Drug Induced Hybrid Electrospun PLA:Cell Derived Extracellular Matrix Scaffolds Support the Survival and Function of Human Primary Hepatocytes

Abstract An exponential increase in liver disease is driving a critical shortage of donor livers for patient transplant. In the UK alone, 58 people died in 2019 while waiting for a donor organ. A solution is sought in the form of tissue-engineered devices which support the survival and function of primary human hepatocytes. Previous work has shown that biofunctionalization of electrospun scaffolds influences hepatocytes. This study assesses the impact of drug-derived ECM on primary human hepatocytes (PHHs); a gold standard research resource. Hepatocytes seeded onto electrospun PLA scaffolds were subjected to drug treatment using histone deacetylase inhibitors. These cells were stripped from the scaffolds to leave behind their ECM. The resulting ECM-PLA scaffolds were seeded with PHHs and cultured for 24/72/120 hours. Scanning electron microscopy (SEM), mechanical and biochemical quantification, histology, and gene expression analyses were performed on the scaffolds. Results demonstrate PHHs are significantly influenced by the drug derived ECM:PLA scaffolds, with alterations in albumin production and gene expression demonstrated. Creating multidimensional scaffolds like these provides a method of creating tailored environments for liver bioengineering and the investigation of cell matrix interactions and is a step on the path to providing lab grown organoids for patient transplant.

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A Systematic Comparison of the Impact of Inflammatory Signaling on Absorption, Distribution, Metabolism, and Excretion Gene Expression and Activity in Primary Human Hepatocytes and HepaRG Cells

Drug Metabolism and Disposition ◽

10.1124/dmd.114.060962 ◽

2014 ◽

Vol 43 (2) ◽

pp. 273-283 ◽

Cited By ~ 43

Author(s):

Marcus Klein ◽

Maria Thomas ◽

Ute Hofmann ◽

Daniel Seehofer ◽

Georg Damm ◽

...

Keyword(s):

Gene Expression ◽

Human Hepatocytes ◽

Inflammatory Signaling ◽

Systematic Comparison ◽

Primary Human Hepatocytes ◽

Heparg Cells ◽

The Impact ◽

Expression And Activity

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Peptide-based scaffolds for the culture and maintenance of primary human hepatocytes

Scientific Reports ◽

10.1038/s41598-021-86016-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Douglas MacPherson ◽

Yaron Bram ◽

Jiwoon Park ◽

Robert E. Schwartz

Keyword(s):

Cytochrome P450 ◽

Human Hepatocytes ◽

Peptide Ligand ◽

3D Scaffold ◽

Primary Human Hepatocytes ◽

Peptide Component ◽

Functional Peptide ◽

Arginine Glycine Aspartic Acid ◽

And Function ◽

Cooperative Assembly

AbstractWe report here the use of a nanofibrous hydrogel as a 3D scaffold for the culture and maintenance of functional primary human hepatocytes. The system is based on the cooperative assembly of a fiber-forming peptide component, fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF), and the integrin-binding functional peptide ligand, Fmoc-arginine-glycine-aspartic acid (Fmoc-RGD) into a nanofibrous gel at physiological pH. This Fmoc-FF/RGD hydrogel was formulated to provide a biomimetic microenvironment with some critical features such as mechanical properties and nanofiber morphology, which were optimized to support hepatocyte culture. The material was shown to support maintenance and function of encapsulated primary human hepatocytes as indicated by actin staining, qRT-PCR, and functional cytochrome P450 assays. The designed gel was shown to outperform Matrigel in cytochrome P450 functional assays. The hydrogel may prove useful for liver development and disease models, as well as providing insights into the design of future implantable scaffolds for the regeneration of liver tissue in patients with liver disease.

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Characterisation of the NRF2 transcriptional network and its response to chemical insult in primary human hepatocytes: implications for prediction of drug-induced liver injury

Archives of Toxicology ◽

10.1007/s00204-018-2354-1 ◽

2018 ◽

Vol 93 (2) ◽

pp. 385-399 ◽

Cited By ~ 6

Author(s):

Ian M. Copple ◽

Wouter den Hollander ◽

Giulia Callegaro ◽

Fiona E. Mutter ◽

James L. Maggs ◽

...

Keyword(s):

Liver Injury ◽

Transcriptional Network ◽

Human Hepatocytes ◽

Drug Induced ◽

Drug Induced Liver Injury ◽

Primary Human Hepatocytes

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Synonymous mutation rs2515641 affects CYP2E1 mRNA and protein expression and susceptibility to drug-induced liver injury

Pharmacogenomics ◽

10.2217/pgs-2019-0151 ◽

2020 ◽

Vol 21 (7) ◽

pp. 459-470

Author(s):

Keguang Chen ◽

Ruichen Guo ◽

Chunmin Wei

Keyword(s):

Gene Expression ◽

Drug Disposition ◽

Lentiviral Vector ◽

Drug Induced ◽

Drug Induced Liver Injury ◽

Cyp2e1 Gene ◽

Recombinant Cells ◽

Mrna And Protein Expression ◽

Cyp2e1 Expression ◽

And Function

Aim: To evaluate whether the synonymous mutant rs2515641 could affect cytochrome P450 2E1 ( CYP2E1) expression and the response to acetaminophen (APAP) or triptolide (TP) treatment. Materials & methods: HepG2 cells were transfected with lentiviral vector containing either CYP2E1-1263C or CYP2E1-1263T. Some of these recombinant cells were then treated with APAP or TP. CYP2E1 gene expression was detected by PCR and western blot. Results: CYP2E1 gene expression decreased significantly both in mRNA and protein level after rs2515641 mutation, indicating that this polymorphism can affect both transcription and translation. Furthermore, rs2515641 mutation dramatically changes the response of CYP2E1 expression to APAP or TP treatment. Conclusion: Rs2515641 significantly changes CYP2E1 expression and function, which would be expected to affect drug disposition and response.

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Evaluation of transcriptomic signature as a valuable tool to study drug-induced cholestasis in primary human hepatocytes

Archives of Toxicology ◽

10.1007/s00204-017-1930-0 ◽

2017 ◽

Vol 91 (8) ◽

pp. 2879-2893 ◽

Cited By ~ 20

Author(s):

Céline Parmentier ◽

Philippe Couttet ◽

Armin Wolf ◽

Thomas Zaccharias ◽

Bruno Heyd ◽

...

Keyword(s):

Study Drug ◽

Human Hepatocytes ◽

Drug Induced ◽

Primary Human Hepatocytes ◽

Transcriptomic Signature

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Circulating miRNA 887 is differentially expressed in ARDS and modulates endothelial function

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00494.2019 ◽

2020 ◽

Vol 318 (6) ◽

pp. L1261-L1269 ◽

Cited By ~ 1

Author(s):

Andrew J. Goodwin ◽

Pengfei Li ◽

Perry V. Halushka ◽

James A. Cook ◽

Aman S. Sumal ◽

...

Keyword(s):

Gene Expression ◽

Cell Function ◽

In Silico Analysis ◽

Leukocyte Migration ◽

Differentially Expressed ◽

Expression Of Genes ◽

Cell Gene Expression ◽

And Function ◽

The Impact

Circulating microRNAs (miRNAs) can be taken up by recipient cells and have been recently associated with the acute respiratory distress syndrome (ARDS). Their role in host predisposition to the syndrome is unknown. The objective of the study was to identify circulating miRNAs associated with the development of sepsis-related ARDS and examine their impact on endothelial cell gene expression and function. We determined miRNA levels in plasma collected from subjects during the first 24 h of admission to a tertiary intensive care unit for sepsis. A miRNA that was differentially expressed between subjects who did and did not develop ARDS was identified and was transfected into human pulmonary microvascular endothelial cells (HPMECs). RNA sequencing, in silico analysis, cytokine expression, and leukocyte migration assays were used to determine the impact of this miRNA on gene expression and cell function. In two cohorts, circulating miR-887-3p levels were elevated in septic patients who developed ARDS compared with those who did not. Transfection of miR-887-3p into HPMECs altered gene expression, including the upregulation of several genes previously associated with ARDS (e.g., CXCL10, CCL5, CX3CL1, VCAM1, CASP1, IL1B, IFNB, and TLR2), and activation of cellular pathways relevant to the response to infection. Functionally, miR-887-3p increased the endothelial release of chemokines and facilitated trans-endothelial leukocyte migration. Circulating miR-887-3p is associated with ARDS in critically ill patients with sepsis. In vitro, miR-887-3p regulates the expression of genes relevant to ARDS and neutrophil tracking. This miRNA may contribute to ARDS pathogenesis and could represent a novel therapeutic target.

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Comparison of basal gene expression profiles and effects of hepatocarcinogens on gene expression in cultured primary human hepatocytes and HepG2 cells

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis ◽

10.1016/j.mrfmmm.2003.11.014 ◽

2004 ◽

Vol 549 (1-2) ◽

pp. 79-99 ◽

Cited By ~ 81

Author(s):

Angela J Harris ◽

Stacey L Dial ◽

Daniel A Casciano

Keyword(s):

Gene Expression ◽

Hepg2 Cells ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Human Hepatocytes ◽

Primary Human Hepatocytes

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Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo

Gut ◽

10.1136/gutjnl-2016-312162 ◽

2017 ◽

Vol 67 (3) ◽

pp. 542-552 ◽

Cited By ~ 58

Author(s):

Lena Allweiss ◽

Tassilo Volz ◽

Katja Giersch ◽

Janine Kah ◽

Giuseppina Raffa ◽

...

Keyword(s):

Acute Infection ◽

In Situ Hybridisation ◽

Human Hepatocytes ◽

Human Hepatocyte ◽

Therapeutic Interventions ◽

Viral Spread ◽

Primary Human Hepatocytes ◽

The Stability ◽

The Impact

ObjectiveThe stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo.MethodsPHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing.ResultsPHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production.ConclusionsWe demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

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