Direct conversion of porcine primary fibroblasts

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, FOXA1and HNF4α2 (3TF-piHeps).

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FIG: MOI determination for hepatic conversion of PKFs. (A) Schematic drawing of lentivirus vector design and cell culture protocol for directed hepatic conversion. (B) Albumin secretion quantification and (C) hepatic markers expression levels of cells directly reprogrammed with different MOIs per lentiviral vector of all 12 TFs, compared to Neg-Ctrl. All data were analyzed using two-way ANOVA, with Bonferroni’s post-test, except for TF gene, that was converted to mixed effects with Tukey’s multiple comparison post-test due to be missing one value. Significance from n = 3 independent values is displayed as *p < 0.05; ****p ≤ 0.0001.

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.

Fráguas-Eggenschwiler, M., Eggenschwiler, R., Söllner, JH. et al. Direct conversion of porcine primary fibroblasts into hepatocyte-like cells.Sci Rep 11, 9334 (2021). https://doi.org/10.1038/s41598-021-88727-1

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