Late stage definitive endodermal differentiation can be defined by Daf1 expression

Abstract

Background: Definitive endoderm (DE) gives rise to the respiratory apparatus and digestive tract. Sox17 and Cxcr4 are useful markers of the DE. Previously, we identified a novel DE marker, Decay accelerating factor 1(Daf1/CD55), by identifying DE specific genes from the expression profile of DE derived from mouse embryonic stem cells (ESCs) by microarray analysis, and in situ hybridization of early embryos. Daf1 is expressed in a subpopulation of E-cadherin + Cxcr4+ DE cells. The characteristics of the Daf1-expressing cells during DE differentiation has not been examined.

Results: In this report, we utilized the ESC differentiation system to examine the characteristics of Daf1-expressing DE cells. We found that Daf1 expression could discriminate late DE from early DE. Early DE cells are Daf1-negative (DE-) and late DE cells are Daf1-positive (DE+). We also found that Daf1+ late DE cells show low proliferative and low cell matrix adhesive characteristics. Furthermore, the purified SOX17(low) early DE cells gave rise to Daf1+ Sox17(high) late DE cells.

Conclusion: Daf1-expressing late definitive endoderm proliferates slowly and show low adhesive capacity.

Keywords: Adhesion; Daf1; Definitive endoderm; In vitro differentiation; Pluripotent stem cell; Proliferation.

Fig. 1

Daf1+ DE cells are less proliferative than Daf1- DE cells. a Mouse ESCs (ESC) were differentiated first to the anterior primitive streak then into the definitive endoderm (DE). Our previously reported microarray analysis of Foxa2, Sox17 and Daf1 expression during DE differentiation are plotted in a time dependent manner. Daf1 expression increased in the DE but not in the APS. ESC; mouse embryonic stem cells, APS; anterior primitive streak, DE; definitive endoderm. b Schematic drawing of the experiment to analyze cell cycle phases of the sorted DE. ES cells were differentiated into DE, then dissociated and sorted for Daf1+/- cells. The sorted cells were immediately analyzed for cell cycle. c Real time-PCR analysis showed that Daf1 expression was enriched in Daf1+ DE. Y axis shows relative gene expression, with 1 equivalent to Daf1 expression level in Daf1 + DE cells. d, e Proliferative properties of Daf1 + DE and Daf1-DE cells were assayed. d Cell proliferation marker, pH3 (M phase), and PCNA (every cell phases except G0) was down-regulated in Daf1 + DE. Analyzed by western blot analysis. e Cell cycle analysis revealed that Daf1 + DE reside longer in the G0/G1 phase with a shorter S and M/G2 phases compared to that of the Daf1-DE cells. Raw data are shown in Additional file 2. Student’s two tailed t-test

Fig. 2

Daf1+ DE cells have lower cell-matrix adhesion than Daf1- DE cells. a A schematic drawing showing experimental design. The sorted DE cells were plated onto matrigel-pre-coated dishes and cultured for 24 h (b, c) or 90 min (d, e). b Transmission images of the cells derived from the Daf1+/-DE cells after cultured for 24 h. c After 24 h culture, the Daf1 + DE cells gave rise to fewer cells. d Cell-matrix adhesion assay, after 90 min culture. Quantification of the attached cells. The adhesion ability of Daf1 + DE cells onto matrigel is weaker than that of the Daf1-DE cells. e Quantification of Cleaved caspase3/7+ apoptotic cells. The Daf1 + DE cells gave rise to a larger proportion of apoptotic cells. f The Integrin expression profiles of the DE cells detected by RT-PCR analysis. Student’s two tailed t-test. Scale bars; 100 μm

Fig. 3

Daf1-DE is the progenitor of Daf1+ DE. a A schematic drawing showing experimental design. b Quantifications of Foxa2+ and Sox17+ cells by immunocytochemistry after 90 min cell-matrix adhesion assay. c, d The proportion of cells expressing Sox17 was higher in the Daf1 + DE as detected by immunocytochemical analysis (c), and the level of Sox17 expression was higher in the Daf1 + DE as detected by western blot (d), of the sorted DE cells (without plating). e Flow cytometry analysis of the descendent cells of Daf1-DE and Daf1 + DE. Left panels: Daf1, Cxcr4 expression profiles of the sorted Daf1+/- DE cells. Right panels: both Daf1-DE and Daf1 + DE acquired Daf1 expression after 24 h. f The scheme of Fig. 3. Sox17^low Daf1-DE differentiated into Sox17^high Daf1 + DE. Student’s two tailed t-test. Scale bar; 100 μm

Fig. 4

Both Daf1+/- could differentiate into pancreatic and intestinal fates. a A schematic drawing showing experimental design. DE was sorted on day 5. The sorted cells were plated onto MEF feeders and cultured with pancreatic and intestinal differentiation medium. Daf1- or Daf1 + DE cells differentiated into Pdx1/GFP+ cells after 4 days culture (b) and Cdx2+ cells after 5 days culture (c). b Arrows depict small colonies of Daf1 + DE-derived Pdx1/GFP+ cells. Numbers depict differentiation ratios: Daf1-DE-derived Pdx1/GFP+ cells; 10.9 %, Daf1 + DE-derived Pdx1/GFP+ cells; 12 % (day 9). c Numbers depict differentiation ratios: Daf1-DE-derived Cdx2+ cells; 18.3 %, Daf1 + DE-derived Cdx2+ cells; 17.1 % (day 10). Scale bar; 100 μm

Fig. 5

Proposed role of Daf1 in DE differentiation. ICM/ESC differentiated into the E-cadherin + Cxcr4 + Foxa2 + Sox17^lowDaf1- early DE. These cells then differentiated into the E-cadherin + Cxcr4 + Foxa2 + SOX17^highDaf1+ late DE. The late DE differentiated into regionalized anterior (pancreatic) and posterior (intestinal) endodermal lineages. Transition from the Daf1-DE to Daf1 + DE is accompanied by restricted cell proliferation and cell-matrix adhesion. ICM; inner cell mass