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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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Related in: MedlinePlus

Immunofluorescent staining of hepatocyte-like cells and the primary hepatocyte. The immunofluorescent staining of CYP3A4, α-fetoprotein and HNF-4α in the cytoplasm of hepatocyte-like cells (A) and the primary hepatocyte (B) were demonstrated with the corresponding phase-contrast pictures over the same fields (10× objective lens). Essentially all hepatocyte-like cells carried all 3 proteins but their staining intensities reached only a half those of the primary hepatocytes.
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Figure 5: Immunofluorescent staining of hepatocyte-like cells and the primary hepatocyte. The immunofluorescent staining of CYP3A4, α-fetoprotein and HNF-4α in the cytoplasm of hepatocyte-like cells (A) and the primary hepatocyte (B) were demonstrated with the corresponding phase-contrast pictures over the same fields (10× objective lens). Essentially all hepatocyte-like cells carried all 3 proteins but their staining intensities reached only a half those of the primary hepatocytes.

Mentions: Using luminescent CYP450-specific substrates, we determined CYP1A1, CYP1A2, CYP2C9 and CYP3A4 isotype activities after the induction with either rifampicin or omeprazole in HepG2, MSC and hepatocyte-like cell. We observed end-point catalytic activity after incubating substrates to the cells using a luminometer. In hepatocyte-like cell, the activity of CYP1A1, CYP1A2 and CYP3A4 was increased to approximately 6-7 folds that of the untreated cell (Figure 4B, C, D). A mild increase in CYP2C9 activity (2 folds) was observed (Figure 4E). The activity of CYP1A2 and CYP3A4 in hepatocyte-like cells was already higher than those in HepG2. The primary hepatocyte provided the highest activities in all CYP450 isotypes. A significant increase in rifampicin-induced CYP3A4 activity was confirmed by the accumulation of CYP450 in hepatocyte-like cell as demonstrated by immunofluorescent staining (Figure 5A). The staining for AFP and hepatocyte nuclear factor 4α confirmed the identity of the hepatocyte-like cell. The corresponding staining to the primary hepatocyte served as the positive controls (Figure 5B). No significant induction was detected in untreated MSCs, but untreated HepG2 and untreated hepatocyte-like cell had low basal level of CYP1A1, CYP1A2, CYP2C9 and CYP3A4 activities.


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)

Immunofluorescent staining of hepatocyte-like cells and the primary hepatocyte. The immunofluorescent staining of CYP3A4, α-fetoprotein and HNF-4α in the cytoplasm of hepatocyte-like cells (A) and the primary hepatocyte (B) were demonstrated with the corresponding phase-contrast pictures over the same fields (10× objective lens). Essentially all hepatocyte-like cells carried all 3 proteins but their staining intensities reached only a half those of the primary hepatocytes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Immunofluorescent staining of hepatocyte-like cells and the primary hepatocyte. The immunofluorescent staining of CYP3A4, α-fetoprotein and HNF-4α in the cytoplasm of hepatocyte-like cells (A) and the primary hepatocyte (B) were demonstrated with the corresponding phase-contrast pictures over the same fields (10× objective lens). Essentially all hepatocyte-like cells carried all 3 proteins but their staining intensities reached only a half those of the primary hepatocytes.
Mentions: Using luminescent CYP450-specific substrates, we determined CYP1A1, CYP1A2, CYP2C9 and CYP3A4 isotype activities after the induction with either rifampicin or omeprazole in HepG2, MSC and hepatocyte-like cell. We observed end-point catalytic activity after incubating substrates to the cells using a luminometer. In hepatocyte-like cell, the activity of CYP1A1, CYP1A2 and CYP3A4 was increased to approximately 6-7 folds that of the untreated cell (Figure 4B, C, D). A mild increase in CYP2C9 activity (2 folds) was observed (Figure 4E). The activity of CYP1A2 and CYP3A4 in hepatocyte-like cells was already higher than those in HepG2. The primary hepatocyte provided the highest activities in all CYP450 isotypes. A significant increase in rifampicin-induced CYP3A4 activity was confirmed by the accumulation of CYP450 in hepatocyte-like cell as demonstrated by immunofluorescent staining (Figure 5A). The staining for AFP and hepatocyte nuclear factor 4α confirmed the identity of the hepatocyte-like cell. The corresponding staining to the primary hepatocyte served as the positive controls (Figure 5B). No significant induction was detected in untreated MSCs, but untreated HepG2 and untreated hepatocyte-like cell had low basal level of CYP1A1, CYP1A2, CYP2C9 and CYP3A4 activities.

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