Differentiation of Monkey Embryonic Stem Cells into Hepatocytes and mRNA Expression of Cytochrome P450 Enzymes Responsible for Drug Metabolism: Comparison of Embryoid Body Formation Conditions and Matrices

Abstract

Examination of drug metabolism using 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. Considerable donor-dependent variations are also problematic. Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts and are capable of differentiating into three embryonic germ layers and germ cells.1) The cells apparently differentiate into various types of mature cells, and are thereby an attractive source for routine access to large numbers of cells that can be used for the development of candidate drug-screening strategies replacing primary cells.2) However, ethical considerations have limited the availability of human ES cells. The phenotype of human ES cells is known to be similar to that of monkey ES cells but differs from that of mouse ES cells with regard to morphology, response to leukemia inhibitory factor, and gene expression patterns. 1,[3][4][5] Research using monkey ES cells is considered to be useful for investigation of differentiation mechanisms in primate ES cells and models of human ES cells. Recently, it has been shown that monkey ES cells can be differentiated into various cell types-including neurons, hematopoietic cells, and pancreatic cells-using growth factors. [6][7][8] Hepatocytes derived from monkey ES cells may be useful for pharmacokinetic examinations such as induction of drug metabolism enzymes and interactions of candidate drugs. To date however, there have been few reported studies describing the differentiation of monkey ES cells into hepatocytes. 9,10) After the emergence of the liver bud from the developing gut tube, the level of hepatic maturation is characterized by the expression of liver-and stage-specific genes. For example, alfa-fetoprotein (AFP) is an early hepatic marker, expressed by hepatoblasts in the liver bud until birth.11,12) The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. In contrast, albumin (ALB), the most abundant protein synthesized by hepatocytes, is initially expressed at lower levels in early fetal hepatocytes but this increases as the hepatocytes mature, reaching a maximum in adult hepatocytes. 13,14) The mRNA expression of cytochrome P450 7A1 (CYP7A1) which is a rate-limiting enzyme in the conversion of cholesterol to bile acids in liver 15) is detected in fetal liver of third trimester of pregnancy.16) After birth, CYP7A1 increases several-fold with age both at the enzyme activity level and the mRNA level. 17,18) The in vitro approaches involve the formation of embryoid bodies (EBs) to mimic the inductive microenvironment required for liver organogenesis [19][20][21] and treatment with specific growth factors and cytokines critical for hepatic differentiation.22) At present, culture systems for ES cells have mainly used gelatin-or collagen-coated plates as the matrix for the maintenance of cells in an undifferentiated state an...

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

“…The effects of RIF on mRNA expression of cmCYP3A4 (3A8) in the cmESC-derived hepatic cells was consistent with the findings reported previously. 7) It was previously reported that cmCYP3A4 (3A8) is induced by RIF through the transcription factor PXR. 29) Our study showed that PXR is expressed in cmESC-derived hepatic cells.…”

“…18) This method was applied to the induction of differentiation of monkey ESCs into hepatic cells. 7) In recent studies, however, a method to add some direct inducing factors to a monolayer culture system of an undifferentiated ESC colony without EB formation has been used extensively. Furthermore, some improved methods for more efficient differentiation into hepatocytes have been reported.…”

“…7) EBs can mimic the inductive microenvironment required for liver organogenesis [8][9][10] and develop into many different cell types in culture. However, this EB formation method is not always appropriate for assays with large numbers of samples, such as high-throughput screening, because the formation of EBs is inefficient.…”

“…4) and maintained according to the method reported previously 7) except that recombinant human bFGF was added to ES medium. Feeder-free dispersed culture was carried out as follows (Fig.…”

Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells*

“…To achieve this goal, researchers have successfully exploited the molecular signals for liver development outlined above to direct the development of mouse (19 -21) and subsequently monkey (22,23) and human (24 -30) ESCs into hepatocyte-like cells (HLCs). Additionally, a few studies have demonstrated that mouse or monkey ESCs can be coaxed to generate HLCs in the presence of murine hepatocytes (31,32) or human liver nonparenchymal cells (33).…”