Limits...
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

Dimethyl sulfoxide (DMSO) promoted endodermal differentiation from 201B7 hiPSCs.Human 201B7 iPS cells were differentiated into definitive endoderm (DE) at a graded concentration of DMSO, and the resultant DE was characterized. (A) Scheme for hiPS cell differentiation. 201B7, hiPS cells, were differentiated into DE cells with Activin and DMSO. Then, BIO and DAPT were added to induce posterior definitive endoderm (Post. DE). (B,C) Flow cytometry analysis. Graded concentrations of DMSO were tested for DE differentiation, and 0.8% DMSO had the strongest effect for potentiating differentiation into CD117+CXCR4+ cells (B). A representative result at 0.8% DMSO is shown (C). (D–F) Immunocytochemical analysis. FOXA2 (green), SOX17 (red), CDX2 (green), DAPI (blue). (D,E) DMSO treatment potentiated DE differentiation, and increased the proportion of FOXA2+SOX17+ endodermal cells (D) and CDX2+SOX17+ posterior endoderm (E). (F) Activin was required for DE (indicated by SOX17+ staining) differentiation. DMSO alone could not induce DE differentiation from hiPS cells. Scale bar; 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663490&req=5

f1: Dimethyl sulfoxide (DMSO) promoted endodermal differentiation from 201B7 hiPSCs.Human 201B7 iPS cells were differentiated into definitive endoderm (DE) at a graded concentration of DMSO, and the resultant DE was characterized. (A) Scheme for hiPS cell differentiation. 201B7, hiPS cells, were differentiated into DE cells with Activin and DMSO. Then, BIO and DAPT were added to induce posterior definitive endoderm (Post. DE). (B,C) Flow cytometry analysis. Graded concentrations of DMSO were tested for DE differentiation, and 0.8% DMSO had the strongest effect for potentiating differentiation into CD117+CXCR4+ cells (B). A representative result at 0.8% DMSO is shown (C). (D–F) Immunocytochemical analysis. FOXA2 (green), SOX17 (red), CDX2 (green), DAPI (blue). (D,E) DMSO treatment potentiated DE differentiation, and increased the proportion of FOXA2+SOX17+ endodermal cells (D) and CDX2+SOX17+ posterior endoderm (E). (F) Activin was required for DE (indicated by SOX17+ staining) differentiation. DMSO alone could not induce DE differentiation from hiPS cells. Scale bar; 50 μm.

Mentions: Previously, in an attempt to establish a screening procedure for chemicals that potentiate DE differentiation, we found that the widely used solvent DMSO itself produced potentiation effects. In this study, we used a hiPS cell line, 201B7, to evaluate DMSO-mediated potentiation of DE differentiation. The experimental scheme is shown in Fig. 1A. 201B7 cells were triggered to adopt DE differentiation in media containing Activin (100 ng/ml) for 4 days, in the presence of graded concentrations of DMSO (0–1.6%), then exposed simultaneously to 6-bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase (GSK)-3β inhibitor, and DAPT, a γ-secretase inhibitor, gave rise to CDX2-expressing posterior DE 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)

Dimethyl sulfoxide (DMSO) promoted endodermal differentiation from 201B7 hiPSCs.Human 201B7 iPS cells were differentiated into definitive endoderm (DE) at a graded concentration of DMSO, and the resultant DE was characterized. (A) Scheme for hiPS cell differentiation. 201B7, hiPS cells, were differentiated into DE cells with Activin and DMSO. Then, BIO and DAPT were added to induce posterior definitive endoderm (Post. DE). (B,C) Flow cytometry analysis. Graded concentrations of DMSO were tested for DE differentiation, and 0.8% DMSO had the strongest effect for potentiating differentiation into CD117+CXCR4+ cells (B). A representative result at 0.8% DMSO is shown (C). (D–F) Immunocytochemical analysis. FOXA2 (green), SOX17 (red), CDX2 (green), DAPI (blue). (D,E) DMSO treatment potentiated DE differentiation, and increased the proportion of FOXA2+SOX17+ endodermal cells (D) and CDX2+SOX17+ posterior endoderm (E). (F) Activin was required for DE (indicated by SOX17+ staining) differentiation. DMSO alone could not induce DE differentiation from hiPS cells. Scale bar; 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Dimethyl sulfoxide (DMSO) promoted endodermal differentiation from 201B7 hiPSCs.Human 201B7 iPS cells were differentiated into definitive endoderm (DE) at a graded concentration of DMSO, and the resultant DE was characterized. (A) Scheme for hiPS cell differentiation. 201B7, hiPS cells, were differentiated into DE cells with Activin and DMSO. Then, BIO and DAPT were added to induce posterior definitive endoderm (Post. DE). (B,C) Flow cytometry analysis. Graded concentrations of DMSO were tested for DE differentiation, and 0.8% DMSO had the strongest effect for potentiating differentiation into CD117+CXCR4+ cells (B). A representative result at 0.8% DMSO is shown (C). (D–F) Immunocytochemical analysis. FOXA2 (green), SOX17 (red), CDX2 (green), DAPI (blue). (D,E) DMSO treatment potentiated DE differentiation, and increased the proportion of FOXA2+SOX17+ endodermal cells (D) and CDX2+SOX17+ posterior endoderm (E). (F) Activin was required for DE (indicated by SOX17+ staining) differentiation. DMSO alone could not induce DE differentiation from hiPS cells. Scale bar; 50 μm.
Mentions: Previously, in an attempt to establish a screening procedure for chemicals that potentiate DE differentiation, we found that the widely used solvent DMSO itself produced potentiation effects. In this study, we used a hiPS cell line, 201B7, to evaluate DMSO-mediated potentiation of DE differentiation. The experimental scheme is shown in Fig. 1A. 201B7 cells were triggered to adopt DE differentiation in media containing Activin (100 ng/ml) for 4 days, in the presence of graded concentrations of DMSO (0–1.6%), then exposed simultaneously to 6-bromoindirubin-3′-oxime (BIO), a glycogen synthase kinase (GSK)-3β inhibitor, and DAPT, a γ-secretase inhibitor, gave rise to CDX2-expressing posterior DE 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