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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 allowed a low concentration of Activin to trigger 201B7 cells to differentiate into DE.The concentration of Activin required to induce DE differentiation from 201B7 iPS cells in the presence of DMSO was examined. (A,B) In the presence of DMSO, Activin can induce DE differentiation at much lower concentrations, as indicated by (A,B) immunocytochemical analysis of SOX17 (red) staining, (C) cell morphology, (D) flow cytometry analysis of CD117 and CXCR4. (E) DMSO is more effective than CHIR99021 or Wnt3a to promote Activin (at 100 or 6.25 ng/ml) mediated DE differentiation. FOXA2+SOX17+ cells were quantified and compared. A100, Activin at 100 ng/ml; A6.25, Activin at 6.25 ng/ml. Scale bar; (A,B) 100 μm, (C) 20 μm.
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f2: DMSO allowed a low concentration of Activin to trigger 201B7 cells to differentiate into DE.The concentration of Activin required to induce DE differentiation from 201B7 iPS cells in the presence of DMSO was examined. (A,B) In the presence of DMSO, Activin can induce DE differentiation at much lower concentrations, as indicated by (A,B) immunocytochemical analysis of SOX17 (red) staining, (C) cell morphology, (D) flow cytometry analysis of CD117 and CXCR4. (E) DMSO is more effective than CHIR99021 or Wnt3a to promote Activin (at 100 or 6.25 ng/ml) mediated DE differentiation. FOXA2+SOX17+ cells were quantified and compared. A100, Activin at 100 ng/ml; A6.25, Activin at 6.25 ng/ml. Scale bar; (A,B) 100 μm, (C) 20 μm.

Mentions: We then examined whether DMSO enabled a low concentration of Activin to induce DE cells from hiPS cells. Activin was used at 100 ng/ml for DE differentiation of human pluripotent stem cells121519293132. We performed differentiation with graded concentrations of Activin (0 ng/ml–100 ng/ml) in the presence or absence of 0.8% DMSO. After triggering DE differentiation of hiPS cells with DE differentiation medium, the number of SOX17+ cells increased in an Activin concentration-dependent manner (Fig. 2A). Without DMSO, very few SOX17+ cells appeared at low Activin concentrations (Fig. 2A). In contrast, in the presence of 0.8% DMSO, SOX17+ cells appeared at low concentrations of Activin (from 0.78 ng/ml) and reached a plateau at 6.25 ng/ml of Activin (Fig. 2B). The induced DE, showing endodermal morphology, was positive for both CD117 and CXCR4 (Fig. 2C,D). Potentiation of DE differentiation by DMSO at low concentration of Activin was also observed with Toe hiPS cells (Supplementary Fig. S2). Next, we divided the 96-h differentiation period into two time windows (the first and second halves to examine the time period of DMSO exposure required (Supplementary Fig. S3). The highest proportion of SOX17+ cells was observed when DMSO was added throughout the 96 h of differentiation. Therefore, continuous exposure to DMSO was required to potentiate DE differentiation (Supplementary Fig. S3).


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 allowed a low concentration of Activin to trigger 201B7 cells to differentiate into DE.The concentration of Activin required to induce DE differentiation from 201B7 iPS cells in the presence of DMSO was examined. (A,B) In the presence of DMSO, Activin can induce DE differentiation at much lower concentrations, as indicated by (A,B) immunocytochemical analysis of SOX17 (red) staining, (C) cell morphology, (D) flow cytometry analysis of CD117 and CXCR4. (E) DMSO is more effective than CHIR99021 or Wnt3a to promote Activin (at 100 or 6.25 ng/ml) mediated DE differentiation. FOXA2+SOX17+ cells were quantified and compared. A100, Activin at 100 ng/ml; A6.25, Activin at 6.25 ng/ml. Scale bar; (A,B) 100 μm, (C) 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: DMSO allowed a low concentration of Activin to trigger 201B7 cells to differentiate into DE.The concentration of Activin required to induce DE differentiation from 201B7 iPS cells in the presence of DMSO was examined. (A,B) In the presence of DMSO, Activin can induce DE differentiation at much lower concentrations, as indicated by (A,B) immunocytochemical analysis of SOX17 (red) staining, (C) cell morphology, (D) flow cytometry analysis of CD117 and CXCR4. (E) DMSO is more effective than CHIR99021 or Wnt3a to promote Activin (at 100 or 6.25 ng/ml) mediated DE differentiation. FOXA2+SOX17+ cells were quantified and compared. A100, Activin at 100 ng/ml; A6.25, Activin at 6.25 ng/ml. Scale bar; (A,B) 100 μm, (C) 20 μm.
Mentions: We then examined whether DMSO enabled a low concentration of Activin to induce DE cells from hiPS cells. Activin was used at 100 ng/ml for DE differentiation of human pluripotent stem cells121519293132. We performed differentiation with graded concentrations of Activin (0 ng/ml–100 ng/ml) in the presence or absence of 0.8% DMSO. After triggering DE differentiation of hiPS cells with DE differentiation medium, the number of SOX17+ cells increased in an Activin concentration-dependent manner (Fig. 2A). Without DMSO, very few SOX17+ cells appeared at low Activin concentrations (Fig. 2A). In contrast, in the presence of 0.8% DMSO, SOX17+ cells appeared at low concentrations of Activin (from 0.78 ng/ml) and reached a plateau at 6.25 ng/ml of Activin (Fig. 2B). The induced DE, showing endodermal morphology, was positive for both CD117 and CXCR4 (Fig. 2C,D). Potentiation of DE differentiation by DMSO at low concentration of Activin was also observed with Toe hiPS cells (Supplementary Fig. S2). Next, we divided the 96-h differentiation period into two time windows (the first and second halves to examine the time period of DMSO exposure required (Supplementary Fig. S3). The highest proportion of SOX17+ cells was observed when DMSO was added throughout the 96 h of differentiation. Therefore, continuous exposure to DMSO was required to potentiate DE differentiation (Supplementary Fig. S3).

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