Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology

Abstract

Background/Aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep) cells directly reprogrammed from mice fibroblast cells were fully characterized. Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α) and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. Results: The expression and function of p-glycoprotein (P-gp), bile salt efflux pump (Bsep), multidrug resistance-associated protein 2 (Mrp2), Na⁺-dependent taurocholate cotransporting polypeptide (Ntcp), and organic anion transporter polypedtides (Oatps) in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α) and treatment with selected inducers. In vitro intrinsic biliary clearances (CL_b,int) of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid) and DPDPE ([D-Pen^2,5] enkephalin hydrate) correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs) (r² = 0.984). Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

Asiatic Acid (AA) Sensitizes Multidrug-Resistant Human Lung Adenocarcinoma A549/DDP Cells to Cisplatin (DDP) via Downregulation of P-Glycoprotein (MDR1) and Its Targets

“…AKT-mediated signaling accounts for fundamental cell functions such as cell proliferation, DNA repair, and antiapoptotic processes [34]. It has been reported that AKT activation occurs in solid tumors frequently [35]. A point mutation in AKT leads to its activation in a PI3K-independent manner [36].…”

Functional Proliferating Human Hepatocytes: In Vitro Hepatocyte Model for Drug Metabolism, Excretion, and Toxicity

“…5B). Three selected cholestatic drugs (rifampin, glibenclamide and ketoconazole) were reported as the inhibitors of BSEP (Ni et al, 2016;Wu et al, 2016). In this study, all of them significantly inhibited d8-TCA biliary excretion in SC-ProliHH-P (Fig.…”

“…A was the concentration or amount of test compound accumulated in the absence (A minus_Ca++ ) or presence (A plus_Ca++ ) of Ca 2+ , which was achieved through adding warm Ca 2+ -free or standard HBSS buffer (Wu et al, 2016).…”