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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. MSCs and Bmi-1/hTERT-immortalized MSCs were visualized (A) after the 2nd passage. The attached cells appeared fibroblast-like, spindle morphology (B) at the 40th passage (12 months after isolation). The MSCs (hMSC) and the TERT-transduced MSCs (hTERT-MSC), or the double TERT/Bmi-1 transduced MSCs (BMI/hTERT-MSC) were studied for cumulative population doubling level (PDL) (C). Flow cytometry analysis confirmed the presence of CD90/CD105 in primary MSCs after isolation (D) and in BMI/hTERT-MSCs (E). All cells were depleted of CD35/CD45 hematopoietic stem cell markers (F). The endogenous and exogenous expression of Bmi-1, TERT in all cell types were studied using quantitative real-time PCR (G). The expression of hepatocyte-selective genes (i.e., albumin (ALB), α-fetoprotein (AFP), cytokeratin18 (CK18), glucose-6-phosphate dehydrogenase (G6PD), hepatocyte nuclear factor (HNF-4α), and tyrosine aminotransferase (TAT)) of BMI/hTERT-MSC after hepatic differentiation was presented as fold change over the untreated MSCs in comparison with HepG2 and the primary hepatocyte (H).
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Figure 1: Characterization of immortalized MSC. MSCs and Bmi-1/hTERT-immortalized MSCs were visualized (A) after the 2nd passage. The attached cells appeared fibroblast-like, spindle morphology (B) at the 40th passage (12 months after isolation). The MSCs (hMSC) and the TERT-transduced MSCs (hTERT-MSC), or the double TERT/Bmi-1 transduced MSCs (BMI/hTERT-MSC) were studied for cumulative population doubling level (PDL) (C). Flow cytometry analysis confirmed the presence of CD90/CD105 in primary MSCs after isolation (D) and in BMI/hTERT-MSCs (E). All cells were depleted of CD35/CD45 hematopoietic stem cell markers (F). The endogenous and exogenous expression of Bmi-1, TERT in all cell types were studied using quantitative real-time PCR (G). The expression of hepatocyte-selective genes (i.e., albumin (ALB), α-fetoprotein (AFP), cytokeratin18 (CK18), glucose-6-phosphate dehydrogenase (G6PD), hepatocyte nuclear factor (HNF-4α), and tyrosine aminotransferase (TAT)) of BMI/hTERT-MSC after hepatic differentiation was presented as fold change over the untreated MSCs in comparison with HepG2 and the primary hepatocyte (H).

Mentions: Cells isolated from bone marrow aspirate displayed a spindle shape upon reaching confluence (Figure 1A). The hTERT/Bmi-1-transduced MSC (BMI1/hTERT-MSC) still maintained fibroblast-like, spindle morphology at 40th passage (Figure 1B) with an exponential growth pattern (Figure 1C). The identity of the studied MSCs was confirmed by the presence of mesenchymal stem cell markers (CD90 and CD105, Figure 1D). MSCs that had gone through immortalization still contained similar levels of CD90 and CD105 (Figure 1E), but was virtually devoid of hematopoietic markers (CD34, CD45, Figure 1F) as determined by a flow cytometer.


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. MSCs and Bmi-1/hTERT-immortalized MSCs were visualized (A) after the 2nd passage. The attached cells appeared fibroblast-like, spindle morphology (B) at the 40th passage (12 months after isolation). The MSCs (hMSC) and the TERT-transduced MSCs (hTERT-MSC), or the double TERT/Bmi-1 transduced MSCs (BMI/hTERT-MSC) were studied for cumulative population doubling level (PDL) (C). Flow cytometry analysis confirmed the presence of CD90/CD105 in primary MSCs after isolation (D) and in BMI/hTERT-MSCs (E). All cells were depleted of CD35/CD45 hematopoietic stem cell markers (F). The endogenous and exogenous expression of Bmi-1, TERT in all cell types were studied using quantitative real-time PCR (G). The expression of hepatocyte-selective genes (i.e., albumin (ALB), α-fetoprotein (AFP), cytokeratin18 (CK18), glucose-6-phosphate dehydrogenase (G6PD), hepatocyte nuclear factor (HNF-4α), and tyrosine aminotransferase (TAT)) of BMI/hTERT-MSC after hepatic differentiation was presented as fold change over the untreated MSCs in comparison with HepG2 and the primary hepatocyte (H).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Characterization of immortalized MSC. MSCs and Bmi-1/hTERT-immortalized MSCs were visualized (A) after the 2nd passage. The attached cells appeared fibroblast-like, spindle morphology (B) at the 40th passage (12 months after isolation). The MSCs (hMSC) and the TERT-transduced MSCs (hTERT-MSC), or the double TERT/Bmi-1 transduced MSCs (BMI/hTERT-MSC) were studied for cumulative population doubling level (PDL) (C). Flow cytometry analysis confirmed the presence of CD90/CD105 in primary MSCs after isolation (D) and in BMI/hTERT-MSCs (E). All cells were depleted of CD35/CD45 hematopoietic stem cell markers (F). The endogenous and exogenous expression of Bmi-1, TERT in all cell types were studied using quantitative real-time PCR (G). The expression of hepatocyte-selective genes (i.e., albumin (ALB), α-fetoprotein (AFP), cytokeratin18 (CK18), glucose-6-phosphate dehydrogenase (G6PD), hepatocyte nuclear factor (HNF-4α), and tyrosine aminotransferase (TAT)) of BMI/hTERT-MSC after hepatic differentiation was presented as fold change over the untreated MSCs in comparison with HepG2 and the primary hepatocyte (H).
Mentions: Cells isolated from bone marrow aspirate displayed a spindle shape upon reaching confluence (Figure 1A). The hTERT/Bmi-1-transduced MSC (BMI1/hTERT-MSC) still maintained fibroblast-like, spindle morphology at 40th passage (Figure 1B) with an exponential growth pattern (Figure 1C). The identity of the studied MSCs was confirmed by the presence of mesenchymal stem cell markers (CD90 and CD105, Figure 1D). MSCs that had gone through immortalization still contained similar levels of CD90 and CD105 (Figure 1E), but was virtually devoid of hematopoietic markers (CD34, CD45, Figure 1F) as determined by a flow cytometer.

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