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Stage-specific regulation of the WNT/?-catenin pathway enhances differentiation of hESCs into hepatocytes.

ABSTRACT: 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.

SUBMITTER: Touboul T

PROVIDER: S-EPMC5010388 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Stage-specific regulation of the WNT/β-catenin pathway enhances differentiation of hESCs into hepatocytes.

Touboul Thomas T Chen Shujuan S To Cuong C CC Mora-Castilla Sergio S Sabatini Karen K Tukey Robert H RH Laurent Louise C LC

Journal of hepatology 20160226 6

<h4>Background & aims</h4>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.<h4>Methods</h4> ...[more]

PMID: 26921690

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Project description:BackgroundManagement of fracture healing with a large bone defect remains a tricky subject in orthopedic trauma. Enhancing osteogenesis of human bone marrow-derived mesenchymal stem cells (hBMSCs) is one of the useful therapeutic strategies for fracture healing. Previous studies have revealed that Apelin may play an important role in bone metabolism. However, its function in the osteogenesis of hBMSCs remains unclear. Therefore, in this study, we investigated the effects and mechanism of Apelin on osteogenic differentiation.MethodsWe investigated the osteogenesis effects of hBMSCs by both exogenous Apelin protein and overexpression Apelin in vitro. Cell proliferation assay was used to assess the effect of Apelin on the proliferation of hBMSCs. ALP staining and Alizarin Red staining were used to evaluate ALP activity and mineral deposition respectively. qPCR and Western blotting analysis were used to detect the expression of target genes and proteins. In vivo, a rat tibial osteotomy model was established; radiographic analysis and histological evaluation were used to confirm the therapeutic effects of Apelin in fracture healing. Statistical significance was determined by two-tailed Student's t test when 2 groups were compared. When more than 2 groups were compared, one-way ANOVA followed by Bonferroni's post-hoc test was used. And two-way ANOVA, followed by Bonferroni multiple comparisons post-hoc test, was performed when the treatment groups at different time points were compared.ResultsThe addition of exogenous Apelin protein or overexpression of Apelin promoted osteoblast differentiation of hBMSCs in vitro. Increased mineral deposits were observed after treatment with extracellular Apelin protein or after the upregulation of Apelin. Moreover, ?-catenin levels were upregulated by Apelin. The enhancement of osteogenic differentiation induced by Apelin was attenuated by specific Wnt/?-catenin signaling pathway inhibitors. In a rat tibial osteotomy model, local injection of exogenous Apelin protein improved bone healing, as demonstrated by imaging and histological analyses.ConclusionsTaken together, these findings indicate that Apelin regulates osteogenic differentiation of hMSCs partly via the Wnt/?-catenin signaling pathway and effectively promotes fracture healing.

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Stage-specific regulation of the WNT/?-catenin pathway enhances differentiation of hESCs into hepatocytes.

Publications

Stage-specific regulation of the WNT/β-catenin pathway enhances differentiation of hESCs into hepatocytes.

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