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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

DMSO promoted a low dose Activin-mediated decrease in Oct4 expression and apoptosis, and increased the G1 population and SMAD2 phosphorylation.The molecular mechanism underlying DMSO potentiation of low dose (6.25 ng/ml) Activin-induced DE differentiation was analysed. (A,B) DMSO decreased the proportion of OCT4-expressing cells induced by Activin at low dose, as analysed by immunocytochemistry. (C) DMSO arrested the cell cycle and decreased cell death, as revealed by flow cytometry to measure DNA quantities using DyeCycle. (D) Flow cytometric analysis of cleaved caspase3 revealed that DMSO inhibited apoptosis, triggered by a low dose of Activin. (E,F) Flow cytometric analysis of phosphorylated SMAD2. DMSO promoted SMAD2 phosphorylation, induced by a low dose Activin. (G) Western blot analysis. DMSO increased pSMAD2 in cells induced by Activin at a low dose. However, Activated β-catenin was unchanged. (H) Suppression of Activin signalling by SB231542 completely inhibited DE differentiation by DMSO, as shown by immunocytochemistry. (I) Wnt and BMP signalling were not required for DE differentiation by DMSO, as revealed by immuncytochemical analysis of SOX17. Scale bar; 50 μm.
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f3: DMSO promoted a low dose Activin-mediated decrease in Oct4 expression and apoptosis, and increased the G1 population and SMAD2 phosphorylation.The molecular mechanism underlying DMSO potentiation of low dose (6.25 ng/ml) Activin-induced DE differentiation was analysed. (A,B) DMSO decreased the proportion of OCT4-expressing cells induced by Activin at low dose, as analysed by immunocytochemistry. (C) DMSO arrested the cell cycle and decreased cell death, as revealed by flow cytometry to measure DNA quantities using DyeCycle. (D) Flow cytometric analysis of cleaved caspase3 revealed that DMSO inhibited apoptosis, triggered by a low dose of Activin. (E,F) Flow cytometric analysis of phosphorylated SMAD2. DMSO promoted SMAD2 phosphorylation, induced by a low dose Activin. (G) Western blot analysis. DMSO increased pSMAD2 in cells induced by Activin at a low dose. However, Activated β-catenin was unchanged. (H) Suppression of Activin signalling by SB231542 completely inhibited DE differentiation by DMSO, as shown by immunocytochemistry. (I) Wnt and BMP signalling were not required for DE differentiation by DMSO, as revealed by immuncytochemical analysis of SOX17. Scale bar; 50 μm.

Mentions: To understand the mechanism underlying the action of DMSO, OCT3/4 expression was examined at 0 h and 96 h after differentiation was triggered by Activin. At 96 h, DMSO treatment further decreased the proportion of OCT4-expressing cells (Fig. 3A,B), as determined by immunocytochemical analysis. Previously, DMSO was reported to induce cell cycle arrest31. We analysed the cell cycle stage of differentiating cells using DyeCycle for DNA content analysis in living cells. Cells differentiated by Activin in the presence of DMSO showed promotion of G1 arrest (Fig. 3C) and a decrease in the proportion of cleaved caspase3+ cells, indicating a decrease in apoptosis (Fig. 3D).


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)

DMSO promoted a low dose Activin-mediated decrease in Oct4 expression and apoptosis, and increased the G1 population and SMAD2 phosphorylation.The molecular mechanism underlying DMSO potentiation of low dose (6.25 ng/ml) Activin-induced DE differentiation was analysed. (A,B) DMSO decreased the proportion of OCT4-expressing cells induced by Activin at low dose, as analysed by immunocytochemistry. (C) DMSO arrested the cell cycle and decreased cell death, as revealed by flow cytometry to measure DNA quantities using DyeCycle. (D) Flow cytometric analysis of cleaved caspase3 revealed that DMSO inhibited apoptosis, triggered by a low dose of Activin. (E,F) Flow cytometric analysis of phosphorylated SMAD2. DMSO promoted SMAD2 phosphorylation, induced by a low dose Activin. (G) Western blot analysis. DMSO increased pSMAD2 in cells induced by Activin at a low dose. However, Activated β-catenin was unchanged. (H) Suppression of Activin signalling by SB231542 completely inhibited DE differentiation by DMSO, as shown by immunocytochemistry. (I) Wnt and BMP signalling were not required for DE differentiation by DMSO, as revealed by immuncytochemical analysis of SOX17. Scale bar; 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: DMSO promoted a low dose Activin-mediated decrease in Oct4 expression and apoptosis, and increased the G1 population and SMAD2 phosphorylation.The molecular mechanism underlying DMSO potentiation of low dose (6.25 ng/ml) Activin-induced DE differentiation was analysed. (A,B) DMSO decreased the proportion of OCT4-expressing cells induced by Activin at low dose, as analysed by immunocytochemistry. (C) DMSO arrested the cell cycle and decreased cell death, as revealed by flow cytometry to measure DNA quantities using DyeCycle. (D) Flow cytometric analysis of cleaved caspase3 revealed that DMSO inhibited apoptosis, triggered by a low dose of Activin. (E,F) Flow cytometric analysis of phosphorylated SMAD2. DMSO promoted SMAD2 phosphorylation, induced by a low dose Activin. (G) Western blot analysis. DMSO increased pSMAD2 in cells induced by Activin at a low dose. However, Activated β-catenin was unchanged. (H) Suppression of Activin signalling by SB231542 completely inhibited DE differentiation by DMSO, as shown by immunocytochemistry. (I) Wnt and BMP signalling were not required for DE differentiation by DMSO, as revealed by immuncytochemical analysis of SOX17. Scale bar; 50 μm.
Mentions: To understand the mechanism underlying the action of DMSO, OCT3/4 expression was examined at 0 h and 96 h after differentiation was triggered by Activin. At 96 h, DMSO treatment further decreased the proportion of OCT4-expressing cells (Fig. 3A,B), as determined by immunocytochemical analysis. Previously, DMSO was reported to induce cell cycle arrest31. We analysed the cell cycle stage of differentiating cells using DyeCycle for DNA content analysis in living cells. Cells differentiated by Activin in the presence of DMSO showed promotion of G1 arrest (Fig. 3C) and a decrease in the proportion of cleaved caspase3+ cells, indicating a decrease in apoptosis (Fig. 3D).

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