Expression of Cytochrome P450 and Transcription Factors during In Vitro Differentiation of Mouse Embryonic Stem Cells into Hepatocytes

Maezawa, Kayoko; Miyazato, Kenji; Matsunaga, Tamihide; Momose, Yasuyuki; Imamura, Tetsuo; Johkura, Kohei; Sasaki, Katsunori; Ohmori, Shigeru

doi:10.2133/dmpk.23.188

Cited by 11 publications

(9 citation statements)

References 32 publications

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“…We have also found that PXR mRNA was expressed only at the late stage in differentiating mouse ES cells. 64) These findings suggest that the cells differentiated on Matrigel reduced-and Matrigel-coated plates demonstrate the characteristics of mature hepatocytes in the CYP3A8 gene expression response following RIF treatment, although cells differentiated on collagen I-coated plate were not sufficiently mature to induce CYP3A8 such as that in hepatoblasts.…”

Section: Discussionmentioning

confidence: 78%

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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

Momose

Matsunaga

Murai

et al. 2009

Biological & Pharmaceutical Bulletin

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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...

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Section: Discussionmentioning

confidence: 78%

“…6). We have previously found that aryl hydrocarbon receptor mRNA was detected in differentiating mouse ES cells throughout the culture period of 36 d. 64) These findings suggest that the enzyme induction process via aryl hydrocarbon receptor is already constructed in the hepatocytes-derived from cmES cells.…”

Section: Discussionmentioning

confidence: 81%

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

Momose

Matsunaga

Murai

et al. 2009

Biological & Pharmaceutical Bulletin

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“…[15][16][17] Many of these methods involve a procedure to differentiate ESCs by EB formation and adhesion culture. EBs differentiate into three embryonic germ layers by suspended cell culture of ESCs, and these cells can then further differentiate into multiple cell types, including hepatocytes, in vitro.…”

Section: Discussionmentioning

confidence: 99%

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

Maruyama

Matsunaga

Yamaori

et al. 2013

Biological & Pharmaceutical Bulletin

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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...

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“…In an independent study, Cyp1a1 was shown to appear earliest — following which AHR, ARNT, and glucocorticoid receptor mRNAs as well — were detected in differentiating mouse ES cells throughout the culture period (Maezawa et al , 2008). The AHR-signaling pathway and sensitivity to PAH-caused toxicity were also described in human ES cells (Kee et al , 2010).…”

Section: Ahr and Cell-signaling Pathwaysmentioning

confidence: 99%

Aryl hydrocarbon receptor (AHR): “pioneer member” of the basic-helix/loop/helix per - Arnt - sim (bHLH/PAS) family of “sensors” of foreign and endogenous signals

Nebert

2017

Progress in Lipid Research

220

191

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The basic-helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family comprises many transcription factors, found throughout all three kingdoms of life; bHLH/PAS members “sense” innumerable intracellular and extracellular “signals” — including endogenous compounds, foreign chemicals, gas molecules, redox potential, photons (light), gravity, heat, and osmotic pressure. These signals then initiate downstream signaling pathways involved in responding to that signal. The term “PAS”, abbreviation for “Per-Arnt-Sim” was first coined in 1991. Although the mouse Arnt gene was not identified until 1991, evidence of its co-transcriptional binding partner, aryl hydrocarbon receptor (AHR), was first reported in 1974 as a “sensor” of foreign chemicals, up-regulating cytochrome P450 family 1 (CYP1) and other enzyme activities that usually metabolize the signaling chemical. Within a few years, AHR was proposed also to participate in inflammation. The mouse [Ah] locus was shown (1973–1989) to be relevant to chemical carcinogenesis, mutagenesis, toxicity and teratogenesis, the mouse Ahr gene was cloned in 1992, and the first Ahr(−/−) knockout mouse line was reported in 1995. After thousands of studies from the early 1970s to present day, we now realize that AHR participates in dozens of signaling pathways involved in critical-life processes, affecting virtually every organ and cell-type in the animal, including many invertebrates.

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Expression of Cytochrome P450 and Transcription Factors during In Vitro Differentiation of Mouse Embryonic Stem Cells into Hepatocytes

Cited by 11 publications

References 32 publications

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

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

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

Aryl hydrocarbon receptor (AHR): “pioneer member” of the basic-helix/loop/helix per - Arnt - sim (bHLH/PAS) family of “sensors” of foreign and endogenous signals

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