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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 Activin and DMSO further differentiated into immature gut epithelium.The ability of DE to differentiate into immature gut epithelium, triggered by a low dose of Activin and DMSO, was characterized. (A) The scheme for hiPS cell differentiation. After DE differentiation with 6.25 ng/ml Activin and DMSO, further incubation with BIO and DAPT induced differentiation into immature gut epithelium. (B–E) BIO and DAPT induced intestinal epithelium. (B) At day 6, SOX17+CDX2+ cells appeared. (C–E) At day 8, CDX2+ cells began to express VILLIN (C) and LGR5 (D,E), as shown by immunocytochemistry (C,D), and flow cytometry (E). Caco2 cells are used as a positive control for VILLIN expression. Scale bar; 50 μm.
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f4: DE induced by 6.25 ng/ml Activin and DMSO further differentiated into immature gut epithelium.The ability of DE to differentiate into immature gut epithelium, triggered by a low dose of Activin and DMSO, was characterized. (A) The scheme for hiPS cell differentiation. After DE differentiation with 6.25 ng/ml Activin and DMSO, further incubation with BIO and DAPT induced differentiation into immature gut epithelium. (B–E) BIO and DAPT induced intestinal epithelium. (B) At day 6, SOX17+CDX2+ cells appeared. (C–E) At day 8, CDX2+ cells began to express VILLIN (C) and LGR5 (D,E), as shown by immunocytochemistry (C,D), and flow cytometry (E). Caco2 cells are used as a positive control for VILLIN expression. Scale bar; 50 μm.

Mentions: To test the differentiation potency of the DE yielded by the present protocol, the DE was further differentiated into the intestinal epithelial lineage by addition of BIO and DAPT and cultured for another 2 to 4 days (Fig. 4A). At day 6, CDX2+SOX17+ posterior DE cells were observed (Fig. 4B, Supplementary Fig. S5). In mouse embryo, Villin is reportedly expressed in the cytoplasm in the immature gut epithelium at E10.58, prior to its later apical expression restricted in the enterocytes. Here, we used VILLIN as a marker for immature gut epithelium. The CDX2+SOX17+ posterior endoderm cells further gave rise to CDX2+VILLIN+ immature gut epithelial cells on day 8 (Fig. 4C, D8), although the expression level was lower than that of the Caco2 cells, a human intestinal cell line showing comparable VILLIN expression to that of the human intestine35. Villin expression in Caco2 cells was also observed in the cytoplasm with fibrous staining, as previously reported3637. Among these VILLIN+ cells, cells positive for LGR5, an intestinal stem cell marker, were observed (Fig. 4D,E). We also confirmed that the DE yielded by this protocol further differentiated into albumin- and CK19-expressing hepatoblasts32, PDX1-expressing pancreatic progenitor38 and SOX2-expressing anterior foregut cells39 (Supplementary Fig. S6), by following the reported protocol4041. These data suggested that this protocol is useful for derivation not only of posterior but also of anterior endodermal lineages.


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 Activin and DMSO further differentiated into immature gut epithelium.The ability of DE to differentiate into immature gut epithelium, triggered by a low dose of Activin and DMSO, was characterized. (A) The scheme for hiPS cell differentiation. After DE differentiation with 6.25 ng/ml Activin and DMSO, further incubation with BIO and DAPT induced differentiation into immature gut epithelium. (B–E) BIO and DAPT induced intestinal epithelium. (B) At day 6, SOX17+CDX2+ cells appeared. (C–E) At day 8, CDX2+ cells began to express VILLIN (C) and LGR5 (D,E), as shown by immunocytochemistry (C,D), and flow cytometry (E). Caco2 cells are used as a positive control for VILLIN expression. Scale bar; 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: DE induced by 6.25 ng/ml Activin and DMSO further differentiated into immature gut epithelium.The ability of DE to differentiate into immature gut epithelium, triggered by a low dose of Activin and DMSO, was characterized. (A) The scheme for hiPS cell differentiation. After DE differentiation with 6.25 ng/ml Activin and DMSO, further incubation with BIO and DAPT induced differentiation into immature gut epithelium. (B–E) BIO and DAPT induced intestinal epithelium. (B) At day 6, SOX17+CDX2+ cells appeared. (C–E) At day 8, CDX2+ cells began to express VILLIN (C) and LGR5 (D,E), as shown by immunocytochemistry (C,D), and flow cytometry (E). Caco2 cells are used as a positive control for VILLIN expression. Scale bar; 50 μm.
Mentions: To test the differentiation potency of the DE yielded by the present protocol, the DE was further differentiated into the intestinal epithelial lineage by addition of BIO and DAPT and cultured for another 2 to 4 days (Fig. 4A). At day 6, CDX2+SOX17+ posterior DE cells were observed (Fig. 4B, Supplementary Fig. S5). In mouse embryo, Villin is reportedly expressed in the cytoplasm in the immature gut epithelium at E10.58, prior to its later apical expression restricted in the enterocytes. Here, we used VILLIN as a marker for immature gut epithelium. The CDX2+SOX17+ posterior endoderm cells further gave rise to CDX2+VILLIN+ immature gut epithelial cells on day 8 (Fig. 4C, D8), although the expression level was lower than that of the Caco2 cells, a human intestinal cell line showing comparable VILLIN expression to that of the human intestine35. Villin expression in Caco2 cells was also observed in the cytoplasm with fibrous staining, as previously reported3637. Among these VILLIN+ cells, cells positive for LGR5, an intestinal stem cell marker, were observed (Fig. 4D,E). We also confirmed that the DE yielded by this protocol further differentiated into albumin- and CK19-expressing hepatoblasts32, PDX1-expressing pancreatic progenitor38 and SOX2-expressing anterior foregut cells39 (Supplementary Fig. S6), by following the reported protocol4041. These data suggested that this protocol is useful for derivation not only of posterior but also of anterior endodermal lineages.

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