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A combined in silico and in vitro study on mouse Serpina1a antitrypsin-deficiency mutants.Eggenschwiler, Reto; Patronov, Atanas; Hegermann, Jan; Fráguas-Eggenschwiler, Mariane; Wu, Guangming; Cortnumme, Leon; Ochs, Matthias; Antes, Iris; Cantz, Tobias; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Springer-Nature, 2019-05-16)Certain point-mutations in the human SERPINA1-gene can cause severe α1-antitrypsin-deficiency (A1AT-D). Affected individuals can suffer from loss-of-function lung-disease and from gain-of-function liver-disease phenotypes. However, age of onset and severity of clinical appearance is heterogeneous amongst carriers, suggesting involvement of additional genetic and environmental factors. The generation of authentic A1AT-D mouse-models has been hampered by the complexity of the mouse Serpina1-gene locus and a model with concurrent lung and liver-disease is still missing. Here, we investigate point-mutations in the mouse Serpina1a antitrypsin-orthologue, which are homolog-equivalent to ones known to cause severe A1AT-D in human. We combine in silico and in vitro methods and we find that analyzed mutations do introduce potential disease-causing properties into Serpina1a. Finally, we show that introduction of the King’s-mutation causes inactivation of neutrophil elastase inhibitory-function in both, mouse and human antitrypsin, while the mouse Z-mutant retains activity. This work paves the path to generation of better A1AT-D mouse-models.
Direct conversion of porcine primary fibroblasts into hepatocyte-like cells.Fráguas-Eggenschwiler, Mariane; Eggenschwiler, Reto; Söllner, Jenny-Helena; Cortnumme, Leon; Vondran, Florian W R; Cantz, Tobias; Ott, Michael; Niemann, Heiner; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Nature Research, 2021-04-29)The pig is an important model organism for biomedical research, mainly due to its extensive genetic, physiological and anatomical similarities with humans. Until date, direct conversion of somatic cells into hepatocyte-like cells (iHeps) has only been achieved in rodents and human cells. Here, we employed lentiviral vectors to screen a panel of 12 hepatic transcription factors (TF) for their potential to convert porcine fibroblasts into hepatocyte-like cells. We demonstrate for the first time, hepatic conversion of porcine somatic cells by over-expression of CEBPα, FOXA1 and HNF4α2 (3TF-piHeps). Reprogrammed 3TF-piHeps display a hepatocyte-like morphology and show functional characteristics of hepatic cells, including albumin secretion, Dil-AcLDL uptake, storage of lipids and glycogen and activity of cytochrome P450 enzymes CYP1A2 and CYP2C33 (CYP2C9 in humans). Moreover, we show that markers of mature hepatocytes are highly expressed in 3TF-piHeps, while fibroblastic markers are reduced. We envision piHeps as useful cell sources for future studies on drug metabolism and toxicity as well as in vitro models for investigation of pig-to-human infectious diseases.