Differentiation of multipotent human skin-derived precursors towards hepatic stellate cell-like cells for modelling liver fibrosis in vitro

Recently, our group showed that hepatic progenitor-like cells derived from multipotent human skin-derived precursors (hSKPs) are valuable for testing of hepatocyte-specific anti-NASH effects of PPAR agonists. However, advanced NASH is often associated with the development of liver fibrosis, a process in which hepatic stellate cells (HSCs) play a pivotal role. Yet, testing of anti-fibrotic drugs is hampered by a lack of adequate human-relevant preclinical models. Here, we apply a publicly available protocol for generation of HSC-like cells from induced pluripotent stem cells (Coll et al. 2018) on hSKPs to differentiate these cells towards hepatic stellate cell-like cells (hSKP-HSCs). Further, we investigate whether the obtained cells can be activated and if these cells respond to the PPAR-α/δ agonist elafibranor.
According to the protocol of Coll et al., hSKPs were sequentially exposed in vitro to BMP4, FGF1, FGF3 (all 20 ng/ml), retinol (5 µM) and palmitic acid (100 µM) for 12 days, rendering hSKP-HSCs. At the end of the differentiation, hSKP-HSCs were exposed for 24 hours to TGF-β (10 ng/ml) with and without elafibranor at subcytotoxic concentration (30 µM). Expression of HSC markers was measured using RT-qPCR and compared to levels in the commercially available human immortalized hepatic stellate cell line LX-2. Protein expression was evaluated using immunocytochemistry.
Differentiation results in star-shaped cells and upregulation of the submesothelial HSC-progenitor marker ALCAM and also of key genes involved in intermediate filament formation (DES) and vitamin A metabolism (LRAT) to levels approaching or exceeding those in LX-2 cells. In addition, hSKP-HSCs express PDGFRβ, a membrane marker of HSCs. Activation of hSKP-HSCs by TGF-β could be demonstrated by upregulation of pro-fibrotic genes ACTA2, COL1A1 and LOXL2. As proof-of-principle, concomitant exposure to elafibranor leads to repression of the aforementioned genes.
In conclusion, hSKPs hold potential to differentiate towards HSC-like cells, expressing specific HSC markers. Upregulation of activation markers upon exposure to TGF-β indicates that hSKP-HSCs respond to pro-fibrotic stimuli. Furthermore, the activated hSKP-HSCs respond to elafibranor, suggesting future possibilities for preclinical drug testing. Nevertheless, further in-depth characterization of hSKP-HSCs is necessary.