Abstract
The derivation of hepatocytes from human induced pluripotent stem cells (hiPSC) is of great interest for applications in pharmacological research. However, full maturation of hiPSC-derived hepatocytes has not yet been achieved in vitro. To improve hepatic differentiation, co-cultivation of hiPSC with human umbilical vein endothelial cells (HUVEC) during hepatic differentiation was investigated in this study. In the first step, different culture media variations based on hepatocyte culture medium (HCM) were tested in HUVEC mono-cultures to establish a suitable culture medium for co-culture experiments. Based on the results, two media variants were selected to differentiate hiPSC-derived definitive endodermal (DE) cells into mature hepatocytes with or without HUVEC addition. DE cells differentiated in mono-cultures in the presence of those media variants showed a significant increase (p < 0.05) in secretion of α-fetoprotein and in activities of cytochrome P450 (CYP) isoenzymes CYP2B6 and CYP3A4 as compared with cells differentiated in unmodified HCM used as control. Co-cultivation with HUVEC did not further improve the differentiation outcome. Thus, it can be concluded that the effect of the used medium outweighed the effect of HUVEC co-culture, emphasizing the importance of the culture medium composition for hiPSC differentiation.
Highlights
Human induced pluripotent stem cells hold great promise for application in cell therapies [1], and in disease research [1,2] and drug toxicity testing with in vitro models [3]
human umbilical vein endothelial cells (HUVEC) were cultured in the presence of 100% endothelial cell growth medium, consisting of basal medium and supplements (EGM complete), 100% hepatocyte culture medium (HCM-I), HCM-I and Endothelial cell growth medium (EGM) complete mixed at a ratio of 1:1 (HCM-I + EGM complete) or HCM-I enriched with endothelial cell growth supplements (HCM-I + EGM supplements)
The curve progressions displaying glucose consumption rates of HUVEC cultivated in EGM complete or HCM-I + EGM complete were similar, showing an increase until day 4 and a slight decrease until day 6 followed by a slow, but stable increase until day 14 (Figure 2A)
Summary
Human induced pluripotent stem cells (hiPSC) hold great promise for application in cell therapies [1], and in disease research [1,2] and drug toxicity testing with in vitro models [3]. Previous in vitro studies on hepatic drug toxicity using hepatocyte-like cells (HLC) differentiated from hiPSC showed promising results [6,7]. Despite these encouraging outcomes, the efficiency of the differentiation process of hiPSC towards functional HLC for further use, including hepatotoxicity assessment, remains low [8]. Several research groups managed to generate hiPSC-derived HLC with 60% [9] up to 80–85% [10] of differentiated cells being characterized by the expression of several hepatic markers, including albumin. Baxter et al characterized human embryonic stem-cell derived HLC as being similar to fetal rather than to adult hepatocytes in terms of their metabolic profile [11]. Further improvement of the differentiation and maturation process is needed to generate fully functional HLC for clinical or in vitro use
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