Generation and Purification of Definitive Endoderm Cells Generated from Pluripotent Stem Cells

Abstract

Differentiation of pluripotent stem cells into cells of the definitive endoderm requires an in vitro gastrulation event. Differentiated somatic cells derived from this germ layer may then be used for cell replacement therapies of degenerative diseases of the liver, lung, and pancreas. Here we describe an endoderm differentiation protocol, which initiates the differentiation from a defined cell number of dispersed single cells and reliably yields in >70-80 % endoderm-committed cells in a short 5-day treatment regimen.

Anterior–Posterior Patterning of Definitive Endoderm Generated from Human Embryonic Stem Cells Depends on the Differential Signaling of Retinoic Acid, Wnt-, and BMP-Signaling

“…The MACS enrichment resulted in a substantially more viable and greater cell population. Thus, we opted for the MACS technique for all further experiments since this technique resulted in a proper purity and good viability after reseeding for further experimentation .…”

“…The MACS enrichment resulted in a substantially more viable and greater cell population. Thus, we opted for the MACS technique for all further experiments since this technique resulted in a proper purity and good viability after reseeding for further experimentation [20,21]. DE-cells were MACS-sorted via the surface antigen CXCR4 to exclude CXCR4-negative cells from further differentiation, which did not adequately respond to the directed differentiation procedure [20,21].…”

“…Thus, we opted for the MACS technique for all further experiments since this technique resulted in a proper purity and good viability after reseeding for further experimentation [20,21]. DE-cells were MACS-sorted via the surface antigen CXCR4 to exclude CXCR4-negative cells from further differentiation, which did not adequately respond to the directed differentiation procedure [20,21]. Differentiation for 4 days with a reliable endoderm differentiation protocol [4] yielded approximately 85% CXCR4-positive DE-cells ( Fig.…”

miRNome Profiling of Purified Endoderm and Mesoderm Differentiated from hESCs Reveals Functions of miR-483-3p and miR-1263 for Cell-Fate Decisions

“…Specific expression of surface proteins was used to purify the desired cell populations from heterogeneously composed differentiation cultures. Pure DE cells were obtained using the specific DE marker CXCR4 ( Davenport et al., 2016 , Diekmann and Naujok, 2016 , Kroon et al., 2008 , Naujok et al., 2014 ). In contrast to a recent publication ( Wang et al., 2011 ), CD49e could not be validated as truly DE specific because it was also detectable upon ME differentiation.…”

“…HUES8 and HES3 cells were cultivated under feeder-free conditions and the differentiation was induced from a defined number of seeded singe cells as described earlier ( Diekmann et al., 2015 , Diekmann and Naujok, 2016 , Naujok et al., 2014 ). Advanced RPMI-1640 supplemented with 1% penicillin-streptomycin, 1% Glutamax, and 0.2% fetal bovine serum was used as base medium and for randomized differentiation.…”