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A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

Ogaki S, Morooka M, Otera K, Kume S - Sci Rep (2015)

Bottom Line: We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells.This increased differentiation into CDX2 + SOX17 + DE cells.The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.

ABSTRACT
The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes.

No MeSH data available.


Related in: MedlinePlus

DE induced by 6.25 ng/ml of Activin and DMSO further differentiated into enterocytes.The ability of DE obtained by treatment with a low dose of Activin and DMSO to differentiate into enterocytes was characterized. (A) hiPS cell-derived cells expressed the intestinal transporter, PEPT1, at day 12. (B) Intestinal epithelium formed a dome-like structure, which resembles enterocyte morphology, at day 15. (C–E) These cells showed alkali phosphatase activity (ALP) (C) and expressed enterocyte markers, by immunocytochemistry (D) or RT-PCR (E). (G) β-Lys-Ala (AMCA) intake assayed at day 15. For primer sequences used, see Supplementary Table S1. The gels were cropped; for full gel images, see Supplementary Fig. 6. Scale bar; (A) 50 μm, (B–D) 100 μm. FI; Fetal Intestine. AI; Adult Intestine.
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f5: DE induced by 6.25 ng/ml of Activin and DMSO further differentiated into enterocytes.The ability of DE obtained by treatment with a low dose of Activin and DMSO to differentiate into enterocytes was characterized. (A) hiPS cell-derived cells expressed the intestinal transporter, PEPT1, at day 12. (B) Intestinal epithelium formed a dome-like structure, which resembles enterocyte morphology, at day 15. (C–E) These cells showed alkali phosphatase activity (ALP) (C) and expressed enterocyte markers, by immunocytochemistry (D) or RT-PCR (E). (G) β-Lys-Ala (AMCA) intake assayed at day 15. For primer sequences used, see Supplementary Table S1. The gels were cropped; for full gel images, see Supplementary Fig. 6. Scale bar; (A) 50 μm, (B–D) 100 μm. FI; Fetal Intestine. AI; Adult Intestine.

Mentions: Next, we examined whether immature gut epithelial cells could differentiate into absorptive enterocytes. At day 12, the differentiated cells expressed an enterocyte-specific transporter, PEPT1 (Fig. 5A). At day 15, cells exhibited a dome-like structure, which is a characteristic feature of enterocytes (Fig. 5B), began to express enterocyte markers such as MDR1, MRP3, OATP2B1, EAAC1, TAUT, CYP3A4. CYP2E1, and CES242 (Fig. 5C–E, Supplementary Fig. S6), and exhibited alkali phosphatase (ALP) activity (Fig. 5C). ALP activity is known as a functional assay for enterocytes43. We also confirmed that hiPS cells-derived enterocytes showed PEPT1 activities by assaying the intake of β-Ala-Lys (AMCA), a fluorescent dipeptides44 (Fig. 5G). These results therefore indicated that the DE cells induced using a low concentration Activin were potent to differentiate into enterocyte-like cells.


A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

Ogaki S, Morooka M, Otera K, Kume S - Sci Rep (2015)

DE induced by 6.25 ng/ml of Activin and DMSO further differentiated into enterocytes.The ability of DE obtained by treatment with a low dose of Activin and DMSO to differentiate into enterocytes was characterized. (A) hiPS cell-derived cells expressed the intestinal transporter, PEPT1, at day 12. (B) Intestinal epithelium formed a dome-like structure, which resembles enterocyte morphology, at day 15. (C–E) These cells showed alkali phosphatase activity (ALP) (C) and expressed enterocyte markers, by immunocytochemistry (D) or RT-PCR (E). (G) β-Lys-Ala (AMCA) intake assayed at day 15. For primer sequences used, see Supplementary Table S1. The gels were cropped; for full gel images, see Supplementary Fig. 6. Scale bar; (A) 50 μm, (B–D) 100 μm. FI; Fetal Intestine. AI; Adult Intestine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4663490&req=5

f5: DE induced by 6.25 ng/ml of Activin and DMSO further differentiated into enterocytes.The ability of DE obtained by treatment with a low dose of Activin and DMSO to differentiate into enterocytes was characterized. (A) hiPS cell-derived cells expressed the intestinal transporter, PEPT1, at day 12. (B) Intestinal epithelium formed a dome-like structure, which resembles enterocyte morphology, at day 15. (C–E) These cells showed alkali phosphatase activity (ALP) (C) and expressed enterocyte markers, by immunocytochemistry (D) or RT-PCR (E). (G) β-Lys-Ala (AMCA) intake assayed at day 15. For primer sequences used, see Supplementary Table S1. The gels were cropped; for full gel images, see Supplementary Fig. 6. Scale bar; (A) 50 μm, (B–D) 100 μm. FI; Fetal Intestine. AI; Adult Intestine.
Mentions: Next, we examined whether immature gut epithelial cells could differentiate into absorptive enterocytes. At day 12, the differentiated cells expressed an enterocyte-specific transporter, PEPT1 (Fig. 5A). At day 15, cells exhibited a dome-like structure, which is a characteristic feature of enterocytes (Fig. 5B), began to express enterocyte markers such as MDR1, MRP3, OATP2B1, EAAC1, TAUT, CYP3A4. CYP2E1, and CES242 (Fig. 5C–E, Supplementary Fig. S6), and exhibited alkali phosphatase (ALP) activity (Fig. 5C). ALP activity is known as a functional assay for enterocytes43. We also confirmed that hiPS cells-derived enterocytes showed PEPT1 activities by assaying the intake of β-Ala-Lys (AMCA), a fluorescent dipeptides44 (Fig. 5G). These results therefore indicated that the DE cells induced using a low concentration Activin were potent to differentiate into enterocyte-like cells.

Bottom Line: We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells.This increased differentiation into CDX2 + SOX17 + DE cells.The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan.

ABSTRACT
The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes.

No MeSH data available.


Related in: MedlinePlus