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Article ; Online: Increased Prevalence of Alpha-1-Antitrypsin Deficiency in Patients with Biliary Tract Cancer and Its Associated Clinicopathological Features

Martin Cornillet / Helen Zemack / Hannes Jansson / Ernesto Sparrelid / Ewa Ellis / Niklas K. Björkström

Cells, Vol 12, Iss 1663, p

2023 Volume 1663

Abstract: Alpha-1 antitrypsin deficiency (A1ATD) is underdiagnosed and associated with liver diseases. Here, we genotyped 130 patients with biliary tract cancer (BTC) scheduled for liver resection and found A1ATD in 10.8% of the patients. A1ATD was found in all ... ...

Abstract	Alpha-1 antitrypsin deficiency (A1ATD) is underdiagnosed and associated with liver diseases. Here, we genotyped 130 patients with biliary tract cancer (BTC) scheduled for liver resection and found A1ATD in 10.8% of the patients. A1ATD was found in all BTC subtypes, and patients had similar clinical features as non-A1ATD BTC, not permitting their identification using clinical routine liver tests. In intrahepatic cholangiocarcinoma (iCCA), the abundance of A1AT protein was increased in the tumor and appeared to be influenced by the genomic alterations. On the one hand, BTC with A1ATD had lower perineural invasion at histopathology and displayed a longer survival, suggesting that a deficiency in this protein is associated with a less aggressive phenotype. On the other hand, iCCA with high A1AT expression had more advanced tumor staging and enriched pathways for complement system and extracellular matrix interactions, indicating that A1AT protein might contribute to a more aggressive phenotype. With increased awareness, screening, and basic studies, A1ATD could represent one more layer of stratification for future targeted therapies in BTC.
Keywords	alpha-1 antitrypsin deficiency ; biliary tract cancer ; survival ; Biology (General) ; QH301-705.5
Subject code	610
Language	English
Publishing date	2023-06-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Gene Editing Correction of a Urea Cycle Defect in Organoid Stem Cell Derived Hepatocyte-like Cells

Mihaela Zabulica / Tomas Jakobsson / Francesco Ravaioli / Massoud Vosough / Roberto Gramignoli / Ewa Ellis / Olav Rooyackers / Stephen C. Strom

International Journal of Molecular Sciences, Vol 22, Iss 3, p

2021 Volume 1217

Abstract: Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver ... ...

Abstract	Urea cycle disorders are enzymopathies resulting from inherited deficiencies in any genes of the cycle. In severe cases, currently available therapies are marginally effective, with liver transplantation being the only definitive treatment. Donor liver availability can limit even this therapy. Identification of novel therapeutics for genetic-based liver diseases requires models that provide measurable hepatic functions and phenotypes. Advances in stem cell and genome editing technologies could provide models for the investigation of cell-based genetic diseases, as well as the platforms for drug discovery. This report demonstrates a practical, and widely applicable, approach that includes the successful reprogramming of somatic cells from a patient with a urea cycle defect, their genetic correction and differentiation into hepatic organoids, and the subsequent demonstration of genetic and phenotypic change in the edited cells consistent with the correction of the defect. While individually rare, there is a large number of other genetic-based liver diseases. The approach described here could be applied to a broad range and a large number of patients with these hepatic diseases where it could serve as an in vitro model, as well as identify successful strategies for corrective cell-based therapy.
Keywords	iPSC ; hepatocytes ; disease modelling ; genome editing ; CRISPR ; urea cycle ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	610
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Procurement and Evaluation of Hepatocytes for Transplantation From Neonatal Donors After Circulatory Death

Emil Bluhme / Ewa Henckel / Roberto Gramignoli / Therese Kjellin / Christina Hammarstedt / Greg Nowak / Ahmad Karadagi / Helene Johansson / Öystein Jynge / Maria Söderström / Björn Fischler / Stephen Strom / Ewa Ellis / Boubou Hallberg / Carl Jorns

Cell Transplantation, Vol

2022 Volume 31

Abstract: Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte ... ...

