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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

Characterization of immortalized MSC-derived hepatocyte-like cells. The TERT/Bmi-1-transduced MSCs had been differentiated into hepatocyte-like cells. The differentiated cell had polygonal shape, granulated cytoplasm and large nucleus (A). After the next passage, the cytoplasmic/nucleolus ratio was further decreased with loose intercellular attachment (B). The glycogen storage activity was demonstrated using Periodic Acid Schiff staining (PAS) with greater than 95% of the population were positive for glycogen (C). After the maintenance in DMEM/F12, 10% FBS in subsequent passages, cells were densely packed with closer intercellular attachment (D). After reaching confluence, cells formed duct-like structure (E). The life span of the immortalized hepatocyte-like cells was beyond 3 months (F) with active division.
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Figure 2: Characterization of immortalized MSC-derived hepatocyte-like cells. The TERT/Bmi-1-transduced MSCs had been differentiated into hepatocyte-like cells. The differentiated cell had polygonal shape, granulated cytoplasm and large nucleus (A). After the next passage, the cytoplasmic/nucleolus ratio was further decreased with loose intercellular attachment (B). The glycogen storage activity was demonstrated using Periodic Acid Schiff staining (PAS) with greater than 95% of the population were positive for glycogen (C). After the maintenance in DMEM/F12, 10% FBS in subsequent passages, cells were densely packed with closer intercellular attachment (D). After reaching confluence, cells formed duct-like structure (E). The life span of the immortalized hepatocyte-like cells was beyond 3 months (F) with active division.

Mentions: After finishing hepatic induction, the hepatocyte-like cells carried the expansion of several basic hepatocyte genes (Figure 1H) with a corresponding polygonal morphology (Figure 2A). Immortalized hepatocyte-like cells at the first passage were loosely attached to adjacent cells (Figure 2B). Up to 70-80% of the hepatocyte-like cells deposited glycogen, especially in densely populated area (PAS assay at passage 4, Figure 2C). After switching to 10% FBS, DMEM/F12 the intercellular attachment was denser with blurring of cell boundary (Figure 2D). At confluence, duct-like structure was observed (Figure 2E). The cells could maintain cell division beyond 3 months (Figure 2F) with sustainable hepatocyte function suitable for drug screening.


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)

Characterization of immortalized MSC-derived hepatocyte-like cells. The TERT/Bmi-1-transduced MSCs had been differentiated into hepatocyte-like cells. The differentiated cell had polygonal shape, granulated cytoplasm and large nucleus (A). After the next passage, the cytoplasmic/nucleolus ratio was further decreased with loose intercellular attachment (B). The glycogen storage activity was demonstrated using Periodic Acid Schiff staining (PAS) with greater than 95% of the population were positive for glycogen (C). After the maintenance in DMEM/F12, 10% FBS in subsequent passages, cells were densely packed with closer intercellular attachment (D). After reaching confluence, cells formed duct-like structure (E). The life span of the immortalized hepatocyte-like cells was beyond 3 months (F) with active division.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Characterization of immortalized MSC-derived hepatocyte-like cells. The TERT/Bmi-1-transduced MSCs had been differentiated into hepatocyte-like cells. The differentiated cell had polygonal shape, granulated cytoplasm and large nucleus (A). After the next passage, the cytoplasmic/nucleolus ratio was further decreased with loose intercellular attachment (B). The glycogen storage activity was demonstrated using Periodic Acid Schiff staining (PAS) with greater than 95% of the population were positive for glycogen (C). After the maintenance in DMEM/F12, 10% FBS in subsequent passages, cells were densely packed with closer intercellular attachment (D). After reaching confluence, cells formed duct-like structure (E). The life span of the immortalized hepatocyte-like cells was beyond 3 months (F) with active division.
Mentions: After finishing hepatic induction, the hepatocyte-like cells carried the expansion of several basic hepatocyte genes (Figure 1H) with a corresponding polygonal morphology (Figure 2A). Immortalized hepatocyte-like cells at the first passage were loosely attached to adjacent cells (Figure 2B). Up to 70-80% of the hepatocyte-like cells deposited glycogen, especially in densely populated area (PAS assay at passage 4, Figure 2C). After switching to 10% FBS, DMEM/F12 the intercellular attachment was denser with blurring of cell boundary (Figure 2D). At confluence, duct-like structure was observed (Figure 2E). The cells could maintain cell division beyond 3 months (Figure 2F) with sustainable hepatocyte function suitable for drug screening.

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