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ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression.

ABSTRACT: It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. However, it is not clear whether specific ECM components such as collagen or fibronectin differentially regulate such processes, especially in 3D scaffolds. In this study, a series of ECM-functionalized inverted colloidal crystal (ICC) microporous scaffolds were fabricated and their influence on Huh-7.5 cell proliferation, morphology, hepatic-specific functions, and patterns of gene expression were compared. Both collagen and fibronectin promoted albumin production and liver-specific gene expression of Huh-7.5 cells, compared with the bare ICC scaffold. Interestingly, cells in the fibronectin-functionalized scaffold exhibited different aggregation patterns to those in the collagen-functionalized scaffold, a variation that could be related to the distinct mRNA expression levels of cell adhesion-related genes. Based on these results, we can conclude that different ECM proteins, such as fibronectin and collagen, indeed play distinct roles in the phenotypic regulation of cells cultured in a 3D environment.

SUBMITTER: Wang Y

PROVIDER: S-EPMC5126637 | biostudies-other | 2016 Nov

REPOSITORIES: biostudies-other

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ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression.

Wang Yan Y Kim Myung Hee MH Shirahama Hitomi H Lee Jae Ho JH Ng Soon Seng SS Glenn Jeffrey S JS Cho Nam-Joon NJ

Scientific reports 20161129

It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. However, it is not clear whether specific ECM components such as collagen or fibronectin differentially regulate such processes, especially in 3D scaffolds. In this study, a series of ECM-functionalized inverted colloidal crystal (ICC) microporous scaffolds were fabricated and their inf ...[more]

PMID: 27897167

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Project description:Mesenchymal stem cells (MSC) and induced pluripotent stem cells (iPSC) have been reported to be able to differentiate to hepatocyte in vitro with varying degree of hepatocyte maturation. A simple method to decellularize liver scaffold has been established by the Department of Histology, Faculty of Medicine, Universitas Indonesia, in SCTE IMERI lab.15 This study aims to evaluate hepatocyte differentiation from iPSCs compared to MSCs derived in our decellularized liver scaffold. The research stages started with iPSC culture, decellularization, seeding cell culture into the scaffold, and differentiation into hepatocytes for 21 days. Hepatocyte differentiation from iPSCs and MSCs in the scaffolds was characterized using hematoxylin-eosin, Masson Trichrome, and immunohistochemistry staining to determine the fraction of the differentiation area. RNA samples were isolated on days 7 and 21. Expression of albumin, CYP450, and CK-19 genes were analyzed using the qRT-PCR method. Electron microscopy images were obtained by SEM. Immunofluorescence examination was done using HNF4-α and CEBPA markers. The results of this study in hepatocyte-differentiated iPSCs compared with hepatocyte-differentiated MSCs in decellularized liver scaffold showed lower adhesion capacity, single-cell-formation and adhered less abundant, decreased trends of albumin, and lower CYP450 expression. Several factors contribute to this result: lower initial seeding number, which causes only a few iPSCs to attach to certain parts of decellularized liver scaffold, and manual syringe injection for recellularization, which abruptly and unevenly creates pattern of single-cell-formation by hepatocyte-differentiated iPSC in the scaffold. Hepatocyte-differentiated MSCs have the advantage of higher adhesion capacity to collagen fiber decellularized liver scaffold. This leads to positive result: increase trends of albumin and higher CYP450 expression. Hepatocyte maturation is shown by diminishing CK-19, which is more prominent in hepatocyte-differentiated iPSCs in decellularized liver scaffold. Confirmation of mature hepatocyte-differentiated iPSCs in decellularized liver scaffold maturation is positive for HNF4-a and CEBPA. The conclusion of this study is hepatocyte-differentiated iPSCs in decellularized liver scaffold is mature with lower cell-ECM adhesion, spatial cell distribution, albumin, and CYP450 expression than hepatocyte-differentiated MSCs in decellularized liver scaffold.

Project description:Objectives: Decellularized extracellular matrix (dECM) is increasingly used in a wide range of regenerative medicine applications and may also offer the potential to support injured myocardium. Here, we evaluated the myocardial gene expression pattern after myocardial infarction (MI) in a standardized rodent LAD-ligation model with and without ventricular stabilization with a customized, cardiac dECM-based scaffold (cdECM). Methods: MI was induced in male Wistar rats by standard LAD-ligation and confirmed 14 days post-intervention by echocardiographic parameters (FAS<40%). Cardiac ECM from donor rats was used to generate individual cdECM-scaffolds (tissue engineered myocardial sleeve, TEMS), which were epicardially implanted after confirmed MI for ventricular stabilization. After 4 and 8 weeks heart function was assessed by echocardiography, rats were sacrificed and explanted hearts were analyzed. In addition to histological analysis, standardized anterior left ventricular wall myocardial tissue samples were assessed by quantitative real-time PCR evaluating the specific gene expression pattern for immunomodulatory (IL-10, TGFBR2, TNFα), pro-angiogenic (VEGFA, FGF2, PGF, PDGFB), pro-survival (HGF, SDF1, IGF1, AKT1), remodeling-associated (TIMP1, MMP2, MMP9) and infarction-specific (NPPA, NPPB) markers. Results: Ventricular stabilization led to integration of the TEMS-scaffold into the myocardial scar with varying degrees of cellular infiltration, as well as significantly improved echocardiographic parameters demonstrating attenuation of maladaptive cardiac remodeling. Further, TEMS implantation after MI altered the myocardial gene expression pattern. Differences in gene expression were most striking after 4 weeks with significantly reduced expression of NPPA (0.36 ± 0.26 vs 0.75 ± 0.40; p < 0.05), NPPB (0.47 ± 0.25 vs 0.91 ± 0.429; p < 0.01), TGFBR2 (0.68 ± 0.16 vs 0.90 ± 0.14; p < 0.01) and PDGFB (0.81 ± 0.13 vs 1.06 ± 0.14; p < 0.01) as well as increased expression of IL-10 (5.93 ± 5.67 vs 1.38 ± 0.60; p < 0.05), PGF (1.48 ± 0.38 vs 1.09 ± 0.25; p < 0.05) and IGF1 (1.67 ± 0.70 vs 1.03 ± 0.42; p < 0.05). However, after 8 weeks differences in the gene expression patterns of remodeling-associated, and pro-angiogenic markers could still be observed between groups. Conclusion: Ventricular stabilization via TEMS implantation after MI did not only led to biological integration of the cdECM-scaffolds into the host tissue and improved functional cardiac parameters, but also altered 4 and 8 week gene expression of infarcted myocardium, possibly contributing to reducing chronic deteriorating effects while increasing the potential for myocardial regeneration.

Dataset Information

ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression.

Publications

ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression.

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