SOX17 and CXCR4 are markers of definitive endoderm systematically used in studies of endoderm differentiation [28] [30].
First, the markers of definitive endoderm (Sox 17 and Foxa 2) were quantified in the different steps of the protocol.
Q-PCR (Figure 3B) and immunostaining (Figure 3C) analyses showed that these cells expressed markers of definitive endoderm including CXCR4, Goosecoid, Mixl1, FoxA2, GATA4, GATA6, Vent-2, WNT3, and N-Cadherin.
To distinguish definitive endoderm from other tissues, transcriptional factors such as Sry-related HMG-box transcription factor Sox17 [14], [14], the mix-like gene MIXL1 [14], [15], [15], and Foxa 2 (previously known as the hepatocyte nuclear factor (HNF) 3β) [17], [18], [19], [19] have been used as markers of definitive endoderm.
Since the work by D'Amour et al. suggested a criteria for accepted markers of definitive endoderm [13], a combination of the increase of markers such as Sox 17, GSC, Foxa 2 and MIXL1 and a decrease in the primitive endoderm markers that are not expressed in definitive endoderm such as Sox 7 needs to be included to support the argument for production of definitive endoderm.
Immunostaining revealed efficient induction in iPS-derived cells of SOX17 and FOXA2, markers of definitive endoderm.
CXCR4 is a marker of definitive endoderm cells and a surface receptor that has been used to purify this population of cells without compromising their viability [33].
CXCR4 has been proposed as a marker of definitive endoderm [52] and the presence of this protein within the urinary bladder has been implicated as an important signaling molecule in mediating normal micturition [53].
The latter, together with CD45 is a marker of definitive hematopoietic progenitor cells (Mikkola et al., 2003).
In addition, EFhd2 is also down-regulated in embryos deficient for the transcription factor Foxa2 and is a marker of definitive endoderm [ 41].
Our in vitro model is distinct from other systems since RA promotes expression of all UPs, together with developmental markers of the definitive endoderm in both a temporal and concentration-dependent manner in vitro.