Abstract	Hepatocyte transplantation is a promising treatment for liver failure and inborn metabolic liver diseases, but progress has been hampered by a scarcity of available organs. Here, hepatocytes isolated from livers procured for a neonatal hepatocyte donation program within a research setting were assessed for metabolic function and suitability for transplantation. Organ donation was considered for infants who died in neonatal intensive care in the Stockholm region during 2015–2021. Inclusion was assessed when a decision to discontinue life-sustaining treatment had been made and hepatectomy performed after declaration of death. Hepatocyte isolation was performed by three-step collagenase perfusion. Hepatocyte viability, yield, and function were assessed using fresh and cryopreserved cells. Engraftment and maturation of cryopreserved neonatal hepatocytes were assessed by transplantation into an immunodeficient mouse model and analysis of the gene expression of phase I, phase II, and liver-specific enzymes and proteins. Twelve livers were procured. Median warm ischemia time (WIT) was 190 [interquartile range (IQR): 80–210] minutes. Median viability was 86% (IQR: 71%–91%). Median yield was 6.9 (IQR: 3.4–12.8) x10 6 viable hepatocytes/g. Transplantation into immunodeficient mice resulted in good engraftment and maturation of hepatocyte-specific proteins and enzymes. A neonatal organ donation program including preterm born infants was found to be feasible. Hepatocytes isolated from neonatal donors had good viability, function, and engraftment despite prolonged WIT. Therefore, neonatal livers should be considered as a donor source for clinical hepatocyte transplantation, even in cases with extended WIT.
Keywords	Medicine ; R
Subject code	610
Language	English
Publishing date	2022-01-01T00:00:00Z
Publisher	SAGE Publishing
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the mouse liver

Franziska Hildebrandt / Alma Andersson / Sami Saarenpää / Ludvig Larsson / Noémi Van Hul / Sachie Kanatani / Jan Masek / Ewa Ellis / Antonio Barragan / Annelie Mollbrink / Emma R. Andersson / Joakim Lundeberg / Johan Ankarklev

Nature Communications, Vol 12, Iss 1, Pp 1-

2021 Volume 14

Abstract: Global transcriptional differences across lobular units in the liver remain unknown. Here the authors perform spatial transcriptomics of liver tissue to delineate transcriptional differences in physical space, confirm lobular zonation along ... ...

Abstract	Global transcriptional differences across lobular units in the liver remain unknown. Here the authors perform spatial transcriptomics of liver tissue to delineate transcriptional differences in physical space, confirm lobular zonation along transcriptional gradients and suggest the presence of previously uncharacterized structures within liver tissue.
Keywords	Science ; Q
Language	English
Publishing date	2021-12-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Maintenance of Hepatic Functions in Primary Human Hepatocytes Cultured on Xeno-Free and Chemical Defined Human Recombinant Laminins.

Masaaki Watanabe / Helen Zemack / Helene Johansson / Louise Hagbard / Carl Jorns / Meng Li / Ewa Ellis

PLoS ONE, Vol 11, Iss 9, p e

2016 Volume 0161383

Abstract: Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of ... ...

Abstract	Refined methods for maintaining specific functions of isolated hepatocytes under xeno-free and chemical defined conditions is of great importance for the development of hepatocyte research and regenerative therapy. Laminins, a large family of heterotrimeric basement membrane adhesion proteins, are highly cell and tissue type specific components of the extracellular matrix and strongly influence the behavior and function of associated cells and/or tissues. However, detailed biological functions of many laminin isoforms are still to be evaluated. In this study, we determined the distribution of laminin isoforms in human liver tissue and isolated primary human hepatocytes by western blot analysis, and investigated the efficacy of different human recombinant laminin isoforms on hepatic functions during culture. Protein expressions of laminin-chain α2, α3, α4, β1, β3, γ1, and γ2 were detected in both isolated human hepatocytes and liver tissue. No α1 and α5 expression could be detected in liver tissue or hepatocytes. Hepatocytes were isolated from five different individual livers, and cultured on human recombinant laminin isoforms -111, -211, -221, -332, -411, -421, -511, and -521 (Biolamina AB), matrigel (extracted from Engelbreth-Holm-Swarm sarcoma), or collagen type IV (Collagen). Hepatocytes cultured on laminin showed characteristic hexagonal shape in a flat cell monolayer. Viability, double stranded DNA concentration, and Ki67 expression for hepatocytes cultured for six days on laminin were comparable to those cultured on EHS and Collagen. Hepatocytes cultured on laminin also displayed production of human albumin, alpha-1-antitrypsin, bile acids, and gene expression of liver-enriched factors, such as hepatocyte nuclear factor 4 alpha, glucose-6-phosphate, cytochrome P450 3A4, and multidrug resistance-associated protein 2. We conclude that all forms of human recombinant laminin tested maintain cell viability and liver-specific functions of primary human hepatocytes, and that recombinant laminin is a promising ...
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2016-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: DUCT reveals architectural mechanisms contributing to bile duct recovery in a mouse model for Alagille syndrome

