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Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers.

Sa-ngiamsuntorn K, Wongkajornsilp A, Kasetsinsombat K, Duangsa-ard S, Nuntakarn L, Borwornpinyo S, Akarasereenont P, Limsrichamrern S, Hongeng S - BMC Biotechnol. (2011)

Bottom Line: Their inducibility outperformed the classical HepG2 cells.The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes.The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand.

ABSTRACT

Background: The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.

Results: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.

Conclusion: The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

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Functional activities of hepatocyte-like cells. The presence of albumin was analyzed using flow cytometer in the immediately differentiated cells (A), the immortalized hepatocyte-like cells (B), and in HepG2 (C). Greater than 80% of the population of hepatocyte-like cells contained albumin (D) that could be maintained at 70% beyond 10 passages. Urea production as represented by diacetyl monoxime test was demonstrated in MSCs, HepG2, and hepatocyte-like cells (E). IMDM medium supplemented with 5 mM NH4Cl served as the control group.
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Figure 3: Functional activities of hepatocyte-like cells. The presence of albumin was analyzed using flow cytometer in the immediately differentiated cells (A), the immortalized hepatocyte-like cells (B), and in HepG2 (C). Greater than 80% of the population of hepatocyte-like cells contained albumin (D) that could be maintained at 70% beyond 10 passages. Urea production as represented by diacetyl monoxime test was demonstrated in MSCs, HepG2, and hepatocyte-like cells (E). IMDM medium supplemented with 5 mM NH4Cl served as the control group.

Mentions: Up to 88% of hepatocyte-like cells (Figure 3A) and immortalized hepatocyte-like cells (Figure 3B), as opposed to 5% of MSCs, contained intracellular albumin. Almost 95% of HepG2 cells contained albumin (Figure 3C). In subsequent passages, hepatocyte-like cells maintained at least 70% of albumin-containing cells (Figure 3D). The functional activity of hepatocyte-like cells at different passages was investigated using urea assay (Figure 3E). The conditioned medium of hepatocyte-like cells contained far higher level of urea than that of MSCs, but was comparable to that of HepG2. Using mRNA levels inherent to MSCs as a reference, the relative expression levels of the corresponding genes in the hepatocyte-like cells, HepG2 cells and the primary human hepatocytes were determined using quantitative real-time PCR. The basal expression patterns for hepatocyte-specific genes at passage 5-9 (e.g., ALB, AFP, CK18, G6PD, HNF-4α and TAT) were varied, depending on the stage of hepatocyte maturation. The AFP expression that is usually presented in hepatic progenitors was detected at higher level than those of the primary human hepatocytes and HepG2 cells (Figure 1H). The observation of cytokeratin18 expression confirmed the differentiation of MSCs into endodermal tissue. Three major hepatocyte genes were up-regulated in hepatocyte-like cells, namely glucose-6-phosphate dehydrogenase (glucose metabolism), HNF-4α (liver development and maturation) and tyrosine aminotransferase (amino acid metabolism). The observation of all hepatocyte specific genes confirmed that the transformed MSCs could actually differentiate into functional hepatocyte-like cells.


Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers.

Sa-ngiamsuntorn K, Wongkajornsilp A, Kasetsinsombat K, Duangsa-ard S, Nuntakarn L, Borwornpinyo S, Akarasereenont P, Limsrichamrern S, Hongeng S - BMC Biotechnol. (2011)

Functional activities of hepatocyte-like cells. The presence of albumin was analyzed using flow cytometer in the immediately differentiated cells (A), the immortalized hepatocyte-like cells (B), and in HepG2 (C). Greater than 80% of the population of hepatocyte-like cells contained albumin (D) that could be maintained at 70% beyond 10 passages. Urea production as represented by diacetyl monoxime test was demonstrated in MSCs, HepG2, and hepatocyte-like cells (E). IMDM medium supplemented with 5 mM NH4Cl served as the control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198927&req=5

Figure 3: Functional activities of hepatocyte-like cells. The presence of albumin was analyzed using flow cytometer in the immediately differentiated cells (A), the immortalized hepatocyte-like cells (B), and in HepG2 (C). Greater than 80% of the population of hepatocyte-like cells contained albumin (D) that could be maintained at 70% beyond 10 passages. Urea production as represented by diacetyl monoxime test was demonstrated in MSCs, HepG2, and hepatocyte-like cells (E). IMDM medium supplemented with 5 mM NH4Cl served as the control group.
Mentions: Up to 88% of hepatocyte-like cells (Figure 3A) and immortalized hepatocyte-like cells (Figure 3B), as opposed to 5% of MSCs, contained intracellular albumin. Almost 95% of HepG2 cells contained albumin (Figure 3C). In subsequent passages, hepatocyte-like cells maintained at least 70% of albumin-containing cells (Figure 3D). The functional activity of hepatocyte-like cells at different passages was investigated using urea assay (Figure 3E). The conditioned medium of hepatocyte-like cells contained far higher level of urea than that of MSCs, but was comparable to that of HepG2. Using mRNA levels inherent to MSCs as a reference, the relative expression levels of the corresponding genes in the hepatocyte-like cells, HepG2 cells and the primary human hepatocytes were determined using quantitative real-time PCR. The basal expression patterns for hepatocyte-specific genes at passage 5-9 (e.g., ALB, AFP, CK18, G6PD, HNF-4α and TAT) were varied, depending on the stage of hepatocyte maturation. The AFP expression that is usually presented in hepatic progenitors was detected at higher level than those of the primary human hepatocytes and HepG2 cells (Figure 1H). The observation of cytokeratin18 expression confirmed the differentiation of MSCs into endodermal tissue. Three major hepatocyte genes were up-regulated in hepatocyte-like cells, namely glucose-6-phosphate dehydrogenase (glucose metabolism), HNF-4α (liver development and maturation) and tyrosine aminotransferase (amino acid metabolism). The observation of all hepatocyte specific genes confirmed that the transformed MSCs could actually differentiate into functional hepatocyte-like cells.

Bottom Line: Their inducibility outperformed the classical HepG2 cells.The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes.The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkoknoi, Bangkok 10700, Thailand.

ABSTRACT

Background: The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450) induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies.

Results: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.

Conclusion: The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.

Show MeSH
Related in: MedlinePlus