Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

doi:10.1038/srep16479

. 2015 Nov 12:5:16479.

doi: 10.1038/srep16479.

Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

Tatsuya Ozawa^{1

2}, Kazuo Takayama^{1

2

3}, Ryota Okamoto^{1

2}, Ryosuke Negoro¹, Fuminori Sakurai^{1

4}, Masashi Tachibana¹, Kenji Kawabata^{5

6}, Hiroyuki Mizuguchi^{1

2

3

7}

Affiliations

¹ Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
² Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
³ iPS Cell-based Research Project on Hepatic Toxicity and Metabolism, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
⁴ Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871, Japan.
⁵ Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
⁶ Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
⁷ Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan.

PMID: 26559489
PMCID: PMC4642303
DOI: 10.1038/srep16479

Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

Tatsuya Ozawa et al. Sci Rep. 2015.

. 2015 Nov 12:5:16479.

doi: 10.1038/srep16479.

Authors

Tatsuya Ozawa^{1

2}, Kazuo Takayama^{1

2

3}, Ryota Okamoto^{1

2}, Ryosuke Negoro¹, Fuminori Sakurai^{1

4}, Masashi Tachibana¹, Kenji Kawabata^{5

6}, Hiroyuki Mizuguchi^{1

2

3

7}

Affiliations

¹ Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
² Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
³ iPS Cell-based Research Project on Hepatic Toxicity and Metabolism, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
⁴ Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871, Japan.
⁵ Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan.
⁶ Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan.
⁷ Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871, Japan.

PMID: 26559489
PMCID: PMC4642303
DOI: 10.1038/srep16479

Abstract

Enterocytes play an important role in drug absorption and metabolism. However, a widely used enterocyte model, Caco-2 cell, has difficulty in evaluating both drug absorption and metabolism because the expression levels of some drug absorption and metabolism-related genes in these cells differ largely from those of human enterocytes. Therefore, we decided to generate the enterocyte-like cells from human induced pluripotent stem (iPS) cells (hiPS-ELCs), which are applicable to drug absorption and metabolism studies. The efficiency of enterocyte differentiation from human iPS cells was significantly improved by using EGF, SB431542, and Wnt3A, and extending the differentiation period. The gene expression levels of cytochrome P450 3A4 (CYP3A4) and peptide transporter 1 in the hiPS-ELCs were higher than those in Caco-2 cells. In addition, CYP3A4 expression in the hiPS-ELCs was induced by treatment with 1, 25-dihydroxyvitamin D3 or rifampicin, which are known to induce CYP3A4 expression, indicating that the hiPS-ELCs have CYP3A4 induction potency. Moreover, the transendothelial electrical resistance (TEER) value of the hiPS-ELC monolayer was approximately 240 Ω*cm(2), suggesting that the hiPS-ELC monolayer could form a barrier. In conclusion, we succeeded in establishing an enterocyte model from human iPS cells which have potential to be applied for drug absorption and metabolism studies.

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Figures

**Figure 1. Promotion of enterocyte differentiation by combination treatment with three compounds and differentiation period extension.**
(A) The procedure for enterocyte differentiation from human iPS cells by treatment of compounds is presented. From day 19 to 24, the human iPS-derived intestinal cells were treated with the test compounds. (B) The gene expression levels of the enterocyte marker *ANPEP* in the test compound-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis on day 24. On the y axis, the gene expression levels in “Control (untreated hiPS-ELCs)” were taken as 1.0. (C) On day 24, the gene expression levels of the enterocyte marker *VILLIN* in the PMA, Wortmannin, SB431542, EGF or Wnt3A-treated human iPS-derived intestinal cells were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in “Control” were taken as 1.0. (D) Temporal gene expression levels of *ANPEP* in the human iPS cell-derived intestinal cells (day 24, 29, and 34) were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Adult Intestine were taken as 1.0. (E) The modified enterocyte differentiation protocol is illustrated. (F) A morphological image of human iPS-derived enterocyte-like cells is represented. Scale bar represents 100 μm. (G) Human iPS cell-derived enterocyte-like cells were assayed for the expression of intestinal marker CDX2 (Red) by immunohistochemistry. Nuclei were stained with DAPI (Blue). Scale bar represents 40 μm. (H) Percentages of VILLIN-positive cells in the SB431542, EGF, and Wnt3A-treated enterocyte-like cells were analyzed by flow cytometry analysis on day 24 and 34. Data are represented as the means ± S.E. (n ≧ 3). Statistical analysis was performed using the unpaired two-tailed student’s t-test. *P < 0.05.

