-focused qPCR arrays and proximity proteomics of selected FOX family members, we determine that most FOX proteins are involved in the regulation of Wnt pathway activity and the expression of Wnt ligands and target genes. We conclude that FOX proteins are common regulators of the Wnt/β-catenin pathway that may control the outcome of Wnt signaling in a tissue-specific manner.
2023-04-19 | PXD038811 | Pride
Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin -driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. Among these, one regards how the Wnt/β-catenin cascade modulates the chromatin behavior: to date, there exists no comprehensive genome-wide annotation of changing chromatin patterns upon Wnt pathway activation. This is important, as shifts in chromatin patterns might underlie how different cells promote diverging gene expression programs in response to Wnt. To address this question, we characterized how Wnt/β-catenin signaling shapes the genome-wide chromatin accessibility landscape in two human cell types, human embryonic kidney cells 293T (HEK293T) and human embryonic stem cells (hESCs), over time. To this end, we treated HEK293T and hESCs with the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days and assessed chromatin accessibility via ATAC-sequencing 4 hours, 24 hours and 3 days after the onset of the stimulation. We found that hESCs respond to Wnt/β-catenin activation by progressively shaping their chromatin accessibility profile in a manner that is consistent with their gradual acquisition of a mesodermal identity: differentiation genes loci open over time, while pluripotency ones close. We refer to this genomic response as plastic. On the other hand, HEK293T, which are known to be highly responsive to Wnt activation, appear more resistant to a long-term Wnt/β-catenin-driven change in cell identity. In this context, the chromatin displays a temporary opening of relevant regions at 4 hours after stimulation, followed by a re-establishment of its pre-stimulation state: we define this transient response as elastic.
2022-09-30 | E-MTAB-12076 | biostudies-arrayexpress
Project description:Wnt/b-catenin signalling is an evolutionarily conserved mechanism for cell-cell communication implicated in both developmental processes and diseases such as cancer. A main part of this signalling pathway is the stabilization and nuclear translocation of b-catenin, driving the transcriptional regulation of target genes. However, while b-catenin target genes traditionally have been through to be collectively regulated upon Wnt pathway stimulation, this appears in contrast with the non-overlapping patterns of Wnt target gene expression in several contexts, including early mammalian embryogenesis. To address the question whether individual cells exhibits diverse responses to Wnt stimulation, we followed Wnt target gene activation in human embryonic stem cells (hESCs) via single cell RNA-sequencing (scRNAseq) after GSK3 inhibition at 4, 24 and 72 hours of stimulation.
2023-02-23 | E-MTAB-12598 | biostudies-arrayexpress
Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin-driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. A first question concerns cell-specificity: how this response is integrated into lineage-specific choices is still unknown. A second question concerns time: it is not known whether β-catenin associates with its targets simultaneously or in a time-dependent fashion. For instance, while TCF/LEF and other components of the Wnt transcriptional complex are constitutively associated with the chromatin, it is β-catenin arrival, upon Wnt induction, that launches target genes transcription. Therefore, discovering the dynamics of the genome-wide β-catenin binding pattern is required to unambiguously define the direct targets of Wnt signaling To address these questions, we realized a time-resolved atlas of β-catenin genome-wide occupancy in two human cell types, human embryonic kidney cells 293T (HEK293T) and human embryonic stem cells (hESCs). To this end, we treated HEK293T and hESCs with the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days, and assessed β-catenin binding via CUT&RUN-LoV-U (Zambanini et al., 2022) 90 minutes, 4 hours, 24 hours and 3 days after the onset of the stimulation. This approach allowed us to establish that β-catenin repositions to different genomic loci along stimulation time, showing that a definition of Wnt target genes must take into account the time-dimension. Moreover, β-catenin physical targets are largely cell-type specific, as only a subset of them is present across the examined contexts.
2022-09-30 | E-MTAB-12077 | biostudies-arrayexpress
Project description:A "Cartes d'Identite des Tumeurs" (CIT) project from the french Ligue Nationale Contre le Cancer (http://cit.ligue-cancer.net). Conditional Knockout mouse model for the tumor suppressor gene Ap, Apc 2lox. The disruption of the Apc gene is realized by injection of an adenovirus encoding the Cre recombinase (AdCre). Homozygous animals injected with a high dose of AdCre showed an activation of the Wnt/beta-catenin signaling in most of the hepatocytes and develop an hepatomegaly and die soon after. Analysis of gene up- and down regulation for liver-indicible APC disruption.
2006-03-13 | E-TABM-81 | biostudies-arrayexpress
Project description:Breast cancer is one of the most common types of cancer in women. One key signaling pathway known to regulate tumor growth, metabolic adaptation, and cellular stress response in breast cancer is Wnt signaling. Breast cancer patients, specifically triple negative breast cancer (TNBC), with upregulated Wnt signaling often have a poor clinical prognosis. However, the effects of Wnt/β-catenin signaling on the nucleolus and the resultant impact on cancer development and progression remain unclear. A notable reduction was observed in the number of nucleoli per nucleus in response to Wnt/β-catenin signaling inhibition in multiple TNBC cell lines. Our comparative proteomic analysis revealed several changes in the composition of the nucleolar proteome of TNBC cells upon inhibition of Wnt signaling. Overall, we demonstrate that Wnt/β-catenin signaling will affects nucleolar functionality and thus influences breast cancer progression. Understanding the role of Wnt signaling in the nucleolus and breast cancer is a critical step towards developing novel therapeutic options for the treatment of breast cancer.
2021-03-05 | PXD021181 | Pride