Simona Hankeova / Jakub Salplachta / Tomas Zikmund / Michaela Kavkova / Noémi Van Hul / Adam Brinek / Veronika Smekalova / Jakub Laznovsky / Feven Dawit / Josef Jaros / Vítězslav Bryja / Urban Lendahl / Ewa Ellis / Antal Nemeth / Björn Fischler / Edouard Hannezo / Jozef Kaiser / Emma Rachel Andersson

eLife, Vol

2021 Volume 10

Abstract: Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, ...

Abstract	Organ function depends on tissues adopting the correct architecture. However, insights into organ architecture are currently hampered by an absence of standardized quantitative 3D analysis. We aimed to develop a robust technology to visualize, digitalize, and segment the architecture of two tubular systems in 3D: double resin casting micro computed tomography (DUCT). As proof of principle, we applied DUCT to a mouse model for Alagille syndrome (Jag1Ndr/Ndr mice), characterized by intrahepatic bile duct paucity, that can spontaneously generate a biliary system in adulthood. DUCT identified increased central biliary branching and peripheral bile duct tortuosity as two compensatory processes occurring in distinct regions of Jag1Ndr/Ndr liver, leading to full reconstitution of wild-type biliary volume and phenotypic recovery. DUCT is thus a powerful new technology for 3D analysis, which can reveal novel phenotypes and provide a standardized method of defining liver architecture in mouse models.
Keywords	Alagille syndrome ; cholangiopathy ; vasculature ; MicroCT ; resin ; Medicine ; R ; Science ; Q ; Biology (General) ; QH301-705.5
Subject code	720
Language	English
Publishing date	2021-02-01T00:00:00Z
Publisher	eLife Sciences Publications Ltd
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Systemic modified messenger RNA for replacement therapy in alpha 1-antitrypsin deficiency

Ahmad Karadagi / Alex G. Cavedon / Helen Zemack / Greg Nowak / Marianne E. Eybye / Xuling Zhu / Eleonora Guadagnin / Rebecca A. White / Lisa M. Rice / Andrea L. Frassetto / Stephen Strom / Carl Jorns / Paolo G. V. Martini / Ewa Ellis

Scientific Reports, Vol 10, Iss 1, Pp 1-

2020 Volume 11

Abstract: Abstract Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes ... ...

Abstract	Abstract Alpha 1-antitrypsin (AAT) deficiency arises from an inherited mutation in the SERPINA1 gene. The disease causes damage in the liver where the majority of the AAT protein is produced. Lack of functioning circulating AAT protein also causes uninhibited elastolytic activity in the lungs leading to AAT deficiency-related emphysema. The only therapy apart from liver transplantation is augmentation with human AAT protein pooled from sera, which is only reserved for patients with advanced lung disease caused by severe AAT deficiency. We tested modified mRNA encoding human AAT in primary human hepatocytes in culture, including hepatocytes from AAT deficient patients. Both expression and functional activity were investigated. Secreted AAT protein increased from 1,14 to 3,43 µg/ml in media from primary human hepatocytes following mRNA treatment as investigated by ELISA and western blot. The translated protein showed activity and protease inhibitory function as measured by elastase activity assay. Also, mRNA formulation in lipid nanoparticles was assessed for systemic delivery in both wild type mice and the NSG-PiZ transgenic mouse model of AAT deficiency. Systemic intravenous delivery of modified mRNA led to hepatic uptake and translation into a functioning protein in mice. These data support the use of systemic mRNA therapy as a potential treatment for AAT deficiency.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2020-04-01T00:00:00Z
Publisher	Nature Publishing Group
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Hypothermic Storage of Human Hepatocytes for Transplantation

