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

Soichiro Ogaki, Mayu Morooka, Kaito Otera, Shoen Kume
2015 Scientific Reports  
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
more » ... 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. Pharmingen). Data were recorded using the BD FACS Diva Software program (BD Pharmingen) and analysed using the FlowJo program (Tree Star). Cell cycle analysis. Cells were dissociated with 0.25% trypsin. Dissociated cells were washed with PBS, then treated with Vybrant DyeCycle Ruby Stain (Life Technologies) for 30 min at 37 °C. Then, cells were analysed by FACSCanto. β-Ala-Lys(AMCA) intake. Cells were treated with 2.4 μ M β -Ala-Lys(AMCA) at 37 °C for 4 hours.
doi:10.1038/srep17297 pmid:26616277 pmcid:PMC4663490 fatcat:pv4p6oevizgcpibojx7odqwv7a