Immortalized Mouse Dermal Fibroblasts-SV40

VPA Induced Primary Porcine Hepatocytes to Reprogram into Hepatic Progenitor Cells In Vitro

Hepatocyte transplantation is considered a feasible and cost-effective alternative to liver transplantation for end-stage liver disease. Human hepatocytes are limited and porcine hepatocytes are a potential alternative. However, the long-term in vitro proliferation of porcine hepatocytes is restricted.

Li's team developed a method to stimulate the in vitro proliferation of primary porcine hepatocytes using Valproic acid (VPA) (Fig. 1a). The growth-stimulating medium was named VPA-HCM, and it contained 0.5 mM VPA added to HCM. Primary pig hepatocytes (PPH) were rapidly proliferated in VPA-HCM (Fig. 1c) and the nucleus-to-cytoplasm ratio was increased at seven days compared to that of primary pig liver cells (Fig. 1b). Immunofluorescence staining showed that compared with freshly isolated pig liver cells, cells cultured in VPA-HCM for seven days expressed significantly higher cell cycle marker (MKI67 and PCNA) and HPC marker (KRT19 and SOX9) and hepatocyte function marker (HNF1A, HNF4A and Albumin) was decreased (Fig. 1d, e). The cell morphology changed from that of a typical hepatocyte to that of a progenitor cell after 7 days of culture in VPA-HCM, and they were called Valproic acid-induced Hepatic Progenitor Cells (VPA-iHPCs). VPA-iHPCs (D7) upregulated HPC and cell cycle genes and downregulated mature hepatocyte function genes in the qPCR assay (Fig. 1f). In summary, VPA-induced the differentiation of primary porcine hepatocytes into high-proliferation HPCs.

Fig. 1. VPA induced primary porcine hepatocytes to reprogram into Hepatic Progenitor Cells in vitro (Li G H, Zeng M, et al., 2024).

Traversal of Porcine Hepatocytes by Plasmodium falciparum Sporozoites

Plasmodium falciparum (Pf) causes severe malaria by infecting human hepatocytes. The molecular mechanisms of host specificity and maturation in non-human hepatocytes are poorly understood. Here, van der Boor et al. used fresh porcine hepatocytes to study Pf sporozoite invasion and development.

Different seeding densities were tested, with viability assessed using brightfield microscopy and MTT from the day of invasion to day five (Fig. 2A and B). Metabolic activity, measured by MTT, increased up to 72 hours post-invasion for all densities and then stabilized (Fig. 2B). Beyond day seven, cell viability decreased, showing clumping under microscopy. An optimal density of 62,500 was selected, as higher densities led to cell clumping rather than monolayer formation, aligning with densities used for primary human hepatocytes. Both human and porcine hepatocytes were infected with NF135.C10 sporozoites two days post-plating. After a three-hour traversal assay, significant FITC dextran-positive cells indicated NF135.C10 sporozoites could traverse porcine hepatocytes, though less efficiently than human ones. Traversal was less in porcine than in human hepatocytes (Fig. 2C and D).

Fig. 2. (A-B) Microscopic images and viability of freshly seeded porcine hepatocytes. (C-D) Traversal of NF135.C10 sporozoites in fresh human and porcine hepatocytes three hours post-invasion of three biological donors (van der Boor SC, van Gemert GJ, et al., 2022).