Abstract
Background: The glycome has emerged as a key regulator of cell fate, partly through its ability to potentiate the action of numerous signaling pathways. We recently demonstrated that a sulfated component of the glycome plays a critical role in promoting the differentiation of embryonic stem cell (ESC)-derived embryoid bodies by modulating downstream growth factors, such as the insulin-like growth factor (IGF) signaling axis. However, the exact components of the glycome which promote ESC differentiation versus stemness remain uncharacterized, due to the lack of a rapid, simple and easily quantifiable methodology. As a proof-of-concept in this study, we utilized a custom-made glycoarray in combination with bioinformatics and molecular biology tools in order to uncover novel glyco-signatures underlying ESC differention in an embryoid body model. A better elucidation of the glycomic transcriptomal signature underlying ESC differentiation would allow us to better manipulate these cells towards a desired lineage. Method: We used a custom-designed Affymetrix microarray, the Glycogene-chip, to screen the transcriptome of differentiating embryoid bodies versus that of undifferentiated ESC. In conjunction with gene ontology, pathway analyses, real-time PCR and immunoblotting, we validated the involvement of the IGF family, and furthermore, uncovered novel differentially regulated genes belonging to the glycoprotein (Angiopoietin-1 and Angiopoietin-like members), sulfotransferase, sulfatase and glycosyltransferase families. Conclusion: These results suggest that the Glycogene-chip, in conjunction with the embryoid body model, provides a fast and reliable tool to uncover novel glycomic signatures that are critical to maitain ESC stemness versus differentiation. In turn, this will allow us to understand the mechanisms governing ESC fate, bringing us one step closer towards finding a new paradigm for the regenerative medicine field.
Highlights
The glycome has emerged as a key regulator of cell fate, partly through its ability to potentiate the action of numerous signaling pathways
These results suggest that the Glycogene-chip, in conjunction with the embryoid body model, provides a fast and reliable tool to uncover novel glycomic signatures that are critical to maitain embryonic stem cell (ESC) stemness versus differentiation
In the realm of using the Glycogene-chip to uncover novel roles of the glycome during ESC differentiation, we focused on several glyco-modifying transcripts that were upregulated to a lesser extent, including sulfatases (SULFs), sulfotransferases (CHST-1 and -2) and the glycoprotein ANGPTL-2
Summary
The glycome has emerged as a key regulator of cell fate, partly through its ability to potentiate the action of numerous signaling pathways. Glycosylation of protein and lipids constitutes one of the most common posttranslational modifications in eukaryotes, resulting in modulation of key developmental functions, including innate immunity, signal transduction and cell differentiation [1,2] These pleitropic effects of the glycome are mainly mediated by the presence of a wide range of glyco-enzymes and their respective isoforms, as well as by the fact that a single glyco-conjugate can simultaneously modulate the activity of numerous growth factors/receptor complexes due to the complexation of various factors onto one conjugate [3,4]. Glyco-conjugates are more potent determinants of cell fate as opposed to the action of a single growth factor/receptor pair Despite these predominant roles of the glycome in governing cell fate, its underlying mechanisms of action remain largely uncharacterized, especially in the stem cell field. These studies have all encountered similar limitations, including false negatives and Shiladitya Sengupta, BWH-HST Center for Biomedical Engineering, Harvard Medical School, 65 Landsdowne Street, Cambridge, MA 02139, USA
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