Differentiation of Monkey Embryonic Stem Cells to Hepatocytes by Feeder-Free Dispersion Culture and Expression Analyses of Cytochrome P450 Enzymes Responsible for Drug Metabolism

Abstract

We reported previously that monkey embryonic stem cells (ESCs) were differentiated into hepatocytes by formation of embryoid bodies (EBs). However, this EB formation method is not always efficient for assays using a large number of samples simultaneously. A dispersion culture system, one of the differentiation methods without EB formation, is able to more efficiently provide a large number of feeder-free undifferentiated cells. A previous study demonstrated the effectiveness of the Rho-associated kinase inhibitor Y-27632 for feeder-free dispersion culture and induction of differentiation of monkey ESCs into neural cells. In the present study, the induction of differentiation of cynomolgus monkey ESCs (cmESCs) into hepatocytes was performed by the dispersion culture method, and the expression and drug inducibility of cytochrome P450 (CYP) enzymes in these hepatocytes were examined. The cmESCs were successfully differentiated into hepatocytes under feeder-free dispersion culture conditions supplemented with Y-27632. The hepatocytes differentiated from cmESCs expressed the mRNAs for three hepatocyte marker genes (α-fetoprotein, albumin, CYP7A1) and several CYP enzymes, as measured by real-time polymerase chain reaction. In particular, the basal expression of cmCYP3A4 (3A8) in these hepatocytes was detected at mRNA and enzyme activity (testosterone 6β-hydroxylation) levels. Furthermore, the expression and activity of cmCYP3A4 (3A8) were significantly upregulated by rifampicin. These results indicated the effectiveness of Y-27632 supplementation for feeder-free dispersed culture and induction of differentiation into hepatocytes, and the expression of functional CYP enzyme(s) in cmESC-derived hepatic cells.Key words embryonic stem cell; differentiation; hepatocyte; monkey; cytochrome P450; feeder-free dispersed culture Investigation of drug metabolism with human hepatocytes is important in the early stages of drug development. However, primary human hepatocytes are short-lived and cannot be maintained in culture over the long term. In addition, there are large donor-dependent variations in drug metabolism. On the other hand, human embryonic stem cells (ESCs) are able to replicate infinitely and differentiate into various types of somatic cells including germ cells.1) Thus, they represent an attractive source to provide large numbers of cells that can be utilized for the development of candidate drug-screening strategies in place of primary cells.2) However, ethical and legal restrictions have limited the availability of human ESCs. The phenotype of human ESCs is known to closely resemble that of monkey ESCs but not mouse ESCs with regard to morphology, leukemia inhibitory factor responsiveness, gene expression profiles, and some disease models.1,3-6) Thus, monkey ESCs are a more suitable model for preclinical research of drug development. In particular, hepatocytes derived from monkey ESCs may be useful for pharmacokinetic studies, such as investigation of drug-drug interactions and the inducibility of drug-metabolizi...

Preparation and characterization of directed, one‐day‐self‐assembled millimeter‐size spheroids of adipose‐derived mesenchymal stem cells

“…Thus, 3D cell aggregates such as multicellular spheroids, mammospheres, or macromass have been utilized as niche‐mimicking models by using suspension culture technologies based on static systems using nonadhesive cell culture dishes and dynamic systems that incorporate spinner flasks with stirred devices or microgravity bioreactors . These culture systems have been used as the models for tumor growth, hepatic metabolism, and for drug screening and enabled cultivation of induced pluripotent stem cells, embryonic stem cells, and somatic stem cells …”

“…5 These culture systems have been used as the models for tumor growth, 6,7 hepatic metabolism, 8,9 and for drug screening and enabled cultivation of induced pluripotent stem cells, embryonic stem cells, and somatic stem cells. [10][11][12] Recent developments in tissue regenerative therapy have shifted the preference to using 3D cell constructs that do not incorporate artificial cell scaffold materials, which can be immunogenic or could transmit infections. 13 Tissue regeneration using cell spheroids have been previously reported.…”

<p>Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells</p>

“…Aravalli et al ESCs from human, rodent, porcine, and monkey species have been differentiated into HLCs. [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49] In this study, we have carried out hepatic differentiation of marmoset ESCs into HLCs and characterized them using a combination of immunohistochemistry, morphological analyses, RT-PCR, and biochemical assays. Activin A-treated marmoset ESCs and primary human hepatocytes (PHHs) were used as controls.…”

“…50 Significantly, our hepatic differentiation protocol is distinct from that of other reports of non-human primates, namely rhesus macaque ESCs 42,43 and cynomolgus ESCs. 48,49 We have used a modified three-step procedure that was previously developed in our laboratory to differentiate porcine iPS cells into HLCs. 13 Each step of this protocol was for a 5-day period, in which the first step was the induction of definitive endoderm by treating marmoset ESCs with activin A, followed by hepatocyte differentiation with growth factors FGF-2 and BMP-4.…”

“…All cells were counterstained with DAPI (shown in blue). that of cynomolgus ESC was either a 27-day 48 or 28-day 49 procedure. Thus, our protocol is significantly shorter in duration and produced mature HLCs within 15 days.…”