Roberto Gramignoli / Kenneth Dorko / Veysel Tahan / Kristen J. Skvorak / Ewa Ellis / Carl Jorns / Bo-Goran Ericzon / Ira J. Fox / Stephen C. Strom Ph.D.

Cell Transplantation, Vol

2014 Volume 23

Abstract: Transplantation of human hepatocytes is gaining recognition as a bridge or an alternative to orthotopic liver transplantation for patients with acute liver failure and genetic defects. Since most patients require multiple cell infusions over an extended ... ...

Abstract	Transplantation of human hepatocytes is gaining recognition as a bridge or an alternative to orthotopic liver transplantation for patients with acute liver failure and genetic defects. Since most patients require multiple cell infusions over an extended period of time, we investigated hepatic functions in cells maintained in University of Wisconsin solution at 4°C up to 72 h. Eleven different assessments of hepatic viability and function were investigated both pre- and posthypothermic storage, including plating efficiency, caspase-3/7 activity, ammonia metabolism, and drug-metabolizing capacity of isolated hepatocytes. Long-term function, basal, and induced cytochrome P450 activities were measured after exposure to prototypical inducing agents. Cells from 47 different human liver specimens were analyzed. Viability significantly decreased in cells cold stored in UW solution, while apoptosis level and plating efficiency were not significantly different from fresh cells. Luminescent and fluorescent methods assessed phases I and II activities both pre- and post-24-72 h of cold preservation. A robust induction (up to 200-fold) of phase I enzymes was observed in cultured cells. Phase II and ammonia metabolism remained stable during hypothermic storage, although the inductive effect of culture on each metabolic activity was eventually lost. Using techniques that characterize 11 measurements of hepatic viability and function from plating efficiency, to ammonia metabolism, to phases I and II drug metabolism, it was determined that while viability decreased, the remaining viable cells in cold-stored suspensions retained critical hepatic functions for up to 48 h at levels not significantly different from those observed in freshly isolated cells.
Keywords	Medicine ; R
Subject code	610
Language	English
Publishing date	2014-09-01T00:00:00Z
Publisher	SAGE Publishing
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Strategies for Short-Term Storage of Hepatocytes for Repeated Clinical Infusions

Carl Jorns M.D. / Roberto Gramignoli / Mohammed Saliem / Helen Zemack / Lisa-Mari Mörk / Bengt Isaksson / Greg Nowak / Bo-Göran Ericzon / Stephen Strom / Ewa Ellis

Cell Transplantation, Vol

2014 Volume 23

Abstract: Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1–2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated ... ...