**Figure 2. Expression analyses of intestinal transporters in the human iPS-derived enterocyte-like cells.**
Human iPS-derived enterocyte-like cells (hiPS-ELCs) were differentiated according to the protocol described in Fig. 1E. (A) The gene expression levels of *PEPT1* in Caco-2 cells, hiPS-ELCs, and Adult Intestine were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Caco-2 cells were taken as 1.0. (B) The hiPS-ELCs were assayed for expression of PEPT1 (Red) by immunohistochemistry. Nuclei were stained with DAPI (Blue). Scale bars represent 40 μm. (**C,D**) The gene expression levels of apical transporters (C) and basolateral transporters (D) in the Caco-2 cells, hiPS-ELCs, and Adult Intestine were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels in Adult Intestine were taken as 1.0. Data are represented as the means ± S.E. (n ≧ 3). Statistical analysis was performed using the unpaired two-tailed student’s t-test. *P < 0.05. (E) After the enterocyte differentiation, the hiPS-ELCs were treated with or without 100 μM captopril for 24 hr. The hiPS-ELCs were treated with D-Ala-Leu-Lys-AMCA (blue) for 4 hr. After the uptake of D-Ala-Leu-Lys-AMCA, the cells were fixed, and stained with anti-VILLIN antibodies (green). Scale bars represent 50 μm.

**Figure 3. CYP3A4 expression level and induction potency in the human iPS-derived enterocyte-like cells.**
Human iPS cell-derived enterocyte-like cells (hiPS-ELCs) were differentiated according to the protocol described in Fig. 1E. (A) The gene expression levels of *CYP3A4* in Caco-2 cells, hiPS-ELCs, and Adult Intestine were measured by real-time RT-PCR analysis. On the y axis, the gene expression level in Caco-2 cells was taken as 1.0. (B) The CYP3A4 protein expression levels in the Caco-2 cells and hiPS-ELCs were measured by western blotting analysis. (C) The CYP3A4 activity levels in the Caco-2 cells and hiPS-ELCs were measured by CYP3A4-Glo assay kit. (**D,E**) The CYP3A4 induction potency was examined in the hiPS-ELCs and Caco-2 cells. The hiPS-ELCs and Caco-2 cells were treated with 100 nM 1,25-dihidroxyvitamin D3 (VD3) (D) or 20 μM rifampicin (RIF) (E) for 24 hr or 48 hr, respectively, and then the gene expression levels of *CYP3A4* were measured by real-time RT-PCR analysis. On the y axis, the gene expression levels of *CYP3A4* in Caco-2 cells treated with DMSO (Solvent) were taken as 1.0. (**F,G**) The gene expression levels of VDR (F) and PXR (G) in the hiPS-ELCs and Caco-2 cells were examined by real-time RT-PCR analysis. On the y axis, the gene expression levels of VDR and PXR in Caco-2 cells were taken as 1.0. The data are represented as the means ± S.E. (n ≧ 3).

**Figure 4. Analysis of barrier formation capacity in the human iPS-derived enterocyte-like cell monolayers.**
(A) The gene expression levels of *ZO-1* in Caco-2 cells, human iPS-derived enterocyte-like cells (hiPS-ELCs) and Adult Intestine were examined by real-time RT-PCR analysis. On the y axis, the gene expression levels in Caco-2 cells were taken as 1.0. (B) Immunostaining analysis of ZO-1 (Green) in the hiPS-ELCs and Caco-2 cells was performed. Nuclei were stained with DAPI (Blue). Scale bars represent 40 μm. (C) TEER values of Caco-2 cell monolayers and hiPS-ELC monolayers were measured by Millicell-ERS. All data are represented as the means ± S.E. (n ≧ 3).

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References

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1. Giacomini K. M. et al. Membrane transporters in drug development. Nat Rev Drug Discov 9, 215–236 (2010). - PMC - PubMed
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[1] Frank R. & Hargreaves R. Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2, 566–580 (2003). - PubMed

[2] Frank R. & Hargreaves R. Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2, 566–580 (2003). - PubMed

[3] Giacomini K. M. et al. Membrane transporters in drug development. Nat Rev Drug Discov 9, 215–236 (2010). - PMC - PubMed

[4] Giacomini K. M. et al. Membrane transporters in drug development. Nat Rev Drug Discov 9, 215–236 (2010). - PMC - PubMed

[5] Doherty M. M. & Charman W. N. The mucosa of the small intestine: how clinically relevant as an organ of drug metabolism ? Clin Pharmacokinet 41, 235–253 (2002). - PubMed

[6] Doherty M. M. & Charman W. N. The mucosa of the small intestine: how clinically relevant as an organ of drug metabolism ? Clin Pharmacokinet 41, 235–253 (2002). - PubMed

[7] Leibach F. H. & Ganapathy V. Peptide transporters in the intestine and the kidney. Annu Rev Nutr 16, 99–119 (1996). - PubMed

[8] Leibach F. H. & Ganapathy V. Peptide transporters in the intestine and the kidney. Annu Rev Nutr 16, 99–119 (1996). - PubMed

[9] Kolars J. C., Awni W. M., Merion R. M. & Watkins P. B. First-pass metabolism of cyclosporin by the gut. Lancet 338, 1488–1490 (1991). - PubMed

[10] Kolars J. C., Awni W. M., Merion R. M. & Watkins P. B. First-pass metabolism of cyclosporin by the gut. Lancet 338, 1488–1490 (1991). - PubMed

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Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

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Generation of enterocyte-like cells from human induced pluripotent stem cells for drug absorption and metabolism studies in human small intestine

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