Abstract	Hepatocyte transplantation is an upcoming treatment for patients with metabolic liver diseases. Repeated cell infusions over 1–2 days improve clinical outcome. Isolated hepatocytes are usually cold stored in preservation solutions between repeated infusions. However, during cold storage isolated hepatocytes undergo cell death. We investigated if tissue preservation and repeated isolations are better than storage of isolated hepatocytes when cold preserving human hepatocytes. Liver tissue obtained from liver surgery or organ donors was divided into two pieces. Hepatocytes were isolated by collagenase digestion. Hepatocytes were analyzed directly after isolation (fresh) or after storage for 48 h at 4°C in University of Wisconsin solution (UW cells). Liver tissue from the same donor was stored at 4°C in UW and hepatocytes were isolated after 48 h (UW tissue cells). Hepatocyte viability and function was evaluated by trypan blue exclusion, plating efficiency, ammonia metabolism, CYP 1A1/2, 2C9, 3A7, and 3A4 activities, phase II conjugation, and apoptosis evaluation by TUNEL assay and caspase-3/7 activities. Hepatocytes stored in UW showed a significantly lower viability compared to fresh cells or hepatocytes isolated from tissue stored for 48 h (54% vs. 71% vs. 79%). Plating efficiency was significantly decreased for cells stored in UW (40%) compared to fresh and UW tissue cells (63% vs. 55%). No significant differences between UW cells and UW tissue cells could be shown for CYP activities or ammonia metabolism. Hepatocytes stored in UW showed a strong increase in TUNEL-positive cells, whereas TUNEL staining in cold-stored liver tissue and hepatocytes isolated after 48 h was unchanged. This observation was confirmed by increased caspase-3/7 activities in UW cells. Although preservation of isolated hepatocytes in UW maintains function, cold storage of liver tissue and repeated hepatocyte isolations is superior to cold storage of isolated hepatocytes in preserving hepatocyte viability and function.
Keywords	Medicine ; R
Language	English
Publishing date	2014-08-01T00:00:00Z
Publisher	SAGE Publishing
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Improved cryopreservation of human hepatocytes using a new xeno free cryoprotectant solution

Mohammed Saliem / Frida Holm / Rosita Bergström Tengzelius / Carl Jorns / Lisa-Mari Nilsson / Bo-Göran Ericzon / Ewa Ellis / Outi Hovatta

World Journal of Hepatology, Vol 4, Iss 5, Pp 176-

2012 Volume 183

Abstract: AIM: To optimize a xeno-free cryopreservation protocol for primary human hepatocytes. METHODS: The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being ... ...

Abstract	AIM: To optimize a xeno-free cryopreservation protocol for primary human hepatocytes. METHODS: The demand for cryopreserved hepatocytes is increasing for both clinical and research purposes. Despite several hepatocyte cryopreservation protocols being available, improvements are urgently needed. We first compared controlled rate freezing to polystyrene box freezing and did not find any significant change between the groups. Using the polystyrene box freezing, we compared two xeno-free freezing solutions for freezing of primary human hepatocytes: a new medium (STEM-CELLBANKER, CB), which contains dimethylsulphoxide (DMSO) and anhydrous dextrose, both permeating and non-permeating cryoprotectants, and the frequently used DMSO - University of Wisconsin (DMSO-UW) medium. The viability of the hepatocytes was assessed by the trypan blue exclusion method as well as a calcein-esterase based live-dead assay before and after cryopreservation. The function of the hepatocytes was evaluated before and after cryopreservation by assessing enzymatic activity of 6 major cytochrome P450 isoforms (CYPs): CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A7. RESULTS: The new cryoprotectant combination preserved hepatocyte viability significantly better than the standard DMSO-UW protocol (P < 0.01). There was no significant difference in viability estimation between both the trypan blue (TB) and the Live-Dead Assay methods. There was a correlation between viability of fresh hepatocytes and the difference in cell viability between CB and DMSO protocols (r2 = 0.69) using the TB method. However, due to high within-group variability in the activities of the major CYPs, any statistical between-group differences were precluded. Cryopreservation of human hepatocytes using the cryoprotectant combination was a simple and xeno-free procedure yielding better hepatocyte viability. Thus, it may be a better alternative to the standard DMSO-UW protocol. Estimating CYP activities did not seem to be a relevant way to compare hepatocyte function between different groups due to high normal variability between different liver samples. CONCLUSION: The cryoprotectant combination may be a better alternative to the standard DMSO-UW protocol in primary human hepatocyte cryopreservation.
Keywords	Human hepatocytes ; Viability ; Cytochrome P540 ; Dimethylsulphoxide ; Cryoprotectant ; Cryopreservation ; Diseases of the digestive system. Gastroenterology ; RC799-869 ; Specialties of internal medicine ; RC581-951 ; Internal medicine ; RC31-1245 ; Medicine ; R ; DOAJ:Gastroenterology ; DOAJ:Medicine (General) ; DOAJ:Health Sciences
Subject code	500
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Baishideng Publishing Group Co., Limited